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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
  • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• the present invention relates to high-throughput isolation and quantification of mRNA from whole blood. More particularly, this invention relates to a method and device for isolating and amplifying mRNA using combinations of leukocyte filters attached to oligo(dT)-immobilized multi-well plates.
• RNA ribonucleic acid
• This information may be of use in clinical practice, to diagnose infections, to detect the presence of cells expressing oncogenes, to detect familial disorders, to monitor the state of host defense mechanisms and to determine the HLA type or other marker of identity.
• RNA exists in three functionally different forms: ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA). Whereas stable rRNA and tRNA are involved in catalytic processes in translation, mRNA molecules carry genetic information.
• RNA Only about 1-5% of the total RNA consists of mRNA, about 15% of tRNA and about 80% of rRNA.
• mRNA is an important diagnostic tool, particularly when it is used to quantitatively observe up- or down-regulation of genes.
• Human peripheral blood is an excellent clinical resource for mRNA analysis.
• the detection of specific chimeric mRNA in blood indicates the presence of abnormal cells and is used in molecular diagnostics for chronic myelogenous leukemia (CML) (Kawasaki E.S., Clark S.S., Coyne M.Y., Smith S.D., Champlin R., Witte O.N., and McCormick F.P. 1988.
• CML chronic myelogenous leukemia
• Micrometastatic cancer cells can also be detected in blood by measuring cancer-specific mRNA, such as carcinoembryonic antigen (CEA) for colon cancer, prostate specific antigen (PSA) for prostate cancer, thyroglobulin for thyroid cancer (Wingo S.T., Ringel M.D., Anderson J.S., Patel A.D., Lukes Y.D., Djuh Y.Y., Solomon B., Nicholson D., Balducci- Silano P ., Levine M.A., Francis G.L., and Tuttle R.M. 1999. Quantitative reverse transcription-PCR measurement of thyroglobulin mRNA in peripheral blood of healthy subjects. Clin. Chem.
• mRNA quantities may change during lengthy isolation processes. While no method exists for the isolation of cancer cells from blood, gene amplification technologies enable the identification and quantification of specific mRNA levels even from a pool of different genes, making whole blood an ideal material for mRNA analysis when gene-specific primers and probes are available.
• RNA extraction methods are available for whole blood applications (de Vries T.J., Fourkour A., Punt C.J., Ruiter D.J., and van Muijen G.N. 2000. Analysis of melanoma cells in peripheral blood by reverse transcription-polymerase chain reaction for tyrosinase and MART-1 after mononuclear cell collection with cell preparation tubes: a comparison with the whole blood guanidinium isothiocyanate RNA isolation method.
• the present invention discloses an efficient high throughput method and device for isolating and quantifying mRNA directly from whole blood, with reproducible recovery, using combinations of leukocyte filters attached to oligo(dT)-immobilized multi-well plates.
• One aspect of the invention includes a method of high throughput quantification of mRNA in whole blood, including the steps of: (a) collecting whole blood; (b) removing erythrocytes and blood components from the whole blood by filtration to yield leukocytes on a filter membrane; (c) subjecting the leukocytes to cell lysis to produce a lysate containing mRNA; (d) transferring the lysate to an oligo(dT)-immobilized plate to capture the mRNA; and (e) quantifying the mRNA.
• an anticoagulant is administered to the whole blood prior to collection of leukocytes.
• filter membranes can be layered together to increase the yield of captured leukocytes.
• the leukocytes that are trapped on the filter membrane are lysed using a lysis buffer to release mRNA from the leukocytes.
• the transfer of lysate to the oligo(dT)-immobilized plate can be accomplished using centrifugation, vacuum aspiration, positive pressure, or washing with ethanol followed by vacuum aspiration.
• the mRNA is quantified by producing cDNA and amplifying the cDNA by PCR.
• Another aspect of the invention includes a device for performing high throughput quantification of mRNA in whole blood, wherein the device includes: (a) a multi-well plate containing: a plurality of sample-delivery wells; a leukocyte-capturing filter underneath the wells; and an mRNA capture zone underneath the filter which contains immobilized oligo(dT); and (b) a vacuum box adapted to receive the filter plate to create a seal between the plate and the box.
• the leukocytes are captured on a plurality of filter membranes that are layered together.
• the vacuum box is adapted to receive a source of vacuum.
• a multi-well supporter is inserted between the vacuum box and the multi-well plates.
• kits which contains: the device for performing high throughput quantification of mRNA in whole blood, heparin, a hypotonic buffer, and a lysis buffer.
• Another aspect of the invention includes a fully automated system for performing high throughput quantification of mRNA in whole blood, including: a robot to apply blood samples, hypotonic buffer, and lysis buffer to the device; an automated vacuum aspirator and centrifuge, and automated PCR machinery.
• FIG. 1 is an exploded drawing of the high throughput mRNA device.
• FIG. 2 depicts the multi-well plate, including the leukocyte filter and oligo-(dT)- immobilized filter wells, of the high throughput mRNA device.
• FIG. 3 is a graph showing the efficiency of leukocyte trapping of fresh and frozen blood samples on filter plates.
• FIG. 4 is a graph showing the effect of number of washes of blood on mRNA quantification.
• FIG. 5 is a graph showing the effect of final treatments of filter plates before cell lysis on mRNA quantification.
• FIG. 6 is a graph showing how lysis buffer inhibits RNase.
• FIG. 7 is a graph showing optimal concentrations of reverse transcriptase for mRNA quantification.
• FIG. 8 is a graph showing optimal values of cDNA for PCR to capture mRNA.
• FIG. 9 is a graph showing the hybridization kinetics of the invention.
• FIG. 10 is a graph showing the linear relationship between whole blood volume used per well and mRNA quantification.
• the present invention allows analysis of larger volumes of unprepared whole blood, provides an efficient means of analyzing mRNA that is derived exclusively from white blood cells; removes rRNA and tRNA, provides consistent mRNA recovery, and is easily adaptable to automation.
• the present invention provides a sensitive quantification system, including: absolute quantification using real time PCR, and excellent reproducibility with coefficients of variation ranging from 20-25%.
• the present invention is applicable to various disease targets (Table I). Table I. Clinical targets
• FIGs 1 and 2 show a preferred structure for implementing the high throughput mRNA quantification of the present invention.
• a vacuum box 10 forms the base of the structure.
• the vacuum box can be made of any material sufficiently strong to withstand vacuum aspiration; however, disposable plastic material is preferred.
• the vacuum box is adapted to receive a source of vacuum in order to perform vacuum aspiration 12.
• a filter plug 14 is located within the vacuum aspirator adapter of the vacuum box.
• the vacuum box 10 preferably has a ledge 16 to mate with a multi-well filter plate 40, or optionally, a multi- well supporter 20.
• the multi-well supporter 20 is optionally provided inside the upper part of the vacuum box so as to support the multi-well filterplate 40.
• One preferred embodiment involves a simple, reproducible, and high throughput method of mRNA quantification from whole blood. The rapid protocol minimizes the secondary induction or degradation of mRNA after blood draw, and the use of 96-well filterplates and microplates allows the simultaneous manipulation of 96 samples. Minimal manipulation during the procedure provides for very small sample-to-sample variation, with coefficient of variation (CV) values of less than 30%, even when PCR is used as a means of quantification.
• CV coefficient of variation
• the method involves preparation of the vacuum box.
• a blood encapsulator such as polyacrylate polymer matrix (Red Z, Safetec) is added to the vacuum box to solidify the blood.
• a multi-well supporter is then placed in the vacuum box.
• a sealing gasket made of silicon-based rubber or other soft plastics is then placed on top of the multi-well plate supporter.
• a filter plug (X-6953, 60 ⁇ Filter Plug HDPE, Porex Products Groups) is placed in the vacuum aspirator adapter of the vacuum box.
• the method involves the preparation of the filter plate. Either glassfiber membranes or leukocyte filter membranes can be used to capture leukocytes.
• multiple-well filterplates are constructed using glassfiber membranes or leukocyte filter membranes to enable the simultaneous processing of multiple blood specimens.
• filters for capturing leukocytes are disclosed in U.S. patent numbers 4,925,572 and 4,880,548, the disclosures of which are hereby incorporated by reference.
• Adsorption of leukocytes on fiber surfaces is generally accepted as the mechanism of leukocyte removal. Since the surface area of a given weight of fibers is inversely proportional to the diameter of the fibers, it is to be expected that finer fibers will have higher capacity and that the quantity as measured by weight of fibers necessary to achieve a desired efficiently will be less if the fibers used are smaller in diameter.
• PBT has been the principal resin used for the development of the products of this invention and is the resin used in the examples. It should be noted, however, that other resins may be found which can be fiberized and collected as mats or webs with fibers as small as 1.5 micrometers or less, and that such products, with their critical wetting surface tensions adjusted as necessary to the optimum range, may be well suited to the fabrication of equally efficient but still smaller leukocyte depletion devices. Similarly, glass fibers, appropriately treated, may be usable to make effective devices.
• the filter plate is placed in the vacuum box.
• multiple filter membranes are layered together to increase the amount of leukocytes captured from whole blood.
• the filter plate is placed upon the plate supporter and the sealing gasket.
• the filter plate is sealed with a plastic adhesive tape (Bio-Rad 223-9444), and the tape is cut to allow access to a desired number of wells.
• each well to which a sample will be added is washed with a hypotonic buffer (200 ⁇ L 5mM Tris, pH 7.4).
• the method preferably involves collecting blood, adding the blood to the multi-well filter plate, and removal of erythrocytes and other non-leukocyte components.
• whole blood can be drawn into blood collection tubes containing anticoagulants, which increase the efficiency of the leukocyte filtering.
• the anticoagulant, heparin is particularly effective in increasing the efficiency of leukocyte filtering.
• the blood sample can be frozen, which removes some of the RNases that destroy mRNA.
• the wells can be washed with a hypotonic buffer. Once blood has been added to the desired number of wells on the filterplate, the blood is filtered through the filter membrane. Filtration can be affected through any technique known to those of skill in the art, such as centrifugation, vacuum aspiration, or positive pressure.
• vacuum aspiration is commenced (with 6 cm Hg) after the blood samples have been added to the filterplate wells.
• Each well is washed several times with a hypotonic buffer (12x with 200 ⁇ L 5mM Tris, pH 7.4).
• each well containing a sample is washed with ethanol (lx with 200 ⁇ L 100% ethanol), which dries the filter membrane and significantly increases the efficiency of leukocyte trapping during vacuum aspiration.
• the vacuum is then applied (20 cmHg for >2 min).
• the method involves cell lysis and hybridization of mRNA to the oligo(dT)- immobilized within the mRNA capture zone.
• Lysis buffer is applied to the filterplate wells (40 ⁇ L/well), and incubation is allowed to occur (room temperature for 20 min) to release mRNA from the trapped leukocytes.
• the multi-well filterplate is sealed in a plastic bag and centrifuged (IEC MultiRF, 2000 rpm, at 4 C, for 1 min). Lysis buffer is then added again (20 ⁇ L/well), followed by centrifugation (IEC MultiRF, 3000 rpm, at 4 C, for 5 min). The multi-well filterplate is then removed from the centrifuge and incubated (room temperature for 2hrs).
• the method involves quantification of mRNA, which in a preferred embodiment entails cDNA synthesis from mRNA and amplification of cDNA using PCR.
• the multi-well filterplate is washed with lysis buffer (150 ⁇ L/well x 3 times, manual) and wash buffer (150 ⁇ L/well x 3 times, manual or BioTek #G4).
• a cDNA synthesis buffer is then added to the multi-well filterplate (40 ⁇ L/well, manual or I&J #6).
• Axymat Amgen AM-96-PCR-RD
• the multi- well filterplate can then be centrifuged (2000 rpm, at 4 C for 1 min).
• PCR primers are added to a 384 well PCR plate, and the cDNA is transferred from the multi-well filterplate to the 384 well PCR plate.
• the PCR plate is centrifuged (2000 rpm, at 4 C for 1 min), and real time PCR is commenced (TaqMan/SYBER).
• the device includes a multi-well filterplate containing: multiple sample-delivery wells, a leukocyte-capturing filter underneath the sample-delivery wells, and an mRNA capture zone under the filter, which contains oligo(dT)-immobilized in the wells of the mRNA capture zone.
• a multi-well filterplate containing: multiple sample-delivery wells, a leukocyte-capturing filter underneath the sample-delivery wells, and an mRNA capture zone under the filter, which contains oligo(dT)-immobilized in the wells of the mRNA capture zone.
• several filtration membranes can be layered together.
• the multi-well plate is fitted upon a vacuum box, which is adapted to receive the plate and to create a seal between the multi-well plate and the vacuum box.
• the vacuum box is adapted to receive a source of vacuum in order to perform vacuum aspiration.
• a multi- well supporter is placed in the vacuum box, below the multi-well filterplate.
• a sealing gasket which can be made from soft plastic such as silicon-based rubber, is inserted between the multi-well supporter and the multi- well filterplate.
• kits for high-throughput quantification of mRNA from whole blood includes: the device for high-throughput quantification of mRNA from whole blood; heparin-containing blood-collection tubes; a hypotonic buffer; and a lysis buffer.
• Another preferred embodiment involves a fully automated system for performing high throughput quantification of mRNA in whole blood, including: robots to apply blood samples, hypotonic buffer, and lysis buffer to the device; an automated vacuum aspirator and centrifuge, and automated PCR machinery.
• Example Various protocols of the method of the present invention were tested and used to quantify ⁇ -actin mRNA and CD4 mRNA from whole blood.
• ACD Three anticoagulants were tested: ACD, EDTA, and heparin, with heparin resulting in the highest percent of leukocyte retention.
• Leukosorb membranes While Leukosorb membranes have been used for ACD blood in transfusion, approximately 15-40% of leukocytes passed through even when four layers of membranes were simultaneously used.
• EDTA blood was tested; the capacity and leukocyte retention was found to be similar to those for ACD. Most notably, however, was that 100% of the leukocytes in heparin blood were trapped on the Leukosorb membranes. The capture of 100% of leukocytes from heparin blood shows the reliability of quantification of mRNA using the present invention.
• glassfiber membranes accepted only 40 ⁇ L of whole blood, even when membranes were washed with hypotonic buffer (50 mM Tris, pH 7.4) to burst erythrocytes.
• hypotonic buffer 50 mM Tris, pH 7.4
• Leukosorb filters accepted significantly larger amounts of whole blood than the glassfiber filters, as indicated in Table II.
• (A, H) represents anticoagulants used in the experiments.
• FIG 5 indicates that while vacuum aspiration resulted in better CD4 mRNA quantification than centrifugation, washing blood samples with ethanol prior to vacuum aspiration yields the most mRNA.
• amplification primers are N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe-N-(tyl)-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
• FIG 6 indicates that lysis buffer plays an important role in RNase inhibition; eosinophils are filled with RNases, which are inactivated by the lysis buffer.
• the multi-well filterplate was then sealed in a plastic bag and centrifuged (IEC MultiRF, 2000 rpm, at 4 C, for 1 min). Lysis buffer was then added again (20 ⁇ L/well), followed by centrifugation (IEC MultiRF, 3000 rpm, at 4 C, for 5 min). The multi-well filterplate was then removed from the centrifuge and incubated at room temperature for two hours to allow hybridization of poly(A)+ RNA tails with the immobilized oligo(dT).
• the multi-well filterplates were then washed three times with 150 ⁇ L Lysis Buffer to remove remaining ribonucleases, followed by three washes with 150 ⁇ L Wash Buffer (BioTek #G4) to remove the Lysis Buffer, which contained some inhibitors of cDNA synthesis.
• the Wash Buffer was completely removed from the multi-well filterplates, and cDNA was synthesized in each well by adding 40 ⁇ L of premixed cDNA buffer.
• the cDNA buffer consists of: 5x First Strand Buffer (Promega M531A, 10 mM dNTP (Promega stock, 20x)), Primer (5 ⁇ M, #24), RNasin (Promega N211A, 40 U/ ⁇ L), M-MLV reverse transcriptase (Promega M170A, 200 U/ ⁇ L), and DEPC water.
• FIG 7 indicates that the optimal concentration of MMLV for mRNA quantification is 0.25 units/well.
• Axymat (Amgen AM-96-PCR-RD) was placed on the multi-well filterplate, which was then placed on a heat block (37 C, VWR) and incubated (>90 min). The multi-well filterplate was then centrifuged (2000 rpm, at 4 C for 1 min). PCR primers were added to a 384-well PCR plate, and the cDNA was transferred from the multi-well filterplate to the 384-well PCR plate.
• FIG 8 indicates that the optimal value of cDNA for PCR is approximately 2 ⁇ L/well. The PCR plate was centrifuged (2000 rpm, at 4 C for 1 min), and real time PCR was commenced (TaqMan/SYBER).
• the method of the current invention has high mRNA specificity; amplification of CD4 mRNA with TaqMan qPCR resulted in undetectable DNA contamination ( ⁇ 10 copies/well).
• the present invention results in low coefficients of variation for mRNA quantification.
• Hybridization for two hours resulted in a coefficient of variation of less than 13%, as compared to traditional coefficients of variation of approximately 300% for mRNA quantification.
• the linear results show that the amount of mRNA that is captured is directly proportional to the volume of whole blood used per well, making the present invention a reliable and reproducible method of quantifying mRNA.

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• 2003
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