Device and method for high-throughput quantification of mRNA from whole blood

1. A method of obtaining mRNA from a leukocyte lysate comprising:

obtaining whole blood;

preparing a plurality of sample wells, each of the sample wells comprising a filter that traps leukocytes in the filter and allows erythrocytes and blood components other than leukocytes in the whole blood to be removed from the filter;

adding an anticoagulant to the whole blood;

adding the whole blood to the sample wells;

removing erythrocytes and blood components other than leukocytes in the whole blood from the filter;

lysing the leukocytes in the samples wells by applying lysis buffer comprising a guanidinium thiocyanate at a concentration in the range of about 1.4M to 1.75M into the sample wells and obtaining a leukocyte lysate comprising mRNA in each of the sample wells;

transferring said leukocyte lysate from each of the sample wells into its corresponding mRNA capture zone in which oligo(dT) is immobilized; and

obtaining said mRNA from said leukocyte lysate by annealing poly-A mRNA in the leukocyte lysate to the oligo(dT).

2. The method of claim 1 , wherein the leukocytes are located on the filter at the time they are lysed.

3. The method of claim 2 , wherein the lysis buffer comprises a detergent.

4. The method of claim 3 , wherein the detergent is Triton X-100, Tween-20, SDS (sodium dodecyl sulfate), sarcosyl, or deoxycholic acid.

5. The method of claim 2 , wherein the lysis buffer further comprises an additional ribonuclease inhibitor.

6. The method of claim 1 , further comprising assaying for the presence or quantity of one or more RNAs in the lysate.

7. The method of claim 6 , wherein said assaying step comprises a Northern blot, RNase protection assay, hybridization reaction, microarray analysis, or reverse transcriptase-polymerase chain reaction analysis.

8. The method of claim 7 , wherein said reverse transcriptase-polymerase chain reaction analysis is real-time RT-PCR or endpoint RT-PCR.

9. The method of claim 1 , wherein the oligo(dT) is immobilized on a plate comprising the plurality of sample wells and the transferring step is performed by vacuum aspiration.

10. The method of claim 1 , wherein the oligo(dT) is immobilized on a plate comprising the plurality of sample wells and the transferring step is performed by centrifugation.

11. A method of obtaining mRNA from a leukocyte lysate comprising:

obtaining whole blood;

preparing a plurality of sample wells, each of the sample wells comprising a filter that traps leukocytes in the filter and allows erythrocytes and blood components other than leukocytes in the whole blood to be removed from the filter;

adding an anticoagulant to the whole blood;

adding the whole blood to the sample wells;

removing erythrocytes and blood components other than leukocytes in the whole blood from the filter; and

lysing the leukocytes in the samples wells by applying lysis buffer comprising guanidine thiocyanate at a concentration in the range of about 1.4M to 1.75M into the sample wells and obtaining a leukocyte lysate comprising mRNA;

extracting the mRNA from the leukocyte lysate and obtaining extracted mRNA;

transferring said extracted mRNA into an mRNA capture zone in which oligo(dT) is immobilized; and

obtaining said mRNA from said leukocyte lysate by annealing said mRNA to the oligo(dT).

12. The method of claim 11 , wherein the extraction is performed via an organic extraction.

13. The method of claim 12 , wherein the organic extraction is a phenol/chloroform extraction.

14. The method of claim 9 , wherein said extracting the mRNA from the leukocyte lysate comprises extracting the lysate with an organic solution, forming organic and aqueous phases; separating the organic and aqueous phases; and isolating the mRNA from the aqueous phase.