Patent ID: 7745180

Claim:
A method of determining a definite quantity of a target mRNA encoding a specific sequence in a blood sample comprising: (a) collecting whole blood; (b) administering an anticoagulant to the whole blood; (c) removing erythrocytes and blood components other than leukocytes from the whole blood and yielding the leukocytes; (d) lysing the leukocytes with a lysis buffer containing a known amount of synthesized poly-A spiked control RNA and producing a lysate comprising total mRNA containing the target mRNA and said synthesized poly-A spiked control RNA, wherein said synthesized poly-A spiked control RNA is non-homologous to the target mRNA from the whole blood and the amount of said synthesized poly-A spiked control RNA in the lysate does not significantly interfere with quantification of the target mRNA; (e) transferring the lysate to an oligo(dT)-immobilized plate and capturing the total mRNA and said synthesized poly-A spiked control RNA on said oligo(dT)-immobilized plate; (f) quantifying the target mRNA and the synthesized poly-A spiked control RNA captured on said oligo(dT)-immobilized plate, thereby obtaining the amount of the target mRNA and the amount of the synthesized poly-A spiked control RNA on said oligo(dT)-immobilized plate respectively; (g) determining the percent recovery of said synthesized poly-A spiked control RNA by dividing the amount of said synthesized poly-A spiked control RNA determined in step (f) by the known amount of said synthesized poly-A spiked control RNA in step (d); and (h) determining the definite quantity of said target mRNA in the blood sample by dividing the amount of said target mRNA determined in step (f) by the percent recovery of said synthesized poly-A spiked control RNA determined in step (g).