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US 7,745,180 B2Granted
Device and method for high-throughput quantification of mRNA from whole blood
AssigneesHitachi Chemical Company, Ltd., Hitachi Chemical Research Center, Inc.
InventorMasato Mitsuhashi
Life of this patent
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Abstract
Disclosed are a method, device kit, and automated system for simple, reproducible, and high-throughput quantification of mRNA from whole blood. More particularly, the method, device, kit and automated system involve combinations of leukocyte filters attached to oligo(dT)-immobilized multi-well plates.
Claims37
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).
6 more elements
A method of high throughput quantification of a target mRNA in a blood sample, comprising the steps of:
- (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 by filtration and yielding leukocytes on a filter membrane;
- (d) lysing the leukocytes on said filter membrane with a lysis buffer comprising antisense primers specific for said target mRNA and producing a lysate comprising total mRNA comprising said target mRNA and said antisense primers wherein said target mRNA hybridizes with said antisense primers and forms a complex comprising said target mRNA and said antisense primers;
- (e) transferring the lysate to an oligo(dT)-immobilized plate to capture the complex comprising said target mRNA and said antisense primers on said oligo(dT)-immobilized plate wherein the oligo (dT) is immobilized on the wells of said oligo(dT)-immobilized plate;
- (f) removing non-hybridized materials from said oligo(dT)-immobilized plate;
- (g) adding a reverse transcriptase to the wells of said oligo(dT)-immobilized plate without addition of further antisense primers, thereby synthesizing cDNAs in a solution of the wells by extension of both the immobilized oligo(dT) and the antisense primers and forming an oligo(dT)-derived cDNA and an antisense primer-derived cDNA, wherein the oligo(dT)-derived cDNA remains immobilized to said plate, and the antisense primer-derived cDNA goes into the solution as a result of displacement by the oligo(dT)-derived cDNA without heat denaturation of said target mRNA and said antisense primer-derived cDNA;
- and
- (h) quantifying the target mRNA in the blood sample by amplifying the antisense primer-derived cDNA from the solution and quantifying the amplified product produced from the antisense primer-derived cDNA.
6 more elements
Dependencies are inferred from the claim text.