JP2005525114A - Apparatus and method for quantifying mRNA from whole blood with high throughput - Google Patents

Abstract

Disclosed are methods, devices, kits and automated systems for quantifying mRNA from whole blood easily, reproducibly and with high throughput. More particularly, the methods, devices, kits and automation systems include a combination of leukocyte filters attached to oligo (dT) immobilized multiwell plates.

Description

[Background of the invention]
[Field of the Invention]
The present invention relates to high-throughput isolation and quantification of mRNA from whole blood. More particularly, the present invention relates to a method and apparatus for isolating and amplifying mRNA using a combination of leukocyte filters attached to oligo (dT) immobilized multiwell plates.

[Description of related applications]
Studies in the field of molecular biology have revealed that the cell's developmental origin and functional activity can be inferred from its ribonucleic acid (RNA) studies. This information can be used to diagnose infection, detect the presence of oncogene-expressing cells, discover familial disorders, monitor the status of host defense mechanisms, and identify the identity of HLA types or other markers. It can be used effectively in clinical practice to investigate. RNA exists in three functionally different forms: ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA). Stable rRNA and tRNA are involved in the catalytic process of translation, and mRNA molecules transmit genetic information. Only about 1-5% of total RNA is composed of mRNA, about 15% is composed of tRNA, and about 80% is composed of rRNA.

mRNA is an important diagnostic tool, especially when used to quantitatively observe gene up-regulation or down-regulation. Human peripheral blood is an excellent clinical material for analyzing mRNA. For example, the detection of specific chimeric mRNAs in the blood indicates the presence of abnormal cells and is used for molecular diagnosis of chronic myelogenous leukemia (CML). This is described in the following document.
Kawasaki, ES, Clark SS, Coyne MY, Smith SD, Champlin R., Witte ON and McCormick FP 1988. Diagnosis of chronic myeloid and acute lymphocytic luekemias by detection of leukemia-specific mRNA sequences amplified in vitro. Proc. Natl. Acad. Sci. USA 85: 5698-5702,
Pachmann K., Zhao S., Schenk T., Kantarjian H., El-Naggar AK, Siciliano MJ, Guo JQ, Arlinghaus RB and Andreeff M. 2001. Expression of bcr-able mRNA in individual chronic myelogenous leukaemia cells as determined by in situ amplification measured by in situ amplification. Br. J. Haematol. 112: 749-59.
By measuring cancer-specific mRNA, micrometastatic cancer cells can also be detected in blood. Cancer-specific mRNAs include carcinoembryonic antigen (CEA) for colon cancer, prostate-specific antigen (PSA) for prostate cancer, thyroglobulin for thyroid cancer (Wingo ST, Ringel MD, Anderson) JS, Patel AD, Lukes YD, Djuh YY, Solomon B., Nicholson D., Balducci-Silano PL, Levine
MA, Francis GL and Tuttle R .. M. 1999. Quantitative reverse transcription-PCR measurement of thyroglobulin mRNA in peripheral blood of healty subject. Clin. Chem. 45: 785-89), and for melanoma Tyrosinase (Perky (Pe
lkey) TJ, Frierson HF Jr. and Bruns DE 1996.
Molecular and immunological detection of circulating tumor cells and micrometastasis from solid tumors
immnological detection of circulating tumor cells and micrometastasis from solid tumors). Clin. Chem. 42: 1369-81).
Furthermore, since the levels of these cancer-specific mRNAs can change after treatment, quantification of specific mRNAs provides a useful indicator during treatment follow-up.

  Since blood contains a larger amount of anucleated red blood cells (approximately 5 million cells / μL) than white blood cells (approximately 5000 leukocytes / μL), granulocytes or lymphocytes from whole blood are usually used as the first stage of mRNA analysis. Is isolated. However, because the collection of specific subsets of leukocytes does not match between different samples, the number of isolated leukocytes is measured for each sample, and the result is not the amount of mRNA per μL of blood, but the mRNA per leukocyte count. Expressed as a quantity. Furthermore, the amount of mRNA can change during a long isolation process. Although there is no method for isolating cancer cells from blood, gene amplification technology allows the identification and quantification of specific mRNA levels even from pools of different genes, so it is gene specific Whole blood is an ideal material for mRNA analysis if primers and gene specific probes are available.

Because whole blood contains large amounts of RNase (from granulocytes) and anucleated red blood cells, it is very difficult to isolate pure mRNA from whole blood. Various RNA extraction methods can be applied to whole blood, but the assay procedure is labor intensive, requires several centrifugations and must be handled carefully to eliminate ribonuclease activity. It is essential. The RNA extraction method includes:
De Vries TJ, Fullkour A., Punt CJ, Ruiter DJ and van Muijen GN 2000. Tyrosinase and MART-1 after mononuclear cell recovery in cell preparation tubes Analysis of Peripheral Blood Melanoma Cells by Reverse Transcription-Polymerase Chain Reaction: Comparison with Whole Blood Guanidinium Isothiocyanate RNA Isolation Method 1 after mononuclear cell collection with cell preparation tubes: a comparison with the whole blood guanidinium isothiocyanate RNA isoation method). Melanoma Research 10: 119-26,
Johansson M., Pisa EK, Tormanen V., Arstrand K. and Kagedal Bl. 2000. Quantitative analysis of tyrosinase transcripts in blood). Clin. Chem. 46: 921-27,
Wingo ST, Ringer MD, Anderson JS, Patel AD, Rooks YD, Jew YY, Solomon B., Nicholson D., Barducchi-Cyrano PL, Levine
MA, Francis GL and Tuttle R .. M. 1999. Quantitative reverse transcription PCR measurement of thyroglobulin mRNA in peripheral blood of healthy subjects. Clin. Chem. 45: 785-89
It is described in.

  As a result, there is a need for a quick and easy method and apparatus for isolating and quantifying large amounts of mRNA from whole blood. Specifically, there is a need for a technique for processing high-throughput mRNA derived from whole blood, which has a series of processes that are reproducible and seamless to gene amplification.

[Summary of Invention]
The present invention provides an efficient high-throughput method for the isolation and quantification of mRNA directly from whole blood with the reproducibility of recovery using a combination of leukocyte filters attached to oligo (dT) immobilized multiwell plates. And an apparatus are disclosed.

  One embodiment of the present invention includes (a) collecting whole blood, (b) removing red blood cells and blood components from the whole blood by filtration, thereby obtaining white blood cells on a filter membrane, (c) Lysing the leukocytes, thereby producing a lysate containing mRNA, (d) transferring the lysate to an oligo (dT) immobilized plate, thereby capturing the mRNA, and (E) including a high-throughput quantification method of mRNA in whole blood, comprising quantifying the mRNA.

  In a preferred embodiment of the method, an anticoagulant is added to the whole blood before collecting the white blood cells. A plurality of filter membranes may be overlaid to increase the yield of captured leukocytes. Leukocytes captured on the filter membrane are lysed using a lysis buffer to release mRNA from the leukocytes. Transfer of the lysate to the oligo (dT) immobilized plate can be performed by centrifugation, by vacuum suction, by positive pressure, or by vacuum suction following an ethanol wash. mRNA is quantified by preparing cDNA and amplifying the cDNA by PCR.

  Another aspect of the invention is: (a) a plurality of sample delivery wells, a leukocyte capture filter at the bottom of the well, and an mRNA capture zone at the bottom of the filter (which has an immobilized oligo (dT)) And (b) a device for quantifying mRNA in whole blood with high throughput, comprising a vacuum box that receives the plate and forms a seal with the plate. In one preferred embodiment of the device, leukocytes are captured on a plurality of superimposed filter membranes. In another preferred embodiment of the apparatus, a vacuum box receives a vacuum source. In another preferred embodiment of the apparatus, a multiwell supporter is sandwiched between the vacuum box and the multiwell plate.

  Another aspect of the present invention includes a kit for high-throughput quantitation of mRNA in whole blood, a kit comprising heparin, hypotonic buffer and lysis buffer.

  Another aspect of the present invention provides high throughput of mRNA in whole blood comprising a blood sample, a robot that applies hypotonic buffer and lysis buffer to the device, an automatic vacuum aspirator and centrifuge, and an automatic PCR machine. Includes a fully automated system for quantifying.

Detailed Description of Preferred Embodiments
The present invention enables the analysis of large volumes of unprepared whole blood and provides an efficient means of analyzing mRNA derived only from leukocytes. The means can remove rRNA and tRNA, consistently recover mRNA, and can be easily adapted to automation. The present invention provides a sensitive quantification system capable of absolute quantification using real-time PCR and having excellent reproducibility with a coefficient of variation in the range of 20 to 25%. Furthermore, the present invention can be applied to various disease targets (Table I).

  The present invention is not limited to any particular mechanical structure. However, FIGS. 1 and 2 show preferred structures for performing high throughput mRNA quantification of the present invention. The vacuum box 10 constitutes the basis of the structure. The vacuum box may be made of any material that is strong enough to withstand vacuum suction, but the material is preferably a disposable plastic material. The vacuum box is adapted to receive a vacuum source 12 for performing vacuum suction. The filter plug 14 is disposed in the vacuum suction device adapter of the vacuum box. The vacuum box 10 preferably has an edge 16 for mating with the multi-well filter plate 40 or, optionally, the multi-well supporter 20. A multiwell support 20 is optionally provided inside the upper portion of the vacuum box to support the multiwell filter plate 40. A sealing gasket 30, preferably made of silicone rubber or other soft plastic, is disposed on the top surface of the multiwell supporter. A multiwell filter plate 40 is disposed on top of the sealing gasket, the filter plate comprising a number of sample wells 46, a multiple leukocyte capture filter 42 below the sample delivery well, and an mRNA capture zone 44 below the filter. Immobilized oligo (dT) is contained in the wells of the mRNA capture zone.

  One preferred embodiment includes a simple and reproducible high-throughput method for quantifying mRNA from whole blood. Because of the rapid protocol, secondary induction or degradation of mRNA after blood collection is minimized and the use of 96 well filter plates and microplates allows 96 samples to be processed simultaneously. Since the number of operations between procedures is minimal, even when PCR is used as a means for quantification, the variation between samples is very small, and the coefficient of variation (CV) value is less than 30%.

In one embodiment, the method includes making a vacuum box. In a preferred embodiment, a blood encapsulant such as a polyacrylate polymer matrix (Red Z, Safetec) is placed in a vacuum box to solidify the blood. The multiwell supporter is then placed in a vacuum box. A silicone rubber or other soft plastic sealing gasket is then placed on the top surface of the multiwell plate supporter. Place the filter plug (X-6953, 60μ Filter Plug HDPE, Porex Products Groups) in the vacuum aspirator adapter of the vacuum box.

The method in this embodiment involves making a filter plate. To capture leukocytes, either a glass fiber membrane or a leukocyte filter membrane can be used. By producing a multiwell filter plate using a glass fiber membrane or a leukocyte filter membrane, a large number of blood samples can be processed simultaneously, and the assay can be simplified. Examples of filters for capturing leukocytes are described in US Pat. Nos. 4,925,572 and 4,880,548, the descriptions of which are hereby incorporated by reference. In general, the adsorption of leukocytes on the fiber surface is considered to be a mechanism for removing leukocytes. The surface area of a given weight of fiber is inversely proportional to its diameter. Thus, if the diameter of the fiber used is small, the thinner the fiber is expected to have a greater adsorption capacity, and the amount required to achieve the desired effectiveness (measured as the weight of the fiber) Less) is expected. Some commonly used fibers (such as polyester, polyamide and acrylic fibers) are sufficiently resistant and available to degradation by the level of alpha radiation required to be graft polymerized Since the monomer has a structure capable of reacting, it is suitable for radiation graft polymerization. PBT is the main resin used in the development of the product of the present invention and is the resin used in this example. However, there can also be other resins that can be fiberized and that can form a mat or fabric consisting of fine fibers of 1.5 micrometers or less. The resin has a critical wetting surface tension adjusted to an optimum range as necessary. It should also be noted that the resin is suitable for the efficient production of filter plates and may be suitable for the production of smaller leukocyte removal devices. Similarly, properly treated glass fibers can be used to make an effective device. CD4 mRNA adsorption is up to 4 times more effective when PBT filters are used, as opposed to when glass fiber filters are used. The filter plate is placed in a vacuum box.
In another preferred embodiment, multiple filter membranes are overlaid to increase the amount of leukocytes captured from whole blood. In a preferred embodiment, the filter plate is placed on a plate supporter and a sealing gasket. In another preferred embodiment, the filter plate is sealed with a plastic adhesive tape (Bio-Rad 223-9444), and the desired number of wells of tape can be cut and made available. In another preferred embodiment, each well to which sample is added is washed with hypotonic buffer (200 μL 5 mM Tris, pH 7.4).

The method preferably includes collection of blood, addition of blood to a multiwell filter plate, and removal of red blood cells and other non-white blood cell components. In a preferred embodiment, whole blood is aspirated into a blood collection tube containing an anticoagulant. This increases the leukocyte filtration efficiency. Heparin, an anticoagulant, is particularly effective in increasing leukocyte filtration efficiency.
In a preferred embodiment, the blood sample may be frozen. Thereby, a part of RNase which destroys mRNA is removed. Wells may be washed with hypotonic buffer. When blood is added to the desired number of wells on the filter plate, the blood is filtered through the filter membrane. The filtration may be performed by any technique known to those skilled in the art such as centrifugation, vacuum suction or positive pressure.

  In one particularly preferred embodiment, vacuum suction is initiated (6 cmHg) after the blood sample is added to the filter plate well. Each well is washed several times with hypotonic buffer (12 times with 200 μL 5 mM Tris, pH 7.4). In another preferred embodiment, each well containing the sample is washed with ethanol (1 × 200 μL 100% ethanol) to dry the filter membrane and significantly increase leukocyte capture efficiency when vacuumed. In another preferred embodiment, a vacuum is then applied (> 20 minutes at 20 cm Hg).

The method includes cell lysis and hybridization of mRNA to oligo (dT) immobilized within the mRNA capture zone. Lysis buffer is added to the filter plate wells (40 μL / well) and incubated (20 minutes at room temperature) to liberate mRNA from the captured leukocytes. In a preferred embodiment, the multiwell filter plate is sealed in a plastic bag and centrifuged (IEC MultiRF, 2000 rpm, 4C for 1 minute). The lysis buffer is then added again (20 μL / well) and then centrifuged (IEC MultiRF, 3000 rpm, 4C for 5 minutes). The multiwell filter plate is then removed from the centrifuge and incubated (2 hours at room temperature).

The method involves quantification of mRNA. A preferred embodiment includes the synthesis of cDNA from mRNA and amplification of the cDNA using PCR. In one preferred embodiment, the multi-well filter plate is washed with lysis buffer (3 times 150 μL / well, manual) and wash buffer (3 times 150 μL / well, manual or BioTek # G4). CDNA synthesis buffer is then added to the multiwell filter plate (40 μL / well, manual or I & J # 6). Axymat (Amgen AM-96-PC
R-RD) may be placed on a multiwell filter plate, which is then placed on a heat block (37C, VWR) and incubated (> 90 minutes). The multiwell filter plate may then be centrifuged (2000 rpm, 4C for 1 minute). PCR primers are added to the 384 well PCR plate and the cDNA is transferred from the multiwell filter plate to the 384 well PCR plate. The PCR plate is centrifuged (2000 rpm, 4C for 1 minute) and real-time PCR is started (TaqMan / SYBER).

  Another preferred embodiment of the invention includes an apparatus for high-throughput quantification of mRNA from whole blood. The apparatus includes a number of sample delivery wells, a leukocyte capture filter below the sample delivery well, and an mRNA capture zone below the filter (the mRNA capture zone is an oligo (dT) immobilized on the mRNA capture zone in the well) A multiwell filter plate. Several filter membranes may be overlaid in order to increase leukocyte collection efficiency. The multi-well plate is mounted on a vacuum box, but the vacuum box receives the multi-well plate, and a seal is formed by the multi-well plate. In a preferred embodiment of the apparatus, the vacuum box receives a vacuum source for performing vacuum suction. In another preferred embodiment, a multiwell supporter is placed below the multiwell filter plate in the vacuum box. In another preferred embodiment of the device, a sealing gasket, which can be made of a soft plastic such as silicone rubber, is sandwiched between the multiwell supporter and the multiwell filter plate.

  Another preferred embodiment includes a kit for high-throughput quantification of mRNA from whole blood. The kit includes a device for high-throughput quantification of mRNA from whole blood, a heparin-containing blood collection tube, a hypotonic buffer and a lysis buffer.

  Another preferred embodiment is for high-level mRNA in whole blood comprising a robot for applying blood samples, hypotonic buffer and lysis buffer to the device, an automatic vacuum aspirator and centrifuge, and an automatic PCR machine. Includes a fully automated system for quantifying throughput.

  Various protocols of the method of the invention were tested and used to quantify β-actin mRNA and CD4 mRNA from whole blood.

Three anticoagulants were tested: ACD, EDTA and heparin, and heparin produced the highest leukocyte retention. A Leukosorb membrane was used to transfer ACD blood, but approximately 15-40% of the leukocytes passed through even when four layers of membrane were used simultaneously. ED
Although TA blood was tested, it was found that the adsorption capacity and leukocyte retention were the same as for ACD blood. Most notably, however, 100% of the white blood cells in heparinized blood have been trapped on the Leukosorb membrane. Capturing 100% of heparinized white blood cells indicates the reliability of mRNA quantification using the present invention. These data indicate that using heparinized blood is very suitable for accurate quantification of mRNA, whereas ACD is required when large volumes of blood are required and less quantitative results are required. Show that blood is useful.

The results using frozen blood samples and fresh blood samples were compared. As shown in FIG. 3, more CD4 mRNA was recovered from fresh blood leaked cells than from frozen sample leaked cells.

  In addition, the effectiveness of the glass fiber filter for holding whole blood was examined by comparing with the holding value of the PBT filter membrane. As shown in Table II, the glass fiber membranes received only 40 μL of whole blood even when the membranes were washed with hypotonic buffer (50 mM Tris, pH 7.4) to destroy erythrocytes. However, Leukosorb filters received significantly higher amounts of whole blood than glass fiber filters, as shown in Table II.

Various times of washing with hypotonic buffer were applied to remove red blood cells and other blood components. As shown in FIG. 4, when the sample was washed at least three times with a hypotonic buffer, the amount of captured CD4 mRNA was more than doubled compared to the case where the sample was not washed. FIG. 4 also shows that the most mRNA was captured when the blood was washed 12 times with hypotonic buffer.
Furthermore, for final CD4 mRNA quantification, various methods for recovering leukocytes were compared, ie, a method by vacuum suction of blood samples, a method by centrifugation, and a method by vacuum suction following ethanol washing. FIG. 5 shows that the vacuum aspiration method yields better CD4 mRNA quantification than the centrifugation method, and the method of washing blood samples with ethanol prior to vacuum aspiration yields the most mRNA. Indicates.

  Leukocytes were captured on glass fiber membranes or Leukosorb membranes and subjected to various times of washing with hypotonic buffer to remove red blood cells and other blood components. To release mRNA from the captured leukocytes, lysis buffer (RNAture) was added to the filter plate (40 μL / well) and the plate was incubated at room temperature for 20 minutes. It is preferable to include an amplification primer in the lysis buffer. FIG. 6 shows that lysis buffer plays an important role in RNase inhibition, and eosinophils are filled with RNase inactivated by lysis buffer. The multiwell filter plate was then sealed in a plastic bag and centrifuged (IEC MultiRF, 2000 rpm, 4C for 1 minute). Lysis buffer was then added again (20 μL / well) followed by centrifugation (IEC MultiRF, 3000 rpm, 4C for 5 minutes). The multiwell filter plate was then removed from the centrifuge and incubated at room temperature for 2 hours to hybridize the poly (A) + RNA tail and the immobilized oligo (dT). The multiwell filter plate is then washed 3 times with 150 μL lysis buffer to remove residual ribonuclease, followed by 3 washes with 150 μL wash buffer (BioTek # G4) to obtain some inhibitor of cDNA synthesis. The containing lysis buffer was removed.

The washing buffer was completely removed from the multiwell filter plate by the last wash described above, and cDNA was synthesized in each well by adding 40 μL of premixed cDNA buffer. The cDNA buffer is 5x first strand buffer (Promega M531A, 10 mM
dNTP (Promega stock, 20 ×)), primer (5 μM, # 24), RNasi
n (Promega N211A, 400 U / μL), M-MLV reverse transcriptase (Promega M170A, 200 U / μL) and DEPC water. FIG. 7 shows that the optimal MMLV concentration for quantifying mRNA is 0.25 units / well.

  Axymat (Amgen AM-96-PCR-RD) was placed on a multiwell filter plate, which was then placed on a heat block (37C, VWR) and incubated (> 90 minutes). The multiwell filter plate was then centrifuged (2000 rpm, 4C for 1 minute). PCR primers were added to the 384 well PCR plate and the cDNA was transferred from the multiwell filter plate to the 384 well PCR plate. FIG. 8 shows that the optimal cDNA value for PCR is approximately 2 μL / well. The PCR plate was centrifuged (2000 rpm, 4C for 1 minute) to start real-time PCR (TaqMan / SYBER). The method of the present invention has high mRNA specificity, and no DNA contamination was detected in CD4 mRNA amplification with TaqMan qPCR (<10 copies / well).

As shown in FIG. 9, the present invention has a small coefficient of variation in mRNA quantification. In the hybridization for 2 hours, the coefficient of variation of the mRNA quantification so far was about 300%, whereas the coefficient of variation was less than 13%. Furthermore, the linear results shown in FIG. 10 show that the amount of mRNA captured is directly proportional to the volume of whole blood used per well, making the present invention reliable and reproducible. It shows that it is a quantitative method of mRNA.

It is an exploded view of a high-throughput mRNA device. FIG. 6 shows a multi-well plate of a high-throughput mRNA device that includes a leukocyte filter and an oligo (dT) immobilized filter well. It is a graph which shows the leukocyte capture | acquisition efficiency on the filter plate of a fresh blood sample and a frozen blood sample. It is a graph which shows the effect of the frequency | count of blood washing | cleaning with respect to mRNA quantification. It is a graph which shows the effect of the final process of the filter plate before cell lysis with respect to mRNA quantification. FIG. 6 is a graph showing the extent to which lysis buffer inhibits RNase. It is a graph which shows the optimal density | concentration of a reverse transcriptase for quantifying mRNA. It is a graph which shows the optimal cDNA value in PCR for capturing mRNA. It is a graph which shows the dynamics of hybridization of this invention. It is a graph which shows the linear relationship between the whole blood volume used per well, and mRNA quantification.

Claims (42)

(A) collecting whole blood;
(B) removing blood components other than red blood cells and white blood cells by filtration from the whole blood to obtain white blood cells on a filter membrane;
(C) lysing the leukocytes on the filter membrane to produce a lysate containing mRNA;
High throughput of specific mRNA in whole blood comprising: (d) transferring the lysate to an oligo (dT) immobilized plate and capturing the mRNA; and (e) quantifying the specific mRNA. Quantitative method.   The method of claim 1, wherein the whole blood is added with an anticoagulant prior to collection of leukocytes.   The method according to claim 1, wherein the whole blood is added with heparin prior to the collection of leukocytes.   The method of claim 1, wherein the whole blood is frozen prior to filtration.   The method of claim 1, wherein the filter membrane is attached to a multiwell filter plate.   The method of claim 1, wherein the filter membrane is a PBT fiber membrane.   6. The method of claim 5, wherein the white blood cells are captured on a plurality of superimposed filter membranes.   The method of claim 1, further comprising washing leukocytes on the filter membrane with a hypotonic buffer to further remove red blood cells and other blood components.   9. The method of claim 8, further comprising the step of drying the filter membrane.   The method of claim 9, wherein the filter membrane is washed with ethanol and vacuumed until the filter membrane is dry.   The method of claim 1, wherein the immobilized plate comprises a multiwell oligo (dT) immobilized plate.   The method of claim 1, wherein transferring the lysate to an oligo (dT) immobilized plate comprises centrifugation.   The method of claim 1, wherein transferring the lysate to an oligo (dT) immobilized plate comprises vacuum suction.   The method of claim 1, wherein transferring the lysate to an oligo (dT) immobilized plate comprises a positive pressure load.   2. The method of claim 1, wherein the quantification of mRNA comprises cDNA synthesis of the specific mRNA and amplification of the resulting cDNA.   The method according to claim 1, wherein the quantified mRNA is a-actin mRNA.   2. The method of claim 1, wherein the quantified mRNA is CD4 mRNA.   The method according to claim 1, wherein mRNA of a translocation gene involved in leukemia is quantified.   The method according to claim 1, wherein mRNA of a cancer-specific gene derived from micrometastatic cancer is quantified.   The method according to claim 1, wherein virus-derived mRNA derived from infected leukocytes is quantified.   21. The method of claim 20, wherein the quantified virus-derived mRNA is HIV.   24. The method of claim 21, wherein the quantification of HIV mRNA is used to diagnose HIV.   21. The method of claim 20, wherein the quantified virus-derived mRNA is CMV.   24. The method of claim 23, wherein quantification of the virus-derived mRNA is used to diagnose CMV.   21. The method of claim 20, wherein quantification of the virus-derived mRNA is used to monitor stored blood for the presence of viral disease.   21. The method of claim 20, wherein quantification of the viral-derived mRNA is used to test antiviral drug sensitivity.   The method according to claim 1, wherein mRNA of an apoptotic gene involved in leukemia is quantified.   2. The method of claim 1, wherein cytokine mRNA is quantified.   The method of claim 1, wherein the quantification of mRNA is used to test side effects of anticancer agents on leukocytes.   The method according to claim 1, wherein mRNA of the DNA repair gene is quantified.   32. The method of claim 30, wherein quantification of the DNA repair gene mRNA is used to test the sensitivity of the DNA repair gene to radiation.   The method according to claim 1, wherein mRNA of the allergen response gene is quantified.   33. The method of claim 32, wherein quantification of the allergen response gene mRNA is used to test for allergen stimulation.   2. The method of claim 1, wherein the mRNA of the donor cell mediated cytokine is quantified.   35. The method of claim 34, wherein quantification of the donor cell mediated cytokine mRNA is used to test graft rejection. (A) i) Multiple sample delivery wells,
ii) a leukocyte capture filter at the bottom of the well; and iii) a multiwell plate at the bottom of the filter with an mRNA capture zone having oligo (dT) immobilized on the top surface thereof; and (b) said A high-throughput mRNA quantification device comprising a vacuum box adapted to receive a plate and create a seal between the plate.   38. The apparatus of claim 36, wherein the vacuum box is adapted to receive a vacuum source.   The apparatus of claim 36, wherein the vacuum box is made of plastic.   The apparatus of claim 36, wherein the seal comprises a plastic-based gasket disposed below the multi-well plate.   37. The apparatus of claim 36, wherein a multiwell supporter is sandwiched between the vacuum box and the multiwell plate.   38. The device of claim 36, wherein the white blood cells are captured on a plurality of superimposed filter membranes. (A) The high-throughput mRNA quantification device according to claim 36,
(B) a hypotonic buffer,
A high-throughput mRNA quantification kit comprising (c) ethanol, and (d) a lysis buffer.

Images