CN101509035A - Lung cancer parting gene sequence and uses thereof - Google Patents

Abstract

The invention discloses a gene sequence for genotyping of lung cancer and a gene chip of the gene sequence. The invention also discloses a method for using the gene sequence to genotype lung cancer samples. The gene sequence used for typing and diagnosing lung cancer of the present invention can quickly and high-throughput screen out genes that may be related to molecular typing of lung cancer, and carry out molecular typing of lung cancer tissues.

Description

Gene sequence and application of lung cancer classification

technical field

The invention relates to a gene detection and analysis method for pathological typing of lung cancer, which belongs to the application field of molecular biology technology.

Background technique

Lung cancer is pathologically divided into several types such as small cell carcinoma, squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. There are also multiple subtypes under the basic type. Different pathological types of lung cancer have differences in tumor biological behavior, sensitivity to radiotherapy, chemotherapy, and molecular targeted drugs, and the prognosis of each type is also different. Therefore, when making a clinical diagnosis of lung cancer, it is very important to clarify its pathological type for formulating treatment plans and analyzing prognosis.

Before the present invention, the pathological classification of lung cancer in the medical field mainly relied on microscopic morphology and immunohistochemical methods. The traditional histopathological method is to fix, dewax, and stain the obtained lesion tissue into sections to distinguish its microstructure under a microscope. This method has many steps, and it needs to wait for one or two days in vain. It is also unavoidable for subjectivity when pathology professionals interpret the slices.

Molecular classification of tumors is from the perspective of systems biology, using modern new high-throughput molecular analysis techniques to classify tumors according to the characteristics of molecular (molecular genetics, molecular biology) changes. Molecular typing is not only related to histomorphological typing, but also a positive complement to the latter. The existing molecular biology knowledge about lung cancer believes that the gene expression profiles of different pathological types of lung cancer are different, that is, different genes have different abundances in different pathological tissues.

DNA chip or DNA microarray is to immobilize a large number of DNA fragments or oligonucleotide fragments of a specific sequence on glass, silicon wafers and other carriers with high density in a matrix through microelectronic technology and microprocessing technology, and the samples to be tested are marked with fluorescent molecules Afterwards, hybridize with large DNA or oligonucleotide fragments on the chip, and obtain a large amount of genetic information through fluorescence scanning and computer analysis. Its outstanding feature is that it can quickly, accurately, and High-throughput detection and analysis can obtain the expression level related to a single gene, and the expression profile of similar or identical gene expression patterns.

Gene expression profiling established by high-throughput cDNA chip and oligonucleotide chip technology has been experimentally used in tumor diagnosis in the past few years, which is a revolutionary change. With the application of these tools, previously indistinct tumor subgroups have been identified. In addition, the development of gene signatures can be used to predict prognosis conclusions, which cannot be drawn from standard histopathology and immunohistochemistry methods, opening up the way to analyze tumors with pathological diagnosis. Through the application example of gene chip, it shows that this technology has a wide range of applications, and it can even improve the ability of histopathological diagnosis and prediction.

The gene expression profiles of small cell lung cancer and non-small cell lung cancer are significantly different: most of the genes that constitute the characteristic expression profile of squamous cell carcinoma are involved in cell detoxification and antioxidant processes (such as GST, carboxyl esterase, etc.), and are often related to bronchial epithelial cells to the environment. It is related to the metabolism and detoxification of carcinogenic factors (such as cigarette ingredients). Adenocarcinoma is characterized by abnormal expression of surfactant-related genes and small airway-related genes (such as Surfactants A2 and B, Mucin1), suggesting the cell origin and pathogenesis of this type of tumor. The characteristic gene expression profiles of large cell lung cancer, such as overexpression of HMGI(Y), TPA, etc., and low expression of E-cad, PAX-8, and r-catenin, etc., reflect epithelial-mesenchymal cell transdifferentiation. In addition, some cases diagnosed as large cell lung cancer by histology can be clearly classified into squamous cell carcinoma or adenocarcinoma group according to the gene expression profile. What is really valuable is the identification of tumor subtypes that cannot be distinguished by histomorphology but have clinical significance through the analysis of gene expression profiles.

Contents of the invention

One of the purposes of the present invention is to provide gene sequences for typing and diagnosing lung cancer, which include

(a) the sequence shown in SEQ ID NO 10 to SEQ ID NO 323;

(b) the complementary sequence of each of the sequences shown in SEQ ID NO 10 to SEQ ID NO 323;

(c) a sequence having at least 70% homology to each of the sequences shown in SEQ ID NO 10 to SEQ ID NO 323.

The second object of the present invention is to use the gene sequence in the field of lung cancer typing and diagnosis, and construct a gene chip for lung cancer genotyping.

A gene chip for lung cancer genotyping that realizes the second purpose of the present invention, which consists of a fixed carrier and an oligonucleotide probe fixed on the fixed carrier, wherein the oligonucleotide probe includes ( a) the sequence shown in SEQ ID NO 10 to SEQ ID NO 323; (b) the complementary sequence of each sequence in the sequence shown in SEQ ID NO 10 to SEQ ID NO 323; (c) with SEQ ID NO 10 to SEQ ID Each of the sequences shown in NO 323 has at least 70% homology. The oligonucleotide probe also includes several or all positive control probes and negative control probes selected from SEQ ID NO 1 to SEQ ID NO 10.

The oligonucleotide gene chip of the present invention is divided into 4 regions, each region is arranged in a matrix of 18×17, and each region contains 9 positive controls, 5 negative controls and 3 blank controls.

The third object of the present invention is to provide a method for genotyping lung cancer samples. This method uses genetic technology to screen genes related to the molecular typing of lung cancer, and provides a valuable basis for the genotyping diagnosis of lung cancer and the subdivision diagnosis of lung squamous cell carcinoma and lung adenocarcinoma subtypes. Provide a reference for realizing individualized treatment of lung cancer cases.

The lung cancer sample genotyping method for realizing the third purpose of the present invention comprises the following steps:

(1) Extract total RNA from 95D cells, lung adenocarcinoma tissue, and lung squamous cell carcinoma tissue, reverse transcribe the total RNA into cDNA, and make fluorescently labeled probes;

(2) The hybridization fluorescent probe prepared above is hybridized with the chip to obtain the hybridization result;

(3) Use the ScanarrayLite laser confocal scanner to scan the chip, and analyze the intensity and ratio of Cy3 and Cy5 fluorescence signals through Genepix 5.1 analysis software, where the difference between Cy5/Cy3 is more than two times and the ratio is greater than 2.0 or less than 0.5, indicating that there are differences in genes The gene expression profiles of lung squamous cell carcinoma and lung adenocarcinoma were analyzed by clustering method.

The gene sequence for lung cancer typing and diagnosis of the present invention can quickly and high-throughput screen out genes that may be related to lung cancer molecular typing, perform molecular typing on lung cancer tissues, provide certain clues, and provide more information for clinical diagnosis .

Description of drawings

Figure 1 is a distribution map of Oligo in each area of the chip. The red line frame is the positive control, the yellow line frame is the blank control, and the blue line frame is the negative control. In the black and white picture, red is black line, yellow and blue are white line;

Fig. 2 is the electrophoresis result of 95D cell total RNA;

Figure 3 is the hybridization result of 95D2 on Block2 of the chip;

Figure 3.1 (a) Cy3-labeled 95D1, (b) Cy5-labeled 95D2, hybridization results on one chip;

Figure 3.2 (a) (b) hybridization results labeled with Cy5;

Figure 4 shows the results of total RNA electrophoresis in some cancer tissues and paracancerous tissues of squamous cell carcinoma and adenocarcinoma;

Figure 4.1 shows the results of total RNA electrophoresis in cancer tissues and paracancerous tissues of the same squamous cell carcinoma patient;

Figure 4.2 shows the results of total RNA electrophoresis in cancer tissues and paracancerous tissues of the same adenocarcinoma patient;

Figure 5 is the result of hybridization of squamous cell carcinoma and its adjacent tissue with the chip, wherein (a) Cy3-adjacent squamous cell carcinoma tissue, (b) overlay of the two, (c) Cy5-squamous cell carcinoma tissue;

Figure 6 is the hybridization result of adenocarcinoma and its paracancerous tissue with the chip, wherein (a) Cy3-addenocarcinoma paracancerous tissue,

(b) Overlay of the two, (c) Cy5-adenocarcinoma tissue;

Figure 7 is a scatter diagram of (a) lung squamous cell carcinoma and its adjacent tissues, (b) lung adenocarcinoma and its adjacent tissues;

Figure 8 shows the results of hybridization between cancer tissues and chips in 7 cases of adenocarcinoma patients, in which (a) Cy3-X1, (b) Cy3-X3,

(c) Cy3-X5, (d) Cy5-X2, (e) Cy5-X4, (f) Cy5-X6, (g) Cy5-X7;

Figure 9 shows the results of hybridization between cancer tissues and chips in 8 cases of squamous cell carcinoma patients, in which (a) Cy3-L1, (b) Cy3-L3,

(c) Cy3-L5, (d) Cy5-L2, (e) Cy5-L4, (f) Cy5-L6, (g) Cy5-L7, (h) Cy5-L8;

Figure 10 is the hybridization result of (a) lung squamous cell carcinoma, (b) lung adenocarcinoma adjacent tissue and chip;

Figure 11 is an unsupervised clustering map of 8 cases of lung squamous cell carcinoma and 7 cases of lung adenocarcinoma and chip, where X represents adenocarcinoma, L represents squamous cell carcinoma, p represents paracancerous tissue, red represents high expression abundance, and yellow represents abundance Medium, green is low abundance. In black and white pictures, red is dark, yellow is light, and green is medium.

Detailed ways

The present invention will be further described below through embodiment.

Example 1 Construction of gene information database related to molecular typing of lung cancer

According to the genes related to molecular typing of lung cancer provided by previous studies and the information in the GenBank database, the BLAST analysis software provided by the National Center for Biotechnology Information (NCBI) was used to find out the conserved sequences of lung cancer related genes, and then the biological software Array Designer 4.2 was used to design 332 Oligo probes with a length of 60bp (mer).

1. Materials

1. Select the source of the target gene:

1.1. Collect 60 lung cancer-related genes, purchased from American Type Culture Collection.

1.2. On the basis of 60 lung cancer-related genes, the lung cancer-related genes were further screened by suppression subtractive hybridization (SSH) and cDNA Microarray.

The experiment is based on immortalized human bronchial epithelial cells (BEP2D) and its R15H20HE (pre-malignant transformation stage), R15H35 cells (malignant transformation stage), hybridized with cDNA chips established with 60 lung cancer-related genes. The differentially expressed gene libraries of BEP2D cells at different stages of malignant transformation were established by using SSH technology, among which A subtractive library (the cDNA of BEP2D cells is the tester, and the cDNA of R15H35 cells is the driver) has 416 clones, and the B subtractive library (the cDNA of R15H20 cells is The cDNA of BEP2D and R15H35 cells is a driver) and there are 301 clones, and the C subtraction library (the cDNA of R15H35 cells is a tester, and the cDNA of BEP2D cells is a driver) has 586 clones. All the clones in the 3 libraries were made on one chip, and 15 cases of lung cancer tissues, 5 cases of lung cancer tissues and 8 other cancer tissues (liver cancer, gastric cancer, esophageal cancer, breast cancer, leukemia, endometrial cancer, etc.) were screened. , 1-3 cases of brain glioma and colon cancer) mRNA expression differences. 208 genes with large differential expression were obtained: 27 cDNA clones with higher expression in lung cancer tissue than lung cancer adjacent tissue, 31 higher expression in lung cancer adjacent tissue than lung cancer tissue, the two were higher than the other 8 cancer tissues: There were 63 in adjacent tissues of lung cancer and 87 in lung cancer tissues.

1.3. Collect genes related to lung cancer pathological type, stage, prognosis and drug resistance from literature reports.

2. BLAST analysis

Find the corresponding full-length DNA sequences of the above genes from GenBank, and perform BLAST analysis on the gene sequences of the 5' non-coding region, 3' non-coding region, and structural proteins and non-structural proteins to ensure that they are consistent with gene sequences of other species. Strong specificity. According to the analysis results, the target fragments of each conserved region of lung cancer-related genes were obtained as candidate sequences for designing Oligo probes.

3. Design of Oligo Probe

Using the biological software Array Designer4.2 to analyze the conserved sequences of lung cancer genes retrieved by BLAST one by one, and design probes with a length of 60bp. In order to reduce non-specific hybridization and ensure the sensitivity and accuracy of the chip, the probe design follows the following five basic principles: ①G+C content is 50%-70%; ②The 50% melting temperature (Tm value) of the probe should be Close to 70°C, fluctuating 5°C; ③Avoid repeating a single base more than 8 times in a row; ④The internal complementary base of the probe molecule is less than 3bp; ⑤The similarity between each probe and the non-target gene must be <70%. The number of consecutive homologous bases is ≤5. When screening candidate probes, various parameters should be controlled within the optimal range as much as possible to ensure that the probes on the same chip get the best hybridization effect under the same hybridization and washing conditions.

The full sequences of lung cancer-related genes were obtained through GenBank search, and these gene sequences were put into the GenBank international database system for BLAST analysis, which confirmed that the sequences of each gene were highly conserved. Therefore, these gene sequences were selected as the target sequences of Oligo probes. Following the basic principles of probe design, 332 specific Oligo (oligonucleotide) probes with a length of 60mer were designed by using ArrayDesigner4.2 software. See Table 1 "Sequences of 332 Oligo Probes" for their sequences.

Example 2 Construction of oligonucleotide gene chips related to molecular typing of lung cancer

1. Reagent preparation

The main reagents required are prepared as follows:

1. PBS solution: Weigh 8g NaCl, 0.2g KCl, 1.44g Na ₂ HPO ₄ , 800ml distilled water, adjust the pH to 7.4 with HCl, add water to 1L, autoclave for later use.

2. 20×SSC: Dissolve 175.3g NaCl and 88.2g sodium citrate in 800ml water, adjust the pH to 7.4, add water to 1L, and autoclave for use.

3. 10% SDS: Dissolve 100g of SDS in 900ml of water, heat to 68°C to aid dissolution, adjust the pH to 7.2 with concentrated hydrochloric acid, add water to make up to 1L, and divide it for use.

4. 50×Denhardt stock solution: 5g of polysucrose, 5g of polyvinylrolidone, 5g of bovine serum albumin (component V) was dissolved in 500ml of water, sterilized by filtration, and set aside.

5. Hybridization solution: the composition is 50% formamide, 5×Denhardt solution, 6×SSC, 0.5% SDS and 100ug/ml, salmon sperm DNA.

2. Experimental method

1. Preparation of Oligonucleotide Probes

All oligonucleotide probe synthesis was performed on a 3400 DNA synthesizer using standard phosphoramidite chemistry. After synthesis, it was deprotected and cleaved with concentrated ammonia water at 55°C for 15 hours, purified with OPC column, and quantified by ultraviolet (UV-2400).

2. Oligonucleotide chip preparation

2.1. Preparation of amino slides: soak the slides with strong alkali ethanol solution, rinse with double distilled water, immerse in lysine in PBS solution to coat, take out and wash with water, centrifuge and dry. Store at room temperature for 2 weeks. Scan the slides to be sampled before sample application, and reject chips with high background signal intensity and poor quality.

2.2. Arrangement of oligonucleotide chip: put the purified probe on the glass slide with a sampler, divide it into 4 areas, and make a matrix of 18×17 in each area. According to the number of fragments to be sampled, the dot matrix of each oligo is determined in advance, and each sample is repeated 3 times on the chip. At the same time, a blank control, a negative control, and a positive control were set up.

2.3. Spotting of the gene chip: The oligonucleotide chip was provided by Beijing Huada Gene Chip, and the oligonucleotide probe was dissolved in 3×SSC to a concentration of 40 pmol/ul. Use the spotarray24 type chip spotter to determine the position and way of spotting on the glass slide according to the pre-array method, and adjust the spotting parameters. The oligonucleotide probes were spotted on the aminated glass slide with a distance of 230um, and placed in a desiccator at room temperature for one week to solidify the oligonucleotides on the slide.

2.4. Post-treatment of the gene chip: hydrate the above-mentioned chip with 1×SSC to make the spots on the chip uniform; fix the thymidine residues on the oligonucleotides on the glass slide and the amino groups on the glass slide by UV cross-linking at 65mJ The covalent bond between; Use 1-methyl-2-pyriglidinone to block the chip to reduce non-specific hybridization points.

3. Preparation of oligonucleotide chips

Put 332 oligos on the aminated slide to make an oligonucleotide chip according to the layout shown in Table 2:

1. Synthesize 332 oligos, including 9 positive controls. In addition, 5 negative controls and 3 blank controls were added, and 4 blocks (Blocks) were set up in a 2×2 array. The arrangement order of the probes in each block is shown in Table 2;

2. Each area is in a 18×17 matrix, and each area contains 9 positive controls, 5 negative controls, and 3 blank controls (see Figure 1);

3. There are 3 replicates of each sample on the chip (3 points replicated laterally).

Table 2 Arrangement order of chip partitions and probes on the chip

Blockl Block2 Block3 Block4

Block1

1-3 4-6 7-9 10-12 13-15 16-18 1 positive 1 positive 2 positive 3 blank negative 1 negative 2 2 oligo22 oligo23 oligo24 oligo25 oligo26 oligo27 3 oligo28 oligo29 oligo30 oligo31 oligo32 oligo33 4 oligo70 oligo71 oligo72 oligo73 oligo74 oligo75 5 oligo76 oligo77 oligo78 oligo79 oligo80 oligo81 6 oligo118 oligo119 oligo120 oligo121 oligo122 oligo123 7 oligo124 oligo125 oligo126 oligo127 oligo128 oligo129 8 positive4 positive 5 positive 6 negative 3 blank oligo166 9 oligo167 oligo168 oligo169 oligo170 oligo171 oligo172 10 oligo173 oligo174 oligo175 oligo176 oligo177 oligo178 11 oligo215 oligo216 oligo217 oligo218 oligo219 oligo220 12 oligo221 oligo222 oligo223 oligo224 oligo225 oligo262 13 oligo263 oligo264 oligo265 oligo266 oligo267 oligo268 14 oligo269 oligo270 oligo271 oligo272 oligo273 oligo307 15 oligo308 oligo309 oligo310 oligo311 oligo312 oligo313 16 oligo314 oligo315 Negative.4 negative5 blank positive7 17 positive 8 positive 9

Block2

1-3 4-6 7-9 10-12 13-15 16-18 1 positive 1 positive 2 positive 3 blank negative1 negative2 2 oligo46 oligo47 oligo48 oligo49 oligo50 oligo51

3 oligo52 oligo53 oligo54 oligo55 oligo56 oligo57 4 oligo94 oligo95 oligo96 oligo97 oligo98 oligo99 5 oligo100 oligo101 oligo102 oligo103 oligo104 oligo105 6 oligo142 oligo143 oligo144 oligo45 oligo146 oligo147 7 oligo148 oligo149 oligo150 oligo151 oligo152 oligo153 8 positive 4 positive 5 positive 6 negative 3 blank oligo190 9 oligo191 oligo192 oligo193 oligo194 oligo195 oligo196 10 oligo197 oligo198 oligo199 oligo200 oligo177 oligo178 11 oligo239 oligo240 oligo241 oligo242 oligo243 oligo244 12 oligo245 oligo246 oligo247 oligo248 oligo249 oligo286 13 oligo287 oligo288 oligo289 oligo290 oligo291 oligo292 14 oligo293 oligo294 oligo295 oligo296 oligo297 oligo325 15 oligo326 oligo327 oligo328 oligo329 oligo330 oligo331 16 oligo332 blank negative 4 negative 5 blank positive 7 17 positive 8 positive 9

Block3

1-3 4-6 7-9 10-12 13-15 16-18 1 positive 1 positive 2 positive 3 blank negative1 negative2 2 oligo10 oligo11 oligo12 oligo13 oligo14 oligo15 3 oligo16 oligo17 oligo18 oligo19 oligo20 oligo21 4 oligo58 oligo59 oligo60 oligo61 oligo62 oligo63 5 oligo64 oligo65 oligo66 oligo67 oligo68 oligo69 6 oligo106 oligo107 oligo108 oligo109 oligo110 oligo111 7 oligo112 oligo113 oligo114 oligo115 oligo116 oligo117 8 positive 4 positive 5 positive 6 negative 3 blank oligo154 9 oligo155 oligo156 oligo157 oligo158 oligo159 oligo160 10 oligo161 oligo162 oligo163 oligo164 oligo165 oligo202 11 oligo203 oligo204 oligo205 oligo206 oligo207 oligo208 12 oligo209 oligo210 oligo211 oligo212 oligo213 oligo250 13 oligo251 oligo252 oligo253 oligo254 oligo255 oligo256 14 oligo257 oligo258 oligo259 oligo260 oligo261 oligo298 15 oligo299 oligo300 oligo301 oligo302 oligo303 oligo304 16 oligo305 oligo306 negative 4 negative 5 blank positive 7 17 positive 8 positive 9

Block4

1-3 4-6 7-9 10-12 13-15 16-18 1 positive 1 positive 2 positive 3 blank negative1 negative 2 oligo34 oligo35 oligo36 oligo37 oligo38 oligo39 3 oligo40 oligo41 oligo42 oligo43 oligo44 oligo45 4 oligo82 oligo83 oligo84 oligo85 oligo86 oligo87

5 oligo88 oligo89 oligo90 oligo91 oligo92 oligo93 6 oligo130 oligo131 oligo132 oligo133 oligo134 oligo135 7 oligo136 oligo137 oligo138 oligo139 oligo140 oligo141 8 positive 4 positive 5 positive 6 negative 3 blank oligo178 9 oligo179 oligo180 oligo181 oligo182 oligo183 oligo184 10 oligo185 oligo186 oligo187 oligo188 oligo189 oligo226 11 oligo227 oligo228 oligo229 oligo230 oligo231 oligo232 12 oligo233 oligo234 oligo235 oligo236 oligo237 oligo274 13 oligo275 oligo276 oligo277 oligo278 oligo279 oligo280 14 oligo281 oligo282 oligo283 oligo284 oligo285 oligo316 15 oligo317 oligo318 oligo319 oligo320 oligo321 oligo322 16 oligo323 oligo324 negative4 negative5 blank positive 7 17 positive 8 positive 9

Example 3 Verification of human highly metastatic lung giant cell carcinoma cell line 95D cell line on oligonucleotide chip

1. Reagent preparation

The main reagents required are prepared as follows:

1.1640 culture medium: 1 bag of RPMI ₁₆₄₀ , dissolved in 800ml of water, add 2g of sodium bicarbonate, add water to 1L, adjust the pH to 7.2 with hydrochloric acid, filter and sterilize for use.

2. 0.25% trypsin: Weigh 0.125g of trypsin, add 50ml of water, filter and sterilize, aliquot and store at -20°C.

3. TAE electrophoresis buffer: use 242g Tris base, 57.1ml glacial acetic acid, 37.2gNa ₂ EDTA2H ₂ O, add water to 1L, make a 50× stock solution, and dilute 50 times when used.

4. DEPC water: Dissolve 500ul DEPC in 500ml distilled water, place at 37°C for 12h, and sterilize by high pressure steam for later use.

5. 10% SDS: Dissolve 100g of SDS in 900ml of water, heat to 68°C to aid dissolution, adjust the pH to 7.2 with concentrated hydrochloric acid, add water to make up to 1L, and divide for use.

6. Hybridization solution (1250 μl): formamide (785 μl), 10% SDS (50 μl), 50×Denhardt (100 μl), 20×SSPE (315 μl).

7. Blocking solution (540 μl): Polydeoxyadenosine nucleotide Polyda (5 μg/v μl (20 μl), tRNA (10 μg/μl, 20 μl).

8. Hybridization chamber solution (1000 μl): Ddwater (610 μl), 20×SSC (160 μl), formamide (230 μl).

9. Washing solution after hybridization: Washing solution A: 1×SSC, 0.2% SDS, Washing solution B: 0.2×SSC, Washing solution C: 0.1×SSC.

2. Recovery, culture and passage of 95D cells

1. Take out the cryopreservation tube containing 95D cells from liquid nitrogen, put it into a beaker filled with 37°C water immediately, and stir it frequently to make it melt into a suspension;

2. Add 5ml of 10% BCS RPMI ₁₆₄₀ complete culture medium, gently blow and beat evenly, centrifuge at 1000rpm×5min, and discard the supernatant;

3. Add 5ml of culture medium again, blow gently to form a suspension, centrifuge at 1000rpm×5min, and discard the supernatant;

4. Add 5ml of culture medium, blow gently to form a single cell suspension, inoculate into a culture bottle, place in a humidified 5% CO ₂ incubator at 37°C, and replace the culture medium after 24 hours.

5. Take out the culture bottle from the incubator, suck out the culture medium, wash it twice with PBS, add 0.5ml of 0.25% trypsin, and incubate at 37°C for 2-3 minutes;

6. Pipet the adherent cells repeatedly, and transfer the cell suspension to a 15ml centrifuge tube. Add 5ml of PBS, centrifuge at 1000rpm×5min, discard the supernatant;

7. Add 5ml PBS again, 1000rpm×5min, discard the supernatant;

8. Add 10ml of 10% BCS RPMI ₁₆₄₀ complete culture solution, blow gently to form a single cell suspension, transfer to 2 culture flasks respectively, and place them in a humidified 5% CO ₂ incubator at 37°C for cultivation.

3. Extraction of total RNA

1. After the cells have been cultured for 24 hours, remove the culture medium;

2. Add TRIzol according to the ratio of 2ml TRIzol reagent per ^10cm2 ;

3. Slowly rotate the culture dish several times to make the TRIzol reagent fully contact the surface of the culture dish for digestion;

4. Transfer to a 1.5ml RNase-free Eppendorf tube treated with DEPC, and place at room temperature for 5 minutes to fully lyse;

5. Then centrifuge at 4°C, 12,000g for 5 minutes, and discard the precipitate;

6. Add 200ul of chloroform (pre-cooled) to each 1ml of TRIzol, add to the homogenate, close the cap of the centrifuge tube tightly, shake and mix well, and place on ice for 15min;

7.4°C, 12000g, centrifuge for 15min;

8. Carefully take the upper aqueous phase and transfer it into a new EP tube;

9. Add 0.5ml of isopropanol (pre-cooled) for every 1ml of TRIzol, gently invert and mix the liquid, and place it at -20°C for 30min;

10.4°C, 12000g, centrifuge for 10min, discard the supernatant;

11. Add 1ml of 75% ethanol for every 1ml of TRIzol, shake the centrifuge tube gently, and suspend the sediment;

12.4°C, 8,000g, centrifuge for 5min, discard the supernatant carefully, and blot dry with absorbent paper;

13. At room temperature, dry the sediment in the ultra-clean bench for 5-10 minutes;

14. After adding RNase-free water to completely dissolve the RNA precipitate, store at -70°C.

Total RNA Identification:

(a) Determination of RNA concentration: add 98 μl of DEPC-treated double distilled water to the microcuvet, then add 2 μl of sample (diluted 50 times), and measure the D ₂₆₀ and OD ₂₈₀ values of each sample with ND-1000 UV spectrophotometer , and the RNA concentration was calculated according to the following formula: RNA amount (μg/μl)=OD ₂₆₀ ×40×dilution factor/1000.

(b) Determination of RNA purity: add 99ul of DEPC-treated double distilled water to the microcuvet, then add 1 μl of sample (diluted 100 times), and measure the OD ₂₆₀ , OD ₂₈₀ and D ₂₃₀ , calculate the OD ₂₆₀ /OD ₂₈₀ ratio. When the OD ₂₆₀ /OD ₂₈₀ is 1.8-2.0, it means that the extracted RNA has high purity and no protein contamination.

(c) Integrity detection of RNA: Use 1% agarose electrophoresis to detect the integrity of the RNA sample. Take 5ul sample and separate the total RNA by agarose electrophoresis. Complete and clear RNA bands of 28S and 18S are observed, and 28S: When 18S=2:1, it means that the RNA sample is intact and not degraded. A reversal of this ratio indicates RNA degradation, since 28S RNA can be characteristically degraded to 18S RNA.

(3) Labeling probes by reverse transcription

1. Direct labeling method

95D cells were labeled with fluorescent Cy3 or Cy5, and 20 μg of total RNA was taken (operated according to the instructions of the first-strand synthesis kit from Invitrogen, USA) for reaction. The reaction steps were as follows:

1. Add the following ingredients to the 1.5ml RNase-free centrifuge tube:

mRNA 20μg

1.2. Place the microcentrifuge tube in a thermal cycler, incubate at 68°C for 10 minutes, then quickly take it out and place it on ice to cool for 5 minutes, then centrifuge gently;

1.3. Add the following components to the tube in a dark room:

components dose RNAsin 0.5μl 5×first strand buffer 4μl

0.1M DTT 2μl Low concentration dNTP mixture (AGT 10mM each, C4mM) 1μl 1mM Cy3-dCTP/Cy5-dCTP 1μl

After mixing and centrifuging briefly, incubate at 42°C for 2 minutes;

1.4. Add 1 μl of SuperScriptII RNaseH reverse transcriptase (200u/μl) to the tube, mix well, and incubate at 42°C in the dark for 2 hours;

Incubate at 68°C for 15 minutes;

1.6. Cool to room temperature, add 25μl 1M HEPES to neutralize NaOH; mix the two reaction solutions (for Cy3 markers and Cy5 markers hybridized on the same chip);

1.7. Replenish water to 500μl, extract once with phenol:chloroform:isoamyl alcohol (25:24:1) extract; centrifuge at 10000rpm for 5min, and take the supernatant;

1.8. Add YM-30 at room temperature and centrifuge at 12000rpm for 8min to purify the probe, so that the final volume is 4μl;

1.9. Add 4.5 μl of 2M ethanolamine to react at room temperature for 15 minutes to terminate the coupling. The product coupled with fluorescein was recovered, purified, concentrated and dried.

Identification and concentration determination of cDNA

Take 1ul of the reverse transcription product and perform electrophoresis on a 1% agarose gel, and estimate the size of the product to determine the quality of the probe. Take 1 μl and dilute it to 100 μl, measure its optical density OD ₂₆₀ and OD ₂₈₀ values with an ND-1000 ultraviolet spectrophotometer, and measure the concentration and fluorescence incorporation rate of the obtained cDNA. Concentration of cDNA: 50 ng/μl.

2. Indirect labeling method

In step (3), do not add Cy3-dCTP/Cy5-dCTP label, first perform reverse transcription; after step (5), each concentrated probe above should be dissolved in 10 μl water respectively, and 1 μl 1M sodium bicarbonate (pH9 .0), then labeled with 1ul Cy3 or Cy5 respectively, and incubated at room temperature for 60 minutes. Other steps are the same as direct labeling method.

(4) Chip hybridization and cleaning

1. hybridization

1.1. Preparation of hybridization solution:

20*SSPE 0.45μl Blocker 0.15μl PolydA 0.30μl

Hybridization Solution 7μl Probe 4μl

Heat to 95°C for 3 minutes, and centrifuge at the fastest speed for 2 minutes.

1.2. Add 12 μl of hybridization system to the chip, mix well and denature at 95°C for 3 minutes, centrifuge at 13,000 rpm for 5 minutes, add the hybridization solution to the chip and incubate for more than 16 hours in a 42°C hybridization box.

2. Cleaning

After hybridization, wash with washing solution A (2×SSC, 0.1% SDS) twice for 5 minutes each, wash solution B (0.2×SSC, 0.1% SDS) twice for 5 minutes each, wash solution C (0.2×SSC) Wash twice for 5 minutes each, centrifuge the chip, and blow dry (dry the liquid on the chip with a hair dryer).

(5) Fluorescent signal detection and result analysis

Place the processed chip on ScanArray3000 (General Scanning Int.) laser confocal fluorescence scanner, scan with Cy3 and Cy5 light respectively, and adjust the scanning intensity of light to achieve the best result. Scanning results were analyzed using Genepix5.1 software to collect data in pictures.

3. Results Analysis

1.95D total cellular RNA

18S RNA and 28S RNA can be clearly seen after 1% agarose gel electrophoresis of the extracted 95D cell total RNA (Fig. 2, wherein the first lane is 95D1; the second lane is 95D2; the third lane is 95D3). The content ratio is about 1:2, and the proper ratio indicates that the RNA is not degraded. Its OD ₂₆₀ /OD ₂₈₀ are all between 1.9-2.0, indicating that the quality is good. The extracted RNA requires no DNA contamination, otherwise it must be treated with DNase enzyme or further purified into mRNA.

2. Gene chip hybridization results

The probes of the 95DD cells labeled by the reverse transcription method were hybridized with the chip, and the results are shown in Figures 3, 3.1 and 3.2: Figure 3 shows the continuous horizontal dots of genes on the same chip, and the average CV value of the gene (variation Coefficient) is 10%; Figure 3.1 shows the hybridization results of 95D1 and 95D2 using indirect labeling method on one chip; Figure 3.2 shows the hybridization results of both using Cy5 labeling, 95D2 using indirect labeling method, and 95D3 using direct labeling method. Figure 3.2 After data analysis, it is found that 322 genes among the 332 genes have high repeatability, and the mutual ratio between 3 repetitions is between 0.5 and 2.0; 6 genes have poor repeatability, and 3 Inter-repeat partial inter-ratio values are out of this range. Therefore, the reproducibility of the 332 gene crosses of 3 replicates was 98.2%.

Embodiment 4 Clinical verification of the oligonucleotide chip of the present invention

In this example, the oligonucleotide chip of the present invention was verified by taking samples from 15 cases of patients with confirmed diagnosis of lung cancer by pathological examination. Among them, there were 8 cases of lung squamous cell carcinoma, all of which were male; 7 cases of lung adenocarcinoma, 4 cases of male and 3 cases of female. 6 cases (4 cases of squamous cell carcinoma and 2 cases of adenocarcinoma) were examined for paracancerous tissues at the same time. Cancer tissues and paracancerous tissues were frozen in liquid nitrogen within 30 minutes after surgical separation. The verification method is as follows.

1. Prepare TAE electrophoresis buffer, DEPC water, 10% SDS, hybridization solution, blocking solution, hybridization chamber solution and post-hybridization cleaning solution according to the method in Example 3.

2. Preparation of hybridization fluorescent probes

1. Extraction and identification of total RNA

Operate according to the TRIZOL Reagent total RNA extraction instructions: wrap the tissue in foil and store it in liquid nitrogen, and pre-cool the equipment to be used in advance.

1.1. Take out the cryopreservation tube from the liquid nitrogen, use the sterilized forceps to clamp out the tissue, quickly crush the tissue with a hammer, add a small amount of liquid nitrogen to grind it quickly, wait until the tissue becomes soft, then add liquid nitrogen, and grind it three times, so that The tissue is thoroughly ground and then transferred to a glass homogenizer;

1.2. Add TRIzol. The dosage is to add 1ml TRIzol reagent per 50-100mg tissue;

1.3. Fully mix TRIaol with the tissue in a homogenizer and homogenize;

1.4. Transfer all the tissue homogenate into a 1.5ml Eppendorf tube that has been treated with DEPC without RNase, and place it at room temperature for 5 minutes to fully lyse it;

1.5. Centrifuge at 4°C and 12,000g for 5 minutes, and discard the precipitate;

1.6. Add 200 μl of chloroform (pre-cooled) to each 1ml of TRIzol, put it into the homogenate, close the cap of the centrifuge tube tightly, shake and mix well, and place on ice for 15 minutes;

1.7.4°C, 12,000g, centrifuge for 15min;

1.8. Carefully take the upper aqueous phase and transfer it into a new EP tube;

1.9. Add 0.5ml of isopropanol (pre-cooled) for every 1ml of TRIzol, gently invert and mix the liquid, and place it at -20°C for 30min;

1. 10.4°C, 12000g, centrifuge for 10min, discard the supernatant;

1.11. Add 1ml of 75% ethanol for every 1ml of TRIzol, shake the centrifuge tube gently, and suspend the sediment;

1. Centrifuge at 8,000g for 5 minutes at 12.4°C, discard the supernatant carefully, and blot dry with absorbent paper;

1.13. At room temperature, dry the sediment in the ultra-clean bench for 5-10 minutes;

1.14. After adding RNase-free water to completely dissolve the RNA precipitate, store at -70°C.

Identification of total RNA: RNA concentration determination and RNA integrity detection were carried out according to the method in Example 3.

2. Labeling probes by reverse transcription

Take 20 μg of total RNA (operated according to the instructions of the first-strand synthesis kit from Invitrogen, USA) for reaction, and the reaction steps are as follows:

2.1.1.5ml RNase-free centrifuge tube add the following components:

mRNA 20μg

2.2. Place the microcentrifuge tube in a thermal cycler, incubate at 68°C for 10 minutes, then quickly take it out and cool it on ice for 5 minutes, then centrifuge gently;

2.3. Add the following components to the tube in a dark room:

components dose RNAsin 0.5μl 5×first strand buffer 4μl 0.1M DTT 2μl Low concentration dNTP mixture (AGT 10mM each, C4mM) 1μl 1mMCy3-dCTP 1μl

After mixing and centrifuging briefly, incubate at 42°C for 2 minutes;

2.4. Add 1 μl of reverse transcriptase (200u/μl) to the tube, mix well and incubate at 42°C in the dark for 2 hours;

孵育68℃，15min；

Incubate at 68°C for 15 minutes;

2.6. Cool to room temperature, add 25μl 1M HEPES to neutralize NaOH;

Mix the two reaction solutions (Cy3 marker and Cy5 marker for hybridization on the same chip);

2.7. Replenish water to 500 μl, extract once with phenol:chloroform:isoamyl alcohol (25:24:1) extract; centrifuge at 10000r/min for 5min, and take the supernatant;

2.8. Add YM-30 at room temperature and centrifuge at 12000r/min for 8min to purify the probe, so that the final volume is 4μl;

2.9. Add 4.5 μl 2M ethanolamine to react at room temperature for 15 minutes to terminate the coupling. The product coupled with fluorescein was recovered, purified, concentrated and dried.

Identification and concentration determination of cDNA

Take 1ul of the reverse transcription product and perform electrophoresis on a 1% agarose gel, and estimate the size of the product to determine the quality of the probe. Take 1 μl and dilute it to 100 μl, measure its optical density OD260 and OD280 values with a UV spectrophotometer, and measure the concentration and fluorescence incorporation rate of the obtained cDNA. Concentration of cDNA: 50 ng/μl.

3. Chip hybridization and cleaning

1. Hybridization method

1.1. The cancer and para-cancerous tissues of the same lung squamous cell carcinoma patient, and the cancerous and para-cancerous tissues of the same lung adenocarcinoma patient were selected to hybridize with the chip to further verify the differences in the expression of the genes initially screened;

1.2. The cancer tissues of 8 squamous cell carcinoma patients were randomly selected to hybridize with the chip, and the consistency among different squamous cell carcinoma patients and the existence of differential genes were checked; the cancer tissues of 7 adenocarcinoma patients were randomly selected to hybridize with the chip, and the Consistency among different adenocarcinomas and differential genes in them;

1.3. Paracancerous tissues of 6 lung cancer patients (2 adenocarcinoma, 4 squamous cell carcinoma) were hybridized with the chip;

2. Hybridization

2.1. Prepare the hybridization solution according to the method in Example 3.

2.2. Add 12 μl of hybridization system to the chip, mix and denature at 95°C for 3 minutes, centrifuge at 13,000 rpm for 5 minutes, add the hybridization solution to the chip and incubate at 42°C for more than 16 hours.

3. After washing the hybridization, use washing solution A (2×SSC, 0.1% SDS) to wash twice for 5 minutes each, wash solution B (0.2×SSC, 0.1% SDS) twice for 5 minutes each, wash solution C (0.2 ×SSC) and washed twice for 5 min each, the chip was centrifuged and dried.

4. Fluorescent signal detection and result analysis

1. Signal detection

Place the hybridized core after rigorous washing on ScanArray3000 (General Scanning Int.), scan with Cy3 and Cy5 light respectively, adjust the scanning intensity of light, and filter background noise to achieve the best results , to obtain images of fluorescent signals. The Cy3/Cy5 raw signal was equalized and corrected with a pre-selected internal reference.

2. Result Analysis

The images were processed with Genepix 5.1 software, and the intensity and ratio of Cy3 and Cy5 fluorescence signals were analyzed to obtain the information of differentially expressed genes in the two tissues. The following conditions are used as the standard of differential expression: (1) the absolute value of the natural logarithm of Cy5/Cy3>0.69 (the signal difference between Cy3 and Cy5 is more than twice); (2) the signal gray value of Cy3/Cy5, compared with its background The value must be greater than 2 times. Cy5/Cy3 ratio<0.5 means down-regulated expression, and ratio>2.0 means up-regulated expression. At the same time, MADS software performed unsupervised clustering analysis on the gene expression profiles of paracancerous tissues of 15 cases of lung cancer and 6 of them (2 cases of adenocarcinoma, 4 cases of squamous cell carcinoma).

Five. Results

1. Extraction of total tissue RNA

The integrity of the RNA of the pathological tissue was identified by electrophoresis. The RNA electrophoresis results of some samples shown in Figure 4, Figure 4.1, and Figure 4.2 show that most of the RNA has good integrity.

2. Expression chip hybridization results

2.1. The same lung squamous cell carcinoma patient's cancer tissue and its adjacent tissue, and the same lung adenocarcinoma patient's cancer tissue and its adjacent tissue were hybridized with the chip (see Figure 5). Signals due to Cy3 fluorescence and Cy5 fluorescence are indicated in green and red, respectively. Therefore, for the two superimposed fluorescent signals at a certain point, if the Cy3 signal is stronger, the point is more green (showing a down-regulation trend), indicating that the gene is highly expressed in cancer tissue and low in paracancerous tissue; if the Cy5 signal is stronger Strong, the point is mostly red (up-regulation trend), which means that the gene is lowly expressed in cancer tissue, but highly expressed in non-cancerous tissue; if the intensity is similar, it is yellow. This figure reflects the difference in the abundance of each gene on the chip between cancer tissue and para-cancer tissue.

2.2. The scatter diagram of the hybridization signal of the gene chip is shown in Fig. 7 . The X-axis coordinates with Cy3 fluorescence intensity, the Y-axis coordinates with Cy5 fluorescence intensity, and each data point represents the hybridization signal of a gene point on the chip. The diagonal line at 45°C is the area where the gene expression is stable, which means that the ratio of Y to X is between 0.5 and 2.0, which basically belongs to non-differential expression. Points away from this line represent that the ratio of Y value to X value is outside the range of 0.5 to 2.0, indicating that the differential expression of the signal on the expression chip is greater than 2 times (probably data with differential expression).

2.3. The squamous cell carcinoma tissues of 8 squamous cell carcinoma patients were hybridized with the chip to check the heterogeneity among cancer tissues of different squamous cell carcinoma patients; For the heterogeneity among cancer tissues, see the hybridization results in Figure 8 and Figure 9.

2.4. The result of hybridization between paracancerous tissue of 6 cases of lung cancer and chip is shown in Fig. 10 .

3. Data Analysis Results

3.1.1 Squamous cell carcinoma tissue and its paracancerous tissue were hybridized with the microarray. There were 4 up-regulated genes and 156 down-regulated genes. See Tables 3 and 4. One case of adenocarcinoma tissue was hybridized with its paracancerous tissue, and 23 genes were up-regulated and 147 genes were down-regulated. See Tables 5 and 6.

3.2. Differentially expressed genes were analyzed by bioinformatics through GeneBank, mainly divided into the following categories: ① Oncogenes: wnt4, c-fos; ② Cell cycle regulatory genes: SPP1, CDK4; ③ Tumor proliferation and metastasis genes: pro-alpha -1type collagen, collagen alpha-2, PAI-1.

3.3. The unsupervised clustering method was used to group 15 samples and 332 genes. The samples were generally described in a tree form, and the length of the branches reflected the correlation of the samples. 5 cases of lung adenocarcinoma and 5 cases of lung squamous cell carcinoma were classified In one group, 2 cases of poorly differentiated squamous cell carcinoma were classified into one group (see Figure 11, where X represents adenocarcinoma, L represents squamous cell carcinoma, and p represents paracancerous tissue).

3.4. The consistency of expression of 332 genes in 7 adenocarcinoma tissues was between 52-92% (see Table 7). For pairwise comparison, a total of 21 comparison groups were obtained. Among them, the similarity between 5 groups (23%, 5/21) is between 50-60%; the similarity between 9 groups (42.9%, 9/21) is between 60-70%; The similarity between 4 groups (19%, 4/21) is between 70-80%, the similarity between 2 groups (9.5%, 2/21) is between 80-90%, and one group ( 5%, 1/21) the similarity between more than 90%.

3.5. The consistency of 332 genes in 8 cases of squamous cell carcinoma tissues was between 55-97% (see Table 8). For pairwise comparison, a total of 28 comparison groups were obtained. Among them, there are 11 groups (39%, 11/28) whose similarity is between 50-60%; there are 8 groups (28.6%, 8/28) whose similarity is between 60-70%; The similarity between 3 groups (10.7%, 3/28) is between 70-80%, and the similarity between 4 groups (14.3%, 4/28) is between 80-90%, and there are 2 groups The similarity between (7.1%, 2/28) is above 90%.

3.6.7 cases of lung adenocarcinoma tissues shared 29 differential genes, 7 of which were different from the squamous cell carcinoma group, such as DCTN2, Stst2, BRCA2, TGFD, etc.; 8 cases of lung squamous cell carcinoma tissues shared 46 differential genes, which were different from the adenocarcinoma group. There are 24 different cancer groups, such as KRT6A, HMGB2, lumican and so on. Both lung adenocarcinoma tissue and lung squamous cell carcinoma tissue expressed Bax, Myb, EGR1 and other 22 genes. (See Table 9)

3.7. In addition to the 15 samples in this example, after a newly collected lung cancer tissue was hybridized with the chip, the similarity with 7 cases of lung adenocarcinoma was 63%, and the similarity with 8 cases of lung squamous cell carcinoma was 49%, and the final pathological diagnosis confirmed Indeed lung adenocarcinoma.

Table 3 Up-regulated genes in lung squamous cell carcinoma and its adjacent tissues

gene number gene name GeneBank Cy5/Cy3 75 pro-alpha-1 type 3 collagen X14420.1 5.20 42 rearranged Ig kappa lightchain variable region X72475.1 4.45 265 topIIB X68060 3.47 200 cDNA from clone DKFZp564H191 AK026634.1 3.22

Table 4 The down-regulated genes (partial) in lung squamous cell carcinoma and its adjacent tissues

gene number gene name GeneBank Cy5/Cy3 148 PAI-1 NM_000602.1 0.01 184 Antigen peptide transporter 2 NM_000544.3 0.02 175 Uracil-DNA glycosylase (UNG) NM_003362.2 0.02 159 wnt-4 NM_030761.3 0.03

Table 5. Genes up-regulated in lung adenocarcinoma and its paracancerous tissues (partial)

gene number gene name GeneBank Cy5/Cy3 238 S100P_AA131149 AA131149.1 23.64 190 SPP1 NM_000582.2 11.07 42 rearranged Ig kappa lightchain variable region X72475.1 6.34 75 pro-alpha-1 type 3 collagen X14420.1 5.03 200 cDNA from clone DKFZp564H191 AK026634.1 2.43 265 topIIB X68060 2.11

Table 6 The genes down-regulated in lung adenocarcinoma and its adjacent tissues (partial)

gene number gene name GeneBank Cy5/Cy3 153 Opioid receptor, kappa 1 NM_000912.3 0.02 28 advanced glycosylation endproduct-specific receptor AGER NM_001136.3 0.03 127 CK19 NM_002276.3 0.03 184 Antigen peptide transporter 2 NM_000544.3 0.05 148 PAI-1 NM_000602.1 0.05 159 wnt-4 NM_030761.3 0.06

Table 7 Results of heterogeneity analysis among 7 adenocarcinoma tissues

X2 X3 X4 X5 X6 X7 X1 67% 64% 64% 52% 57% 55% X2 60% 74% 55% 67% 92%

X3 82% 65% 77% 61% X4 66% 75% 76% X5 87% 57% X6 65%

Table 8 Results of heterogeneity analysis among 8 squamous cell carcinoma tissues

L2 L3 L4 L5 L6 L7 L8 L1 97% 50% 82% 52% 57% 61% 55% L2 55% 86% 63% 55% 70% 52% L3 62% 67% 88% 75% 89% L4 65% 58% 59% 51% L5 61% 56% 53% L6 65% 93% L7 65%

Table 9. Shared differential genes between adenocarcinoma and squamous cell carcinoma and differential genes between them

gene number gene name Adenocarcinoma Shared Differential Genes Differential genes between adenocarcinoma and squamous cell carcinoma squamous cell carcinoma shared differential genes 14 Solute carrier family 27 √ 16 Zd36c11.s1 (TGFBR2) √ 36 P50a(DCTN2)(PIGPC1) √ 61 CENPM (LAMB2) √ 160 Stat2 √ 201 BRCA2 √ 331 TGF √ 192 Alpha-2-macroglobulin √ 207 Bax √ 248 Beta-2-microglobulin √ 199 cDNA from clone DKFZ564H191 √ 223 Collagen alpha-2 √ 246 HLA-A √ 247 HLA-B √ 114 FLJ22883 fis √

212 MDR √ 213 Myb √ 42 Rearranged Ig kappa light chain variable region √ 101 RPL23 √ 119 LOC154336 √ 200 TI-227H √ 65 UBC √ 291 CX43 √ 294 EGR1 √ 304 KLF6 √ 321 TGF √ 256 GST-π √ 257 HSPB1 √ 259 MDM2 √ 70 LAMB2 √ 208 CDK4 √ 137 c-fos √ 127 Ck19 √ 17 HMGB2 √ 111 Zeta1 √ 108 EEF1A1 √ 228 KRT6A √ 131 lunican √ 26 NCI-CGAP-Lu1 √ 135 N-myc √ 49 ODC1 √ 92 POLR2G √ 53 RB-binding protein √ 224 Small cell lungcar cinomaepithelial antigen √ 312 Mmp9 √ 292 Cx43 √

295 Cx45 √ 316 P63 √ 270 LRP √ 271 P16 √ 258 HSPB2 √ 260 MGMT √

Table 1. Sequences of 332 Oligo probes

oligo name 1 name 2 sequence 1 βactin__255 βactin__255 CGGAGCGTGGTTACTCGTTCACGACCACGGCCGAGCGCGAAATTGTGCGGGACATCAAGG 2 Gapdh97__1423 Gapdh97__1423 AGCACTTTTACGGGTGGGGGGAGGGAGTGTTCTGCTGGTCTCCAATTACCAAGAATTCTC 3 positive control NM__001916.2 AGGCTCTAGCTCTGGGCCCTCCTTCAGCCCCCATCATGGGAATAAATTAATTTTCTCAAT 4 positive control NM__003528.2 GAATCCAAATGAGCTCTAGACATTATCACAGACTGAATAGATCTTAACTGTCTCCTACAT 5 positive control NM__018955.2 TCTGCACTATAGCCATTTGCCCCAACTTAAGTTTAGAAATTACAAGTTTCAGTAATAGCT 6 positive control NM__005566.1 AGTCACATCCTGGGATCCAGTGTATAAATCCAATATCATGTCTTGTGCATAATTCTTCCA 7 positive control NM__005387 GCTGAAAGCTAGACCTTTGGTATTTTCCATGCTATAATTCTTATGGCTGCTGAAATGTGT 8 positive control NM__002046.3 CCCCTTGAAGAGGGGAGGGGCCTAGGGAGCCGCACCTTGTCATGTACCATCAATAAAGTA 9 positive control NM__001967.3 AGCAAATCCTAGGTAGGGTTTAATCCCCAGTAAAATTGCCATATTGCACATGTCTTAATGA 10 Cycloph640__726 Cycloph640__726 AACGGAGAGATGATTCTGAGACACCACGAGGACATGCAGGTGGAGGAGTGCGGATGCCTG 11 MGC82420proein EST__33325__1721 AACTGGGCCTGTGGAGTTGCCCGTGTCCCAGGGGTAGTCGAGGCCGTACGGGATTGATAA 12 HPRT1__1584 HPRT1__1584 CCAGGAATACACATAAAGAGCAAAAATGCTATTCTACAATATCCTCATCTTACCAGCTCAC 13 SLC6A8 HSU36341__16256 TCTCCACGCTGCCTCTGGAGGTCAGGAGAAAACTATGTGGCTTCCCTAACACAGACAGGG 14 Fatty acid transport NM__024330.1 CACTATTTTGTAATAAATGTGGCTGGAGCTGATCCAGCTGTCTCTGACCTACAAAAAAAAA 15 FYC01 N M__024513.1 GGTGGAAAACTTTTGTATCCCTAAGCATATTATTTATAGTGTCTGCCATGCCATGTGGA 16 Zd36c11.s1 W68630__195 TCTAGACTGGTAGAGCCGAGCCACCGGTGAGAAGCAANGGACAGCAGCAGGAAGAGCCAT

17 HMGB2 NM__002129.2 GCAGGTTGTAGCTTTTTGATGGGCTACTCATACAGTTAGATTTTACAGCTTCTGATGTTG 18 CDH5 NM__001795.2 CCCTCACACTGCAAGGGATTGTAGATAACACTGACTTGTTTGTTTTAACCAATAACTAGC 19 Ze22f06.r1 AA010777.1 CCAAGGCGGGNCCCGGGGCCTTTGGCTTGGGCAAATAAAGCGTTAAAGCCCCGCAAAAAAAA 20 KRT5 NM__000424.2 CCCCACCCCAATCCCAAATTTTGTTTTGGTTCTAACTACCTCCAGAATGTGTTCAATAAA twenty one TRIM29 NM__01201.2 GAGGCTGCCCTCCTCCTTCTGATTACCCCCCCATGTTGCATATCAGGGTGCTCAAGGATTGG twenty two COX7A1 NM__001864.2 AGCCTGGGGGGCCTGAGAGACTTGAACAAGTGTCAATAAACGCTGGCCTCTGAAAAAAAAA twenty three TGF-betaI IR alpha D50683.1 GTTCTACTTGCTTGGGTCCTTATCGGAGCTCCACTCCTTTGGGTCACATGTCAAAAAAAA twenty four top2A NM__001067.2 GACTCGGGGACAACATTTGATCCAAGATCTTAAATGTTATATTGATAACCATGCTCAGCA 25 PECAM1 NM__00442.2 GCTGAAGGCTGAAAGACAGGAAGATGGTGCCATCAGCAAATATTATTCTTAATTGAAAAC 26 NCI__CGAP__LU1 AA522674.1 TGGGTCATAGTCCATTCCACTCTGCTCTCAAAGAAGAGCACGCAAGAGAGAAACTGGGAAG 27 HomohapienscDNAclone AA524029.1 CAGTCTTCGATGACCCATGAGNTGTTTGGCGCTCAGCTTCATCGGCGGGCCTCTCTGGCT 28 AGER NM__001136.3 TTCCAGCCACGGGCCCCAGGAAAGCCGTGCTGTCAGCCATCAGCATCATCGAACCAGGCGA 29 FCN3 NM__003665.2 CAAGGGTGAGCCAGGAGATCCAGTGAACCTGCTCCGGTGCCAGGAAGGCCCCAGAAACTG 30 Cytokeratin 17 HSCYTOK17__2438 CCTGGCCCCTACCCACTTTAAATTGCCTGCTTCTCCTGCAGCGGTGACCACCCGTCAGGT 31 DLC1 NM__006094.3 CTTGATGTGCAGAAAGAAGCCGGACACCATGATCCTAACACAAATTGAAGCCAAGGAAGC 32 PCNA NM__002592.2 AAATGCTTGCTGACCTGGGCCAGAGCTCTTCCCTTACGCAAGTCTCAGCCGGTCGTCGCG 33 MCM2 NM__004526.2 CCTGTCTGTTTCCCCCACTCTCTTATTTGTGCATTCGGTTTGGTTTCTGTAGTTTTTAATTT 34 MCM6 NM__005915.4 CTCTTTCCCTGGAATCAACTTGTATGAAACCTTGTCAAAATGTACTCCCAAGTATGTAC 35 FHL1 NM__001449.3 GGAGTGGTTAGAGAAGCTGATGCCTCATTTTCTACATTCTGTCATTAGCTATTATCATCTA 36 DCTN2 NM__006400.3 GTGGCTGACTGTAATACTGTACAACTGTTTCTGACCATTAAATGCTGTTGTACTCTGAAA 37 DST NM__001723.3 TGCTACCATAAACATTTTGGAGGTGCATCTGCTATGTGACATGGTAAATATGGTTAAGTG 38 TRAPPCI NM__021210.2 TTATCCCCTGCCTAATGCTGCGAGAAGCACAGAATAAACTTTTTGTCACTCTCAAAAAAA

39 ALG14 NM__144988.2 GGCGTCTGACAGTCCTGAGAATTATTGATGGTAAGGAATAAAAAATGTACAGATGACTCA 40 MAGE-1 HUMMAG1A__1153 TTTGGGCTTTGGGAAACTGCAGTTCCTTCTGGGGGAGCTGATTGTAATGATCTTGGGTGG 41 HomosapienscDNAclone AI472209.1 GAACTAGATCACTCACTGGAAATCAGAAAGCATTCAGTCAGTCTGATAATGATCACAGTT 42 I.114 HSICKLV54__196 TTCTTCATCTTCCGCCATCTGATGAGCAGTTGAAATCTGGACTGCCTCTGTTGTGTGCCTG 43 PT1.3__03__F03.rtumorl AI525592.1 TTAAAAAAANCCCGGGTAAAANATNNNNGNAAAGNTGGGGGGAAAACCCAAANTGGGAGGGN 44 S-100L AI539439.1 CATCATGGATCTGTGGCTGTGTACTTGTTTTTCTCTCAGCCTCACCCCCACATGGTGAGCT 45 ABCA3 NM__001089 AGGACACATTGAATACGTTGTGACCATCCAGAAAATAAATGCTGAGGGGACACAAAAAAA 46 CKS2 NM__001827.1 CACAGCAACAGAGCTCAGTTAAATGCAACTGCAAGTAGGTTACTGTAAGATGTTTAAGAT 47 DSG3 NM__001944.1 GCTCACTGTATTGGGCTAATAATTTGGCACTTATTAGCTTCTCTCATAAACTGATCACGA 48 GPC1 NM__002081 CAGTGTCAGCGGGTGACGTGTGTTCTTTTGAGTCCTTGTATGAATAAAAGGCTGGAAACC 49 ODC1 NM__002539.1 GTAGCTTGTACAATGGCAGAATGGGCCAAAAGCTTAGTGTTGTGACCTGTTTTTAAAATA 50 PGC NM__002630.1 TGTCTTTCCACTCTGGATTCAGCCTTCTTTTTCTGGACTCTGGACTTTCTCTAATAATAA 51 RB1 NM__000321.1 TCAAGGGCTTACTATTTCTGGGTCTTTTGCTACTAAGTTCACATTAGAATTAGTGCCAGA 52 RBBP4 NM__005610.1 ACAGACTTTATTTCAGCTATCCCTCTATAATAGGTACCACCGATAATGCTATTAGCCCA 53 RB-binding protein RB-bindin__protein__56 CGTCGTCGGAGGCTGAAAAAGCCCAAGGTGCTGCCGTTGCCCGTACAACTCGGACTTGCT 54 TK1 NM__003258.1 TCTCTTCAAGTGTGGAGAGGGCAGGGTCCACGCCTCTGCTGTAGCTTATGAAATTAACTA 55 TYMS NM__001071.1 GGGAGCTGAGTAACACCATCGATCATGATGTAGAGTGTGGTTATGAACTTTATAGTTGTT 56 CLEC3B NM__003278.1 TGCGCTTGGAGCCTCTTTTTGCAAATAAAGTTGGTGCACGTTCGCGGAGAGGAAAAAAAAA 57 LPL NM__000237.1 TCTCACAGAGCCAACTCACTCTTATGAAATGGGCTTTAACAAAACAAGAAAGAAACGTAC 58 A.tricolor Chlorop-last rbcl X52980.1 GCTGCTTGTGAAGTATGGAAGGAAATCAAATTTGAATTCCCGGCAATGGATACAATTTAG 59 C10orf116 NM__006829.2 CCCCCTCATTAAAATTCACGTTCCCACCCTGTGTCCACTTCATGATTCCTCGCAAGCTGG 60 SLIT2 NM__004787.1 TCTTTGGAAAAGGTTGTATACTTCTTGACCATGTGGGACTAATGAATGCTTCATAGTGGA

61 CENPM NM__024053.3 GCCCACACCTATCAAAGCCCCCTGCTCTACTGTGACCTGGAGGTGGAAGGCTTTAGGGCC 62 glutaredox in X76648.1 GCACGAGGCAATACCTGCAACTGAGGATTCTTCCCGGGGAGACCGCAGCCCATCGGCATG 63 ZWINT NM__032997.2 CATGCTTAGCCCACCCAGAGCTAACCCCTGTCTTTCTTCCCCAGGCCTCTGACTTCCCTCA 64 RBPMS NM__006867.2 TAGATCCGAATAAGATCCGAATAAGAATATGTAATGGACCAGGCGCAGTGCCTCACGCCT 65 UBC NM__021009.3 GGGGGTGTCTAAGTTTCCCCTTTTAAGGTTTCAACAAATTTCATTGCACTTTCCTTTCAA 66 ControlplasmidpNIAysic-7 DQ023293.1 CAGATGCTACACAATTAGGCTTGTACATATTGTCGTTAGAACGCGGCTACAATTAATACA 67 KIAA0101 NM__014736.4 TGAAGAGGCAGGAAGCAGTGGCTTAGGAAAAGCAAAGAGAAAAGCATGTCCTTTGCAACC 68 DOC1 NM__014890.1 TCTGAAACTAACTGTTGCCTGCATAGTCATATCGAGTGTGCACTTACTGTATATCTTTTC 69 BRCA2 NM__000059.2 AGGATGAATATGAAGAGTGGTGTTTCCTTTTGAGCAATTCTTCATCCTTAAGTCAGCATG 70 LAMB2 NM__002292.2 GCACTGAAGAGCTCTTGGCCCGGCAGGGCCCCCAATAAACCAGTGTGAACCCCCAAAAAA 71 Atcab1-613__1167 Atcab1-613__1167 GCAGACGCAGCAGCTGGGACCCCCTCCCAACCTCAATGCCCTGCCATTAAATCCGCAAACA 72 Histone(H2A.Z) M37583.1 GGTTTTGTTCAGACCTTATTTCCACTCTGGTGGATAAGTTCAATAAAGGTCATATCCCAA 73 H2A.Z HUMHIS2AZ__404 GTTCCTGCATGGGCGGCATGAACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACT 74 XMP and YMP U52100.1 GCTGGGTTGCTTCTGCTGCAGTACAGAATCCACATTCAGATAACCATTTTGTATATAATC 75 Pro-alpha-1type3collgen X14420.1 GGGGCTGCTTTTGTCACACTAGCATAGAGAATGTGTTGAAATTTAACTTTGTAAGCTTGTGT 76 AF070648.1 GTTCTACATAGATGCTTAGTCCCTCATGCAAATCAATTACTGGTCCAAAAAGATTGCTGAA 77 Acetyl-Coenzyme A BC009753.2 CGGTGGGCAGAGAGCCTACTAGGAAATGTGCAGAATAAACTATTTTTTGAAGGAAAAAAA 78 P53-related KETgene Y16961.1 GGGGAGCCAGAAGCCAATCTACAATCTCTTTTTTTGTCAGGACATGCAATAAAATTTA 79 CIP2 L25876.1 ACCAGTCGTTATCAACTTGAATGTAAATGTACATGTGCAGATATTCCTAAAGTGCCTTCG 80 Lung amiloridesensitive Na+channel protein X76180.1 CATCATCCTGCTTGGAGCAAGTAGGCAGGAGCTCAATAAATGTTTGTTGCATGAAAAAAA 81 MFAP4 L38486.1 GGTGTCCTACCCCGAACTCCAAAAATTACACCTGGAGTCAGGTGCAGAAGGGAACCTTGT

82 COL7A1 L0287.1 GCCTGTGATGACATGGTGCTGATTCTGGGGGGCATTAAAGCTGCTGTTTTAAAAGGCAAA 83 KIAA0796 protein AB018339.2 GCAAAGTTTCTCAGAATGGAGACATTTCTCATTGAAGAAATGATAGAGAAGTCTCAAGAAGG 84 DKFZp564p187 AL037969 TTACGGATAGAGGATGAGGAAAACTTCTCTACCGAGATTTAACCCATATGTTCTGCCCAGC 85 MRPS14 NM__022100.1 GAAAGTCTCCATCTAAAGAGGAAAAGTTTGTTGGAACAGTATATATTCCCTATTTTTATG 86 NET1 NM__005863.2 AGTGGAAATGATAATCAGAAAAGACTGTAAATAGATCCATCCAAATGATTTCTCTGTACA 87 YWHAZ NM__003406.2 GGACTACGACGTCCCTCAAAACCTTGCTTCTAGGAGATAAAAAGAACATCCAGTCATGGAT 88 Vt95d02.r1 AA734744.1 TGGAAAGTATAAAAGTGTTGCATTATTGCATCCCCCTCCCACAACCTTCAGGCAACTAGAG 89 Xp22 BAC GSHB 526D21 AC003037.1 AATAACAGTGAAGGGAGAGTCTGCTCTTCTGCTCTAGAATTTTCTCAGCATCGACGTCAAG 90 COII M25171.1 AAGCTAACTTAGCATTAACCTTTTAAGTTAAAGATTAAGAGAACCAACACCTCTTTACAG 91 USP8 NM__005154.2 GCAAAGCAATTTTTCTGTTGGCTTTGGGCTGTATTTGTGCACTAAAATCTTTATTCTAAAA 92 POLR2G NM__002696.1 AGCTTAACTACTGCCTCCTCATTTTTCAGTATGTGTTCTAAGTATAAAAAGTCCTTTGGT 93 Vector Pucp26 U07168.1 ATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAA 94 RAPD with thprimeroperon OPH4 AJ244005.1 CACACACTTTGTAAAAAAACAATCAGGTGACTAAACATATTAACAGGTTGCTGGTTTATAA 95 NUCKS1 NM__022731.2 CTCTTTGCCAGCCCTCCCCTTTCCCAATGAAAGCCATGTCAAATTAATCACTGAAAAAAA 96 PACc l one RP4693M11frome4q24.3 AC006146.2 ATCTGATATTTAAAGTCACAGGATATAGGATTACAAAATAAGTAGGCCAGGCACAATGGC 97 CASP4 NM__001225.3 TTTACTTTCATTTTTGACTCTGAGGCTCTTTCCAACGCTGTAAAAAAGGACAGAGGCTGT 98 CAPZA1 NM__006135.2 CCACACTATTCTTCTGTATCAGGTAGTCTAATAGAAATATACCTGTTTTGTTCTAAAATTG 99 KIAA1557protein AB046777.1 GAGAACTCGTCTCATTACTTTATAGTCATGTCATGTATGTTTTTTTAACCATGAAATGAC 100 NARS XM__008783.1 AGTTGATTTCTCCACCAAATGCATGTCATGTATTCTCAATAGGCTGTATTCCCAGCAGTC 101 RPL23 XM__008149.2 ACTTGTGGCCCCGGATTGCATCCAATGCTGGCAGCATTGCATGATTTCTCCAGTATATTG 102 Ribosomal S51052 Ribosomal S51052 TCGTAAGAACTGAGAATTTGATTTTTTTTCATCTGATGAAGATTTCTTTTATTTCTCATT 103 HDAC1 XM__001767.5 CTCCTAATTCTGCAGGTGGAGGTTGCTAGTCTAGTT

TCCTTTTTGAGATACTATTTTCAT 104 NFE2L2 XM__002548.7 CACCATTTGGGCTAGTTTCTGTGTAAGTGTAAATACTACAAAAACTTATTTATACTGTTC 105 ARC21 AF006086.1 GGGTGATTTGAAATTTTCTGCAGCATTAAAGCTGGCGCTTAATAAGAATAAGTAATAATA 106 GRY-RBP AF037448.1 TGACCTAATTTGGGTTTTCAAAACCATTTTGGCTACTTGTATTCTTTATGCTGTTGTTTA 107 KCNJ2 AF153820.1 TTCCCATTTGCTGTCTTTTTGTATCTAAAGTCTTCCTATTGTACTGCACAAACCATGGAT 108 EEF1AA XM__004398.1 CATTGATGCCCCAGGACACAGAGACTTTATCAAAAACATGATTACAGGGACATCTCAGGC 109 3'UTR U26032.1 CCAGTTGTATTTAATTTATTCATTATCCCTAGATAGCTATTAAGATACTTAGATTAGAC 110 KIAA1235 AB033061.1 TTTATTCAGTATAAAATCTTTATATGTTCCACATGTTAAGAATAAATGTACATTAAATCT 111 CCT6A NM__001762.3 TGACATAAAATTCCATGTTAGATAAGCATATGTTACTTACCTTGTTATTAAATATTTCTT 112 PRO2975 AF119911.1 AACAAAATCCCAGATCATATAGATGTTTACAGTGATTACATTTTATCTAAGCAACATACAT 113 DNMT3A NM__022552.3 AGATATGAGATATATATAAAAGGTACTGTTAACTACTGTACAACCCGACTTCATAATG 114 FLJ2288fis AK026536.1 TAACACTCACAAACAAAACTAACTAATACTAACATCTCAGACGCTCAGGAAATAGAAACCG 115 TP53 M1312.1 CCCTCCTTCTCCCTTTTTATATCCCATTTTTATATCGATCTCTTATTTTACAATAAAACT 116 TMP21 XM__007423.4 AGTGAGCTTTGAACTGTAGATAACTCTTAAAGAAAATGTCATTTTAGACAATTAAAATA 117 SRP9 NM__003133.2 TACATACTGTGTACCTAATTATGTATAGCAGTGTAGTCTCAATTATATCTGAAAGTAATT 118 MEMD Y10183.1 TTGGTGTGGGAGATCAAAGGTTTAAAGTCTAACTTCTAAGATATATTTGCAGAAAGAAGC 119 LOC154336 XM__002255.5 CAGAAGACAAGTAACTGTATGAGTTAATAAAAGACATGAACTCTTACTGCTTCAACAAAC 120 c-erbB2 NM__001005862.1 AGTGTGGGGGGTCCTTCTCACACCCACTTTGTCCATTTGCAAATATATTTTGGAAAACA 121 c-Myc NM__002467.3 GACTGAAAGATTTAGCCATAATGTAAACTGCCTCAAATTGGACTTTGGGCATAAAAGAAC 122 CD44v6 NM__000610.3 CCACTGTTATTGTTACTTTGACTTTTCAGAGCACACCCTTCCTCTGGTTTTTGTATATTTT 123 VEGF NM__001025366.1 GGAAAAGATATTAAACATCACGTCTTTGTCTCTAGTGCAGTTTTTCGAGATATTCCGTAGT 124 TPA NM__000930.2 GGCTTCAGCATATTTATAGCAATCCATGTTAGTTTTACTTTCTGTTGCCACAACCCCTGT 125 NSE NM__001975.2 CCACAGTTGCCACCACCTCTGTGGCATTGAAATGAG

CACCTCCATTAAAGTCTGAATCAG 126 ProGRP NM__002091.3 ATTCAAGGCCCCGAGCTGTTACCATTCACAATAAAAGCTTAAACACATTGTCCAAAAAAA 127 CK19 NM__002276.3 CCTTACCCCCGGCTCTTCTCCTGACCTGCCAATAAAAATTTATGGTCCAAGGGAAAAAAA 128 C-MER NM__006343.2 CCATGGCCCCAAAGCACCAGATGAATGTTGTTAAGTAAGCTGTCATTAAAAATACATAAT 129 Trk NM__001007792.1 TCCTGCTTCTCTAGGCAAGGTCCCGTCATAGCAATTATATTTTATTATCCCTTGAAAAAAA 130 NTS NM__006183.3 CCTAGCCTGCATATCATTCACCCAAAAACAAAGTAATAGCGCCTCTTTTATTATTTTGACT 131 Lumican NM__002345.3 GGTGGTAGATATTGAGGCCAAGAATATTGCAAAATACATGAAGCTTCATGCACTTAAAGA 132 Cathepsin 02 NM__000396.2 CTTCCTAAGTTCCCTTCTATATCCTCAAGGTAGAAATGTCTATGTTTTCTACTCCAATTC 133 N-cadherin NM__001792.2 ACCAGAATATAAATGATACACCCTCTGACCCCCAGCGTTCTGAATAAAATGCTAATTTTGGA 134 Max NM__145113.1 ACTGCCGAAGAATTGTCTTGCAAGTTAAGGCTTCCCCCTTTACTATAAGACTATAAATAA 135 N-Myc NM__004688.1 AGTGTTCTCTAGGTCAACCTCACATAGCATACTTTGAAGAATAGACTTAACAGAATCATG 136 TGF-β NM__000660.3 CACTATTCCTTTGCCCGGCATCAAGGCACAGGGGACCAGTGGGGAACACTACTGTAGTTA 137 c-fos NM__00525.2 TCATGCATTGTTGAGGTGGTCTGAATGTTCTGACATTAACAGTTTTCCATGAAAACGTTT 138 Ras NM__005402.2 GATGCCACTACCCGATTTGTTTATTTGCAATTTGAGCCATTTAAAGACCAATAAACTTCC 139 Kras NM__033360.2 CCAGTTGTTTTCATCATTTGGTTGCGCTGACCTAGGAATGTTGGTCATATCAAACATTA 140 Hras-1 H-ras-131171 AACTGCCAATCCCAGAGGACAAGACTGGGAAGTCCCTGCAGGGAGAGCCCATCCCCGCAC 141 Bcl-2 NM__000633.2 GCACTTTGTTTGTGTTGTTGGAAAAAGTCACATTGCCATTAAACTTTCCTTGTCTGTCTA 142 Apoptin NM__012234.3 CGAGCAGCATGTAATTCTTTTTTCTTCCTGGACATAAAGATAGCTTGAGTAAACTGTTCTA 143 EGFR NM__005228.3 AAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATC 144 P53 NM__000546.2 ATGTTCCGAGAGCTGAATGAGGCCTTGGAACTCAAGGATGCCCAGGCTGGGAAGGAGCCA 145 Hrk NM__003806.1 GAAATCCAGCTGCAGAAACAGACACCCCAATGCTATTTACATACAGCTCTATATATATAA 146 Smad4 NM__005359.3 GACTGTACCACAGACACAATATGTATGCTTTTTACCTAGCTGGTAGCATAAATAAAACTG

147 PAI-1 NM__000602.1 AAACCTACCAGCCAGAGGGTAGGGCACAAAGATGGATGTAATGCACTTTGGGAGGCCAAG 148 Caspase-5 NM__004347.1 CCTCTGTCAACATCAAAGAGCACATTTACCAGTATAGCTTGCATAGTCAATATTTGGTAT 149 Thrombospondinl NM__003246.2 AGTCAGTGTTGTACATAGCATAAAAACTCTGCAGAGAAGTATTCCCAATAAGGAAATAGC 150 Retinoic acid receptor beta2 NM__000965.2 ACAATGCTTTCTATGTTCATATACTGTTTACCTTTTTTCCATGGACTTCTCCTGGCAAAGAA 151 P107 NM__002895.2 CCATCTCAGCCTGATGTCACAACCCAATAAATGGGGCACTTTCTTCTTTTGTAAACTATT 152 Retion blastoma-like protein2 NM__005611.2 AGTTGAATTGAGGGGATTAATATGAAAACTTATGACCCTCTTCCTTTAGGAGGGAGTTATC 153 Opioid receptor kappa1 NM__000912.3 CGGTATAACCCAGCTTCCTTAAGGCAATTTTCTTCTCTGAAACAAGAATATACTCATATG 154 Fragile histidine triad NM__002012.1 CCCCCTGTCTCCTTTATTCGGCAGCCTTATGATTAAACTTCCTTCTCTGCTGCAAAAAAAA 155 Met proto-onco gene NM__000245.2 ACCTGCAGCTGAACTGAATGGTACTTCGTATGTTAATAGTTGTTCTGATAAATCATGCAA 156 ATMs NM__000051.3 CCCAAAGTGTTTACCTAATCAGTAGGTTCACAAACTCTTGGTCATTATAGTATATGCCTA 157 Colorectal mutant cancer protein NM__002387.1 GAGTCTTTATGTCCATAAGCTACGGGTAGGTCTATTTTTCAGAGCATGATACAAATTCCAC 158 V-raf-1 NM__002880.2 TCTAGTCACATCATGTGTATACAAGGAAGCCAGGAATACAGGTTTTCTTGATGATTTGGG 159 Wnt-4 NM__030761.3 TATCTGTGTCCGTTTTTCTACTTGCAGACCTAAGGTGGAGTAACAAGGAGTATTACCACC 160 Stat2 NM__005419.2 CAGAAAGGGGCTCTTCTGAGCAGAAATGGCTAATAAACTTTGTGCTGATCTGGAAAAAAA 161 Hepatocyt growth facto NM__000601.4 TCTGAAGCACCCCACCAATACAACTGTCTTTTACATGAAGATTTCAGAGAATGTGGAATTT 162 Insulin-like growth factor2 NM__000612.2 AGCAGCATCTTCAAACATGTACAAAAATCGATTGGCTTTAAACACCCTTCATACCCTCC 163 Insulin-like growth factor1 NM__000618.2 GCTACTTTGAAACCAGAAAATAATGACTGGCCATTCGTTACATCTGTCTTAGTTGAAAAG 164 Insulin-like growth factor binding pro-tein NM__002178.2 AAGAGTGTTGGTGTTGGCTGGGGTGTCAATAAAGCTGTGCTTGGGGTCGCTGAAAAAAAAA 165 Glial growth factor2 NM__013957.6 GGCTGTCTAGTGTAATTGCTAACCAAGACCCTATTGCTGTATAAAACCTAAATAAACACA 166 Cyclin dependent kinase inhibitor 1c NM__000076.1 GCACGAGAAGGTACACTGGTCCCAAAGTGTAAAAGCTTTAAGAGTCATTTATAAAATGT 167 MLH1 NM__000249.2 GGTAGCACTTAAGACTTATACTTGCCTTCTGATAGTATTCCCTTTATACACAGTGGATTGA

168 Smad7 NM__005904.2 CCTATGGGTGTTATCACCTAGCTGAATGTTTTTCTAAAGGAGTTTTATGTTCCATTAAACG 169 Smad1 NM__005900.2 CTTATGCTCTCGTACATTGAGTACTTTTTATTCCAAAACTAGTGGGTTTTTCCTACTGGAA 170 Smad5 NM__001001419.1 ACTGTAGGTCTGCTGCTTATTTGTATTTGTTGTGCTTCTGTTTATGTTGTAGAAGCTGAA 171 MSH3 NM__002439.2 GTTCAGATAATTGGCAACTGGGTGAATCTGGCAGGAATCTATCCATTGAACTAAAATAAT 172 XPC NM__005053.2 CCCACCCCAGAACAGAACCGTGTCTCTGATAAAGGTTTTGAAGTGAATAAAGTTTTAAAA 173 RAD50/DNA NM__005732.2 GGGGGGGGTCCTAAATGTAATCACGAGTAAGATTAAGAGCAAATCAATTCTAGTCATATA 174 UNG NM__003362.2 CTGTTAGTCGGGTTAGAGTTGGCTCTACGCGAGGTTTGTTAATAAAAGTTTGTTAAAAGT 175 POLQ NM__199420.2 TGTACTTTGTGATCCCAATCAGAGGGATGGAGCTAATCTTTTTGCTGTTGAAATAAAATG 176 XRCCS NM__00543.1.1 TCGTTGCAGCACTGCCCATCAGTGACAGACTGGATTAAAAAAATGTGGTACATACACACC 177 DNA-PKcs DNA-PKcs__6659 CCTTCTAACACTTGTATTTGGAGGCTCTTCTGTGATTTTGAGAAGTATACTCTTGAGTGT 178 TIP60 NM__182710.1 GGTGGGTGCTGGCTGCAAAAATTTCTGGCTTCTCTTACCCCTATTGCCCCCGGCAATAAA 179 Phosphatase2AB56-beta NM__006244.2 CAGTAATCATGGTCTACTCCCTCTTTCCGTGGCTGGGGGTAGACTTAATAAAGAGAGAAAT 180 Adenomatosis poLyposis coli NM__000038.3 ACCATCTACTTCTGTTGTTTTTCCCAGGCTTCCATAAACAATGGAGATACATGCAAAAAAA 181 Antigen peptide Transporter 1 NM__000593.5 AGGCATATGCTGGCCCATAAACACCCTGTAGGTTCTTGATATTTATAATAAAATTGGTGT 182 Atrial natriureTic precursor NM__006172.1 AAGGTAGAACCTCACCTGTTACTGAAAGTGGTTTGAAAGTGAATAAACTTCAGCACCATG 183 Antigen petideTransporter 2 NM__000544.3 TCTTGCTCAGTATTTTCGCAAACAGCAGCTGTCTAAGTTCACTGTTCTGAACTTTATTTT 184 Cytochrom P450 Subfamily I NM__000499.2 TGTAATAGGGAAAATTCTAACCCAGGTATCCAAAAAATGTGTAAGAACAACTACCTGAGCT 185 Elanogasterflightless-I NM__002018.2 CCCTCACTTCTTCCGCTAGGGATAAAGCAGATGTGGATGCCCTTTAAGAGATATTAAATGC 186 Neurofibromin 2 NM__181826.1 GAACCACCCCCACGGCTCACTCCTTGGTCTGGGGCCACCTTCTTGCCCTTTCTTTTTTTT 187 ALK-1 NM__000020.1 ATGGTGCCCTATCTGGGTAGAGGTAGTGTGAGTGTGGTGTGTGCTGGGGATGGGCAGCTG 188 EGF NM__001963.2 CCCCTACAGAATTTTCCCTCTTGGTGTGATTGCACAGAATTTGTATGTATTTCAGTTAC 189 Macmarcks NM__023009.4 CCATTCTCTTCCACCTGGTTTTACTGTAACATTTGGAAAAGGAATAAATGTCGTCCCTTT

190 SPP1 NM__000582.2 GTGGTGTCAATTGCTTATTTGTTTTTCCCACGGTTGTCCAGCAATTAATAAAACATAACCT 191 Collagen binding protein NM__00513.1 ACGTCCAAGAAAAACTGCAGGAAAGCCTATTTTGTTCTAGTATTAACAGGTACTAGAAG 192 Alpha-2-macroglobulin NM__000014.4 TCTCAGAGCTCCACAGAAGACACGTGTTTTTGTATCTTTAAAGACTTGATGAATAAACAC 193 Bleomycin hydrolase NM__000386.2 GCAGGAAAAGGTGATATCCATTCTTTCTGATAACTAGATGGTGCTGAGAAGCTTTTGAAT 194 Similar coenzme Asynthase AK025736.1 TAGAAAGCAAGAATGCTTTCAAGAATGCTACAGACAGGCCGGGCGTGAGCCACTGTGCCC 195 DNA binding protein 1 AJ277077 ACTAGAGACCTGGGAATGAACTGAAGGTGAAACAAAGCTTCGAATCACTAGTGAATTCGC 196 FLJ21288 fis AK024941.1 TGGGTCTCAGACATGTCTACAAAATATGGGTACTATTTTTATGCCCGTGTATTTCACATT 197 DKFZp762P233 AB037773.1 ATTGCCTCAGCAGGTGTGAAGCGTGTGCTTTAGTTTCGTGGGAGGCCTGGCATCCCCGAG 198 DKFZp564M0264 AL117621.1 GTACAGTACTTGGAATTGTGTTCTTTATGGTTGTAGTGTTGGTAAAGCACTAATATGCAG 199 DKFZp564H191 AL110126.1 GCTGCTCATACTGCAGTCAATTACTGGAAGCGGATATATTTCCTATGCAAAAACTGTTTA 200 TI-227H AK026634.1 GATATCATCTCAACTTAGTATTATACCCACACCCACCCAAGAACAGGGTTTGTTAAAAAA 201 BRCA2 NM__000059.1 GTAATTTATTAACTAATCAAGAAAAACATCTTTGGCTGAGCTCGGTGGCTCATGCCTGTA 202 BLM NM__000057.1 CTTTCTTGTTTGTCAGCATCTGACCATCTGTGACTATAAAGCTGTTATTCTTGTTATACC 203 Ash1 NM__018489.1 GCCCCCATGGGGTCAGAAGGGCACGGTAGTTCTTGCAATTATTTTTGTTTTACCCCTTCAT 204 Malignant brainTumor (1(3)mbt) AL031681.16 TGTTCATTCATCTTTCTGTCCACTGCCCCCATTTATTCCCATAGATGCCAGAGAGATTT 205 AL033520.16 AL033520.16 TTTGGCTTCATGGCACATAATATTTTCACTCACCGCCTTGCTGAGCACTTGTTGGCTGAC 206 Brag 1 BRAG1__2288 GATGGACCCATAAGAGTGACGCCTGTGGAGCGCGTGCTCTTCCTCTGCAGCCAAAAAAAA 207 Bax Bax__414 TGCCTCTGGAATTGCTCAAGTTCATTGATGACCCTCTGACCCTAGCTCTTTCCTTTTTTT 208 CDK4 CDK4__676 TCCCTCTTGTCCCTTTTCCCTACCTTTATATTTGGGGTCCTTTTTTATACAGGAAAAACAA 209 CEA CEA__1736 TCAGTCACTACTCTTGCTGCAGTTATGAAAAGAATGAGGCCAAGTCTGATGAAAATAAAC 210 DK6 inhibitor p18 K6inhibitorp18 101 CACAAGTGTCTGTAGGGGTGAAGGGAGCCGGGACCGGTGATTTGAGGGGGACCAGCTACA 211 IL-1alpha IL-1alpha__ CAATCAAGTCCTACAAGCTAAAATTAGATGAACTCA

1358 ACTTTGACAACCATGAGACCACTG 212 IL-1alpha IL-1alpha__2405 CTTACAGAACCATCTGGCTTCATTCTTCTTGGACTTGATCCTGCTGATTCTTGCATTTCC 213 Myb Myb__1599 TGAACTTTTGAGATATGACGGTGTACTTACTGCCTTGTAGCAAAATAAAGATGTGCCCTT 214 PDGF-B PDGF-B__1606 CAAAACCTGCTTCCTTCAGTTTGTAAAGTCGGTGATTATATTTTGGGGGCTTTCCTTTT 215 PDFA NM__033023.3 TTGTAAAGGTGAAGTTTGTATGTTTACTTAATATTACCTGTTTTGTATACCTGAGAGCCT 216 MYCL1 MYCL1__1533 GGGCAATTTCCTGTTGCAAGGGTCTGATTTATTTTGAAAGGCAAGTTCACCTGAAATTTT 217 Xeroderma pigMentosun groupE XerodermapigMen-tosungroupE__2069 CTTCTTCGGAAAGAAGAAGGTGGGAGGATGTGAATTGTTAGTTTCTGAGTTTTACCAAAT 218 c-jun c-jun__1582 AGGTGCTTATTCTCAAAAGCAGGAATTGGTGGCAGATTTTACAAAAGATGTATCTCTTCCAA 219 Elongation fact Or 1-alpha Elongationfact Or1-alpha__1669 CATATACTGAGATTGAGATTAACTTCCTGTGAAACCCAGTGTCTTAGACAACTGTGGCTT 220 Mda-6 Mda-6__1032 GCTCCTCCCCACAATGCTGAATATACAGCAGGTGCTCAATAAATGATTCTTAGTGACTTTA 221 TFIIH TFIIH AACTCAGGTGTTTTTTATTTACGCGTCAGGGCTTTTCTTGTTTAATAAGTTATGATAGCT 222 C-YES1 C-YES1 TGGTGTATATGTTTTAAAAATACTCTCATATAGTCACACTTTAAATTAAGCCTTATATTAG 223 Collagen alpha-2 Collagenalpha-2 TTGTTCATAATACAAAGGTGCTAATTAATAGTATTTCAGTACTTGAAGAATGTTGATGG 224 SCLCEA SCLCEA TAGAACACTTTTTGTTATCAGTCAATATTTTTAGATAGTTAGAACCTGGTCCTAAGCCTA 225 CDC42 CDC42 TGGAGAAGGGGTGACTACAGTCAAACCAGGTGATAAAGTCATCCCGCTCTTTACTCCTCCA 226 a-tu-1331 NM__138923.1 TTAGCCTTTTTAAAAGTAGTAAGTAAATGATAATAAAATCACCCTCTCCTAATCTTCCTGG 227 UBE2C NM__007019.2 GCCTGGACATCCTGAAGGAAAAGTGGTCTGCCCTGTATGATGTCAGGACCATTCTGCTCT 228 KRT6A NM__005554 CCTCTCCCTCAGGTTGCCTGTCCTCTCCTGGCCTCCAGTCTCCCCTGCTGTCCCAGGTAGA 229 MSLN NM__005823.4 ATTCGCAAGTGGAATGTGACGTCCCTGGAGACCCTGAAGGCTTTGCTTGAAGTCAACAAA 230 TSPAN4 NM__003271.4 GGTGGGAGGGGCCTCCAGCACTTTTTATATTTACGTATTTCCAAAGCAGTGTTCACACG 231 CACNA2D2 NM__006030.2 GCTCAATGGAGAATATAAACATATACAGAAAAATATATATTTTAAGTTTAAAAAACAGAA 232 N2A3 U97105.1 CTTTAAGGAAGTGTCAAACAAATAGAAAAATCTGGA

AGAATTTACTAAGTGTAATAAATT 233 EST__AL117561 AL117561 ATGTGTTTAACTTTGTACTGGTAATTTTCTGTATATTTGGAATATTTGGGTTAAAAATA 234 X52195 AI983204 AGAATGCTCTCAAAGAGCTGAATTATGATGACTTGTAGGTATTGATTAGATGAGAACACCA 235 TEK L06139.1 GATAGTGGATTTGCCAGATCATATAGAAGTAAACAGTGGTAAATTAATCCCATTTGCAA 236 EST__AL049313 AL049313.1 AGGATCCAGTCATCTGAAGTATTTTATGTAGAGTTAGAGTTTCACAATATTGACTATAC 237 LOC64116__AL049963 AL049963 AACACTAATAATAAATTATTCCAAGTCTTTAAGGAGCCAAAGAAAAAAAAGATTTTCCACA 238 S100P__AA131149 AA131149 ATTCCCCTAAGGCTGAGCCTGCTCATGTACCCTCTGATTAATAAATGCTTATGAAATGATA 239 WW, C2 and coiled-coil domain contain-ing 2 BC053873.1 TCATTTTTAGGGGGTTCTATCTATAATTTATGGTAAATAGTTGGGAGGCAAAGGGAAGAA 240 KIAA0828__AB020635 AB020635 TGTGTGTTAGGTTATTTATTTATTAAAATCAAGAATCCTGTGCCTGTAGTGTTGGCCCAG 241 NM__005719.2 CCAGACCGGAAGCGCTCCGCTGTACCTGGATCCTGCTCCTCTGGGTTGAAACCCGGGCGC 242 HACL1 NM__012260.2 CCTGTCCGTCAGAGGTGCAGTACCAGAGGTGGCGTGCTGCCGATTTCGCGTTTGCCTTGC 243 FLJ21807 fis AK025460.1 TTTATGCTGTTACAGGAGAAGTGACATACTTATATATGTTTATATTAGCAAGGTCT 244 MRP5 U83661.2 TTGCTGTACNANAAATCTGGTNTTGCTMTTMNACTGTTAGGAAGAATTANCATTTCATTC 245 CYP2D6 NM__001025161.1 CCTAGTCCCCAGCCTGCTCCCTAGCCAGAGGCTCTAATGTACAATAAAGCAATGTGGTAG 246 HLA-A NM__002116.5 CTTCCTTCCCCTATTAAAATTAGAACCTTAGTATAAATTTACTTTTCTCAAATTCTTGCCAT 247 HLA-B NM__005514.5 TTACTTCCCTACTGAAAATAAGAATCTGAATATAAATTTGTTTTTCTCAAATATTTGCTAT 248 B2M NM__004048.2 GCTTGCTTTTTAATATTGATATGCTTATACACTTACACTTTATGCACAAAATGTAGGGTT 249 EST(gb|BE167844.1| EST(gb|BE167844.1| TGCTGGTAGGTTTAAGAAAGGAATAGAAAGTTTTTTGAAATCCTTTTATTTCTQACCAGTC 250 LSm3 gb|AF182289.1 AACTGTGACTACTATAGAAATTGATGAAGAAACATATGAAGAGATATATAAATCAACGAA 251 Kidney 2 pooled tumors gb|AA393850.1 TAAGACATAATATATAAAAAATATGTACAATCCATGGTTTGTGCAGTACAATAGGAAGACT 252 transamidas AF022913.1 TCTCTTGTATGAATTGGAAATAAGTATAAGGAAACTAAATTTGAATCAACTATTAATTTTT 253 GRAINY-HEAD AK02844.1 AGTCTGCCTGTCTGCATTGTACATAGTGTTTATAATATTGTAATAATATATTTACCTGT 254 COX-2 COX-2 GGAATTTGGGTTGTGTATGCGAATGTTTCAGTGCCT

CAGACAAATGTGTATTTAACTTAT 255 ER ER CGGGATGCTCGAGCACCTGTAAACAATTTTCTCAACCTATTTGATGTTCAAATAAAGAAT 256 GST-π GST-π CTCTGAGCGGGAGGCAGAGTTTGCCTTCCTTTTCTCAGGACCAATAAAATTTCTAAGAGA 257 HSPB1 HSPB1 CCCCCGCCACCTGTGTGTTCTTTTGATACATTTATCTTCTGTTTTTCCAAATAAAGTTC 258 HSPB2 HSPB2 GTCATAGCCTTGGTTTAGTTTTGGGTGGAGCTGAATAAACCCAAATTTCAGGGCAAAAAA 259 MDM2 MDM2 CCATCAACTTCTAGTAGCATTATTTAGCAGCCAAGAAGATGTGAAAGAGTTTGAAAGG 260 MGMT MGMT AACACTGCATCGGATGCGGGGCGTGGAGGCACCGCTGTATTAAAGGAAGTGGCAGTGTCC 261 PR PR CAGGCCACACTTTAAAAACAAACACACAAACAACAAAAAACGGGTATTTCTAGTCATCTTG 262 PS2 PS2 GGCTCACAACACAGATTGACTGCTCTGACTTTGACTACTCAAAATTGGCCTAAAAATTAA 263 PTEN PTEN AGCGTTTTTTTTCTTTGAAGATTTATGATGCACTTATTCAATAGCTGTCAGCCGTTCCAC 264 topI IB__X68060 topII__X68060 GCTGTGTCTGGATTAGGTGTGATtGGACGAGACAGATACGGAGTTTTTCCACTCAGGGGC 265 TP TP CTCAGTAAGAAACTCGTGGAGGGGCTGTCCGCTCTGGTGGTGGACGTTAAGTTCGGAGGG 266 TYMS TYMS GAGCTGAGTAACACCATCGATCATGATGTAGAGTGTGGTTATGAACTTTATAGTTGTTTT 267 c-kit/CD117 c-kit/CD117 ATGTTGGTGTTCCACGTGTTTTATTCCTGTATGTTGTCCAATTGTTGACAGTTCTGAAGA 268 Nm23B Nm23B CACCTCAGGGTGAACTGGAAAACTCGACCGCACTTTAGTGCCAGGACCATGGCCAACCTG 269 AR AR ACAGGTAGTTAAGCCCCCCCAAAACAAGACGGAAAGTGAAAATACTTCAGATAAACCCAA 270 LRP LRP AGCTACCTGCACTTCTAGATTCATCTTCTTGTGAGCCCTGGGCTTAGGAGTCACCATGGC 271 P16 P16 AAAATGTAAAAAAAGAAAAACACCGCTTCTGCCTTTTCACTGTGTTGGAGTTTTCTGGAGT 272 P21 P21 TGGCCGCCCCCTGCCCCCAGCCTCTGGCATTAGAATTATTTAAACAAAAACTAGGCGGT 273 Nm23A Nm23A TGCTTCCTCCCAGCATAGGATTCATTGAGTTGGTTACTTCATATTGTTGCATTGCTTTTT 274 ADM ADM GAAGGAAACACCGAGTCTCTGTATAATCTATTTACATAAAATGGGTGATATGCGAACAGC 275 AKtAKtl AKtAKtl GCCTCCTGGCCAGACGCTGCCGCTGCCGCTGCACCACGGCGTTTTTTTACAACATTCAAC 276 Ang1 Ang1 GCTTTCTTCATAGCAGGTAAGCCTCTCCTTCTAAAAA

CTTCTCAACTGTTTTCATTTAAGG 277 Agg2 Agg2 ACAGATCATCTTGGAACTGCATTCTTCTGAGCACTGTTTATACACTGTGTAAATACCCAT 278 BRCA1 BRCA1 GGTCTTTCCCTAGCCCCCCCAGTGTGCAAGGGCAGTGAAGACTTGATTGTACAAAATACGT 279 BRMS1 BRMS1 TCAGAAGAGAAAATCGGATGACAGGCGGACCCACAGGCCCCTCAGGGTCTGCCCAGCCAG 280 BcrAb1 BcrAb1 TGGGAACGGCTGATGTGGACTGTCTTTTTCATTTTTTTCTCTCTGGAGCCCCTCCTCCCC 281 CD34 CD34 GCCCCACAGGAGAAAGGCTGGGCGAAGACCCTTATTTACACGGAAAACGGTGGAGGCCAGG 282 CDKN1B CDKN1B GCATGTTTTGTGCATTTGTAAATGCTGTGTTGGGTAGAATAGGTTTTCCCCCTTTTTGTT 283 CHP2 CHP2 CCCCTTTTGGCTGTATAGCAAAAGTGTTTTAATCCACGGTTGTGCCTTATTGTTCCATTAA 284 CK14 CK14 CTGGACCCTGCTTCACCCTCACCCCCTCCTGGCAATCAATACAGCTTTCATTTATCTGAGTT 285 CK20 CK20 ACTCAGAGGAGCTGCAAATTGCCTGCAAAAATGAAATCCAGTGAGCACTAGAATATTTAA 286 CK7 CK7 TCAGAATAGCTTCCAATAAAAGCAGCCTCATTCTGAGGCCTGAGTGATCCACGTGAAAAAAA 287 CXCL12 CXCL12 TCCAGGAGAAGGCCAATTCCTATACGCAGCGTGCTTTAAAAAATAAATAAAGAAACAACTC 288 CXCR4 CXCR4 TCCTTTGCCTCTTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGG 289 CX26 CX26 CTCCACATACTTCAGTGAGGGTAAGTATTTCCTGTTGTCAAGAATAGCATTGTAAAAGC 290 CX32 CX32 TCCCCAAGGCCTCTCTCTGCCTGAAATGTTACACATTAAACAGGATTTTACAGCCAAAAA 291 CX43 CX43 GCATGTAATGTAGACCTAGTCCATCAGATCATGTGTTCTGGAGAGTGTTTCTTTATTCAAT 292 CX45 CX45 GCAATAATACAGTTGCAGAACTTTACATTTGTGTCTTCCAGATCTGGAGAAGAACAGACA 293 DNMT3a DNMT3a ACCCTCTCTTTGAGGGGGGGCTCTGTCAGACATGCCGGGATCGCTTCCTTGAGCTGTTTT 294 EGR1 EGR1 GCTTATAAACACATTGAATGCGCTTTATTGCCCATGGGATATGTGGTGTATATCCTTCCA 295 ER ER GCAGCAATCCATGCGCCTGGCTAACCTCCTGATGCTCCTGTCCCACGTCAGGCATGCGAG 296 ERCC1 NM__001983 AGGCCAGAAGTTGGAGACCAGCCTGGGCAAGTGGACACCTCATTTTTACTAAAAAATAAAA 297 Ecad Ecad CGAATATAGTTCTGTGTAGAGAATGTCACTGTAGTTTTGAGTGTATACATGTGTGGGTGC 298 Endoglin Endoglin TGTCCTGGGCCTGCCCCTGTGTATTCACCACCAATA

AATCAGACCATGAAACCTGAAAAA 299 HIN1 HIN1 GGGAGGACCGTCCATCCCCTTCCCCCGGCCCCTTCAATAAACGTGGTTAAGAGCAAAAA 300 HMLH1 HMLH1 CTGTGGGATGTGTTTCTTCTTTCTCTGTATTCCGATACAAAGTGTTGTATCAAAGTGTGAT 301 HSP90 HSP90 GATGGTGACAAGAAGAAGAAGAAGAAGATTAAGGAAAAGTACATCGATCAAGAAGAGCTC 302 IFN IFN GGTGCTTCCAAATATTGTTGACAACTGTGACTGTACCCAAATGGAAAGTAACTCATTTGTGT 303 IL4 IL4 CAGACATCTTTGCTGCCTCCAAGAACACAACTGAGAAGGAAACCTTCTGCAGGGCTGCGA 304 KLF6 KLF6 AACCTCAGCCCACTCTCAGTCAAGGAGGGCAGAAAATAAAAGATGACATCACTGCCAAAA 305 LMP1 LMP1 AACACACACACACACACACGTGGGGATAGCTGGACATGTCAGCATGTTAAGTAAAAGGAG 306 LPA2 LPA2 TGAAGCAGTCCACGGGGAGGGGATGATACAAGGAGTAAACCTTTCTTTACACTCTAAAAA 307 LPA3 LPA3 TCCATCAGCCGCCGGAGGACACCCATGAAGCTAATGAAGACGGTGATGACTGTCTTAGGG 308 LPLUNC1 LPLUNC1 TGAGCTCTATAGACCATCCCTCTCTGCAATCAATAAACACTTGCCTGTGATGCCTGCAAA 309 Layilin Layilin CAGAGTGTTAGTCTGTGTCTACAGCCCTTCCTCTGCATGTGGCCACAGGGGACCTTTTTT 310 MLCK MLCK TGGTTATGGCTGCTGCTTCCTGTGGATGAAAGTGGCTGTAAGAGCTCCTAGACGTTTCTG 311 MMP7 MMP7 CTCCTTTATGGTGTGACTGTGTCTTATTCATCTATACTTGCAGTGGGTAGATGTCAATAA 312 MMP9 MMP9 GGGCCCCTCTCTTCTCACCTTTGTTTTTTGTTGGAGTGTTTCTAATAAACTTGGATTCTCT 313 MSI MSI GATCATTTGAGCTCATGAGGGCCCAGGAGGTCAAGGCTTCAGTGAGCCATGATCATGCCA 314 MT1MMP MT1MMP TAGGGGAAATGGGGTGAACGGTGCTGGCAGTTCGGCTAGATTTCTGTCTTGTTTGTTTTT 315 Nup88 Nup88 CTCACACCTGAACTGAACACCATTGAAGGCTTAAACCCATATTGTAAAACAGGTAGAATT 316 P63 P63 GGGAGCCAGAAGCCAATCTACAATCTCTTTTTGTTTGCCAGGACATGCAATAAAATTTAA 317 PDP95 PDP95 GCTACTTTGGTTTCTGTCTGTAAGTTAAGACCTTGGATGTGGTTTAATTGTTTGTCCTCA 318 S100A4 S100A4 GCGCTTCTTCTTTCTTGGTTTGATCCTGACTGCTGTCATGGCGTGCCCTCTGGAGAAGGC 319 SCF SCF GACAGCAGTAGCAGTAATAGGAAGGCCAAAAATCCCCCTGGAGACTCCAGCCTACACTGG 320 Survivin Survivin GTAGAGAAGCTGGAAAAAAATGGTTTTGTCTTCAAC

TCCTTTGCATGCCAGGCGGTGATG 321 TGF TGF CAGGCTTTCTGAAAGATCTAGTGGTTCACACAGAGAGAGAGAGTACTGAAAAAGCAAC 322 TIMP1 TIMP1 GTTCCCACTCCCATCTTTCTTCCGGACAATGAAATAAAGAGTTACCCACCCAGCAGAAAAA 323 TRAIL TRAIL ACCACTAAAAGATCGCAGTTTGCCTGGTGCAGTGGCTCACACCTGTAATCCCAACATTTTT 324 Tiaml Tiaml CCATGTAGCCCACCCAACTTTGTCATTTTTCAACAACATCAGAAAGAATTGATCAGAATCCC 325 vim vim CGCAAGATAGATTTGGAATAGGAATAAGCTCTAGTTCTTAACAACCGACACTCCTACAAG 326 XRCC1 XRCC1 CACACACACACACACACACACACACACACACACACGATGCATTTAATAAAGATGAGTTGGTT 327 bFGF bFGF GGAGCTGGTAACTGATGAAATCTTTTCCCACCTTTTCTCTTCAGGAAATATAAGTGGTTT 328 Plk1 Plk1 CCCCACCATATGAATTGTACAGAATATTTCTATTGAATTCGGAACTGTCCTTTCCTTGGC 329 MAGEA3 MAGEA3 CAGCATCCAGGTTTATGAATGACAGTAGTCACACATAGTGCTGTTTATATAGTTTAGGAG 330 PAOs PAOs TGAGACACCAGATGCTCACGGAGATGCTGGACACATAAAGCAAGTTACAGCCACAAAAAA 331 PRL3 PRL3 TTAGACCCCGGGGCAGTCAGGTGCTCCGGACACCCGAAGGCAATAAAACAGGAGCCGTGA 332 HGF HGF TGTCTGAAGCACCCACCAATACAACTGTCTTTTACATGAAGATTTCAGAGAATGTGGAAT

Several English abbreviations are used in this manual. The Chinese meanings of English abbreviations commonly used in the field of biology are as follows:

English Abbreviation Full English Name Chinese Meaning

DNA Deoxyribonucleic acid Deoxyribonucleic acid

RNA Ribonucleic acid Ribonucleic acid

cDNA Complementary DNA Complementary DNA

PBS Phosphate buffered saline Phosphate buffered saline

SDS Sodium dodecyl sulphate Sodium dodecyl sulfate

SSC Sodium Chloride Sodium Citrate Buffer Saline-Sodium Citrate Buffer

DEPC Diethyl pyrocarbonate Diethyl pyrocarbonate

RNase Ribonuclease Ribonuclease

DTT Dithiothreitol Dithiothreitol

Tris Trishydroxymethyl aminomethane

GTP Guanosine triphosphate Guanosine triphosphate

DMSO Dimothyl sulfoxide Dimethyl sulfoxide

HEPES hydroxyethyl piperazineethanesulfonic acid hydroxyethylpiperazineethanesulfonic acid

BCS Bovine calf serum Bovine calf serum

RPMI1640 Rosewell park memorial institute RPMI1640 culture medium

                                               

OPC oligonucleotide purification kit catr dge

Bp Base pair Base pair

CV Coefficient to fvariation Coefficient of variation

OD Optical density Optical density

rpm Revolution per minute Revolution per minute

Lung Cancer Typing Gene Sequence List

sequence listing

<110> Chinese People's Liberation Army General Hospital

<120> Gene sequence and application of lung cancer typing

<140>200810212208.4

<141>2008-09-05

<160>332

<170>PatentIn version 3.1

<210>1

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>1

<210>2

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>2

<210>3

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>3

<210>4

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>4

<210>5

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>5

<210>6

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>6

<210>7

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>7

<210>8

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>8

<210>9

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>9

<210>10

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>10

<210>11

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>11

<210>12

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>12

<210>13

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>13

<210>14

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>14

<210>15

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>15

<210>16

<211>60

<212>DNA

<213> Human (Homo sapiens)

<220>

<221>misc_feature

<222>(38)..(38)

<223> n is a, c, g, or t

<400>16

<210>17

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>17

<210>18

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>18

<210>19

<211>60

<212>DNA

<213> Human (Homo sapiens)

<220>

<221>misc_feature

<222>(11)..(11)

<223> n is a, c, g, or t

<400>19

<210>20

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>20

<210>21

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>21

<210>22

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>22

<210>23

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>23

<210>24

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>24

<210>25

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>25

<210>26

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>26

<210>27

<211>60

<212>DNA

<213> Human (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(22)

<223> n is a, c, g, or t

<400>27

<210>28

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>28

<210>29

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>29

<210>30

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>30

<210>31

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>31

<210>32

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>32

<210>33

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>33

<210>34

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>34

<210>35

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>35

<210>36

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>36

<210>37

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>37

<210>38

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>38

<210>39

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>39

<210>40

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>40

<210>41

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>41

<210>42

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>42

<210>43

<211>60

<212>DNA

<213> Human (Homo sapiens)

<220>

<221>misc_feature

<222>(9)..(9)

<223> n is a, c, g, or t

<220>

<221>misc_feature

<222>(21)..(21)

<223> n is a, c, g, or t

<220>

<221>misc_feature

<222>(24)..(26)

<223> n is a, c, g, or t

<220>

<221>misc_feature

<222>(28)..(28)

<223> n is a, c, g, or t

<220>

<221>misc_feature

<222>(33)..(33)

<223> n is a, c, g, or t

<220>

<221>misc_feature

<222>(51)..(51)

<223> n is a, c, g, or t

<220>

<221>misc_feature

<222>(60)..(60)

<223> n is a, c, g, or t

<400>43

<210>44

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>44

<210>45

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>45

<210>46

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>46

<210>47

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>47

<210>48

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>48

<210>49

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>49

<210>50

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>50

<210>51

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>51

<210>52

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>52

<210>53

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>53

<210>54

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>54

<210>55

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>55

<210>56

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>56

<210>57

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>57

<210>58

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>58

<210>59

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>59

<210>60

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>60

<210>61

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>61

<210>62

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>62

<210>63

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>63

<210>64

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>64

<210>65

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>65

<210>66

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>66

<210>67

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>67

<210>68

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>68

<210>69

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>69

<210>70

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>70

<210>71

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>71

<210>72

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>72

<210>73

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>73

<210>74

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>74

<210>75

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>75

<210>76

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>76

<210>77

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>77

<210>78

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>78

<210>79

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>79

<210>80

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>80

<210>81

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>81

<210>82

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>82

<210>83

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>83

<210>84

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>84

<210>85

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>85

<210>86

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>86

<210>87

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>87

<210>88

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>88

<210>89

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>89

<210>90

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>90

<210>91

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>91

<210>92

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>92

<210>93

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>93

<210>94

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>94

<210>95

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>95

<210>96

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>96

<210>97

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>97

<210>98

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>98

<210>99

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>99

<210>100

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>100

<210>101

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>101

<210>102

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>102

<210>103

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>103

<210>104

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>104

<210>105

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>105

<210>106

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>106

<210>107

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>107

<210>108

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>108

<210>109

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>109

<210>110

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>110

<210>111

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>111

<210>112

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>112

<210>113

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>113

<210>114

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>114

<210>115

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>115

<210>116

<211>59

<212>DNA

<213> Human (Homo sapiens)

<400>116

<210>117

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>117

<210>118

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>118

<210>119

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>119

<210>120

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>120

<210>121

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>121

<210>122

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>122

<210>123

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>123

<210>124

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>124

<210>125

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>125

<210>126

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>126

<210>127

<211>60

<213> Human (Homo sapiens)

<400>127

<210>128

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>128

<210>129

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>129

<210>130

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>130

<210>131

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>131

<210>132

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>132

<210>133

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>133

<210>134

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>134

<210>135

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>135

<210>136

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>136

<210>137

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>137

<210>138

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>138

<210>139

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>139

<210>140

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>140

<210>141

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>141

<210>142

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>142

<210>143

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>143

<210>144

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>144

<210>145

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>145

<210>146

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>146

<210>147

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>147

<210>148

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>148

<210>149

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>149

<210>150

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>150

<210>151

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>151

<210>152

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>152

<210>153

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>153

<210>154

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>154

<210>155

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>155

<210>156

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>156

<210>157

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>157

<210>158

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>158

<210>159

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>159

<210>160

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>160

<210>161

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>161

<210>162

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>162

<210>163

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>163

<210>164

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>164

<210>165

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>165

<210>166

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>166

<210>167

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>167

<210>168

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>168

<210>169

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>169

<210>170

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>170

<210>171

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>171

<210>172

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>172

<210>173

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>173

<210>174

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>174

<210>175

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>175

<210>176

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>176

<210>177

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>177

<210>178

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>178

<210>179

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>179

<210>180

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>180

<210>181

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>181

<210>182

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>182

<210>183

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>183

<210>184

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>184

<210>185

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>185

<210>186

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>186

<210>187

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>187

<210>188

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>188

<210>189

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>189

<210>190

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>190

<210>191

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>191

<210>192

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>192

<210>193

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>193

<210>194

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>194

<210>195

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>195

<210>196

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>196

<210>197

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>197

<210>198

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>198

<210>199

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>199

<210>200

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>200

<210>201

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>201

<210>202

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>202

<210>203

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>203

<210>204

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>204

<210>205

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>205

<210>206

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>206

<210>207

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>207

<210>208

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>208

<210>209

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>209

<210>210

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>210

<210>211

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>211

<210>212

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>212

<210>213

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>213

<210>214

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>214

<210>215

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>215

<210>216

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>216

<210>217

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>217

<210>218

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>218

<210>219

<212>DNA

<213> Human (Homo sapiens)

<400>219

<210>220

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>220

<210>221

<211>60

<213> Human (Homo sapiens)

<400>221

<210>222

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>222

<210>223

<211>59

<212>DNA

<213> Human (Homo sapiens)

<400>223

<210>224

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>224

<210>225

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>225

<210>226

<211>59

<212>DNA

<213> Human (Homo sapiens)

<400>226

<210>227

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>227

<210>228

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>228

<210>229

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>229

<210>230

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>230

<210>231

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>231

<210>232

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>232

<210>233

<211>59

<212>DNA

<213> Human (Homo sapiens)

<400>233

<210>234

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>234

<210>235

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>235

<210>236

<211>59

<212>DNA

<213> Human (Homo sapiens)

<400>236

<210>237

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>237

<210>238

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>238

<210>239

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>239

<210>240

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>240

<210>241

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>241

<210>242

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>242

<210>243

<211>56

<212>DNA

<213> Human (Homo sapiens)

<400>242

<210>244

<211>60

<212>DNA

<213> Human (Homo sapiens)

<220>

<221>misc_feature

<222>(10)..(10)

<223> n is a, c, g, or t

<220>

<221>misc_feature

<222>(12)..(12)

<223> n is a, c, g, or t

<220>

<221>misc_feature

<222>(22)..(22)

<223> n is a, c, g, or t

<220>

<221>misc_feature

<222>(32)..(32)

<223> n is a, c, g, or t

<220>

<221>misc_feature

<222>(50)..(50)

<223> n is a, c, g, or t

<400>244

<210>245

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>245

<210>246

<211>60

<212>DNA

<213> Human (Homo sapi ens)

<400>246

<210>247

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>247

<210>248

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>248

<210>249

<211>60

<212>DNA

<213> Human (Homo sapiens)

<220>

<221>modified_base

<222>(53)..(53)

<223>q is queuosine

<400>249

<210>250

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>250

<210>251

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>251

<210>252

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>252

<210>253

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>253

<210>254

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>254

<210>255

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>255

<210>256

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>256

<210>257

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>257

<210>258

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>258

<210>259

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>259

<210>260

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>260

<210>261

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>261

<210>262

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>262

<210>263

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>263

<210>264

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>264

<210>265

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>265

<210>266

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>266

<210>267

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>267

<210>268

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>268

<210>269

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>269

<210>270

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>270

<210>271

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>271

<210>272

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>272

<210>273

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>273

<210>274

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>274

<210>275

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>275

<210>276

<211>60

<212>DNA

<213> Human (Homo sapi ens)

<400>276

<210>277

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>277

<210>278

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>278

<210>279

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>279

<210>280

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>280

<210>281

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>281

<210>282

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>282

<210>283

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>283

<210>284

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>284

<210>285

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>285

<210>286

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>286

<210>287

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>287

<210>288

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>288

<210>289

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>289

<210>290

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>290

<210>291

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>291

<210>292

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>292

<210>293

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>293

<210>294

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>294

<210>295

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>295

<210>296

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>296

<210>297

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>297

<210>298

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>298

<210>299

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>299

<210>300

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>300

<210>301

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>301

<210>302

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>302

<210>303

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>303

<210>304

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>304

<210>305

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>305

<210>306

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>306

<210>307

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>307

<210>308

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>308

<210>309

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>309

<210>310

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>310

<210>311

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>311

<210>312

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>312

<210>313

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>313

<210>314

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>314

<210>315

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>315

<210>316

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>316

<210>317

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>317

<210>318

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>318

<210>319

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>319

<210>320

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>320

<210>321

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>321

<210>322

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>322

<210>323

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>323

<210>324

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>324

<210>325

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>325

<210>326

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>326

<210>327

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>327

<210>328

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>328

<210>329

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>329

<210>330

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>330

<210>331

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>331

<210>332

<211>60

<212>DNA

<213> Human (Homo sapiens)

<400>332

Claims (8)

1. the gene sequence that is used for genotyping of lung cancer, is characterized in that, described sequence comprises: (a) the sequence shown in SEQ ID NO 10 to SEQ ID NO 323; (b) the complement of each of the sequences shown in SEQ ID NO 10 to SEQ ID NO 323; (c) a sequence having at least 70% homology to each of the sequences shown in SEQ ID NO 10 to SEQ ID NO 323. 2. A gene chip for genotyping of lung cancer, characterized in that it comprises a fixed carrier and an oligonucleotide probe fixed on the fixed carrier, wherein said oligonucleotide probe comprises the selected from claim 1 Several or all gene sequences. 3. gene chip as claimed in claim 2, described oligonucleotide probe also comprises several or all positive control probes and negative control probes selected from SEQ ID NO 1 to SEQ ID NO 10. 4. The gene chip according to claim 2, which is divided into 4 regions, each region is arranged in a matrix of 18×17, and each region contains 9 positive controls, 5 negative controls and 3 blank controls. 5. The gene chip according to any one of claims 2-4, wherein the fixed carrier is selected from aminated glass slides. 6. A method for genotyping a lung cancer sample, comprising the following steps: (1) Extract RNA from lung cancer samples, reverse transcribe the total RNA into cDNA, and make fluorescently labeled probes; (2) Hybridizing the fluorescently labeled probe prepared above with the oligonucleotide biochip to obtain a hybridization result; (3) The chip was scanned with a laser confocal scanner, and the obtained images were analyzed with computer software, and the correlation analysis was performed on the gene expression profiles of lung cancer by clustering method. 7. The method of claim 6, wherein the lung cancer samples comprise lung adenocarcinoma samples and lung squamous cell carcinoma samples. 8. The method of claim 6, wherein the fluorescent labels include Cy3 and Cy5.

Images