# US 7,745,180 B2 · Device and method for high-throughput quantification of mRNA from whole blood

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- Number: US 7,745,180 B2
- Document ID: US7745180B2
- Status: Granted
- Kind: B2
- Application number: 10/796,298
- Filed: 2004-03-09
- Published: 2004-12-30
- Granted: 2010-06-29
- Earliest claimed: 2002-04-24
- Assignees: Hitachi Chemical Company, Ltd.; Hitachi Chemical Research Center, Inc.
- Inventors: Masato Mitsuhashi
- Web page: https://patentgrep.com/patent/US7745180B2

## CPC

- C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers}
- C12N 1/066: by physical processes
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6846: {Common amplification features}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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.

## Claims

### Claim 1

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).

### Claim 2 (depends on 1)

The method of claim 1, wherein the erythrocytes and blood components are removed by filtration to yield the leukocytes on a filter membrane.

### Claim 3 (depends on 2)

The method of claim 2, wherein the filter membrane is attached to a multi-well filter plate.

### Claim 4 (depends on 2)

The method of claim 2, wherein the filter membrane is a polybutylene terephthalate (PBT) fibrous membrane.

### Claim 5 (depends on 2)

The method of claim 2, additionally comprising washing the leukocytes on the filter membrane with hypotonic buffer to further remove erythrocytes and other blood components.

### Claim 6 (depends on 5)

The method of claim 5, additionally comprising drying the filter membrane.

### Claim 7 (depends on 6)

The method of claim 6, wherein the filter membrane is washed with ethanol and subjected to vacuum aspiration until the filter membrane is dry.

### Claim 8 (depends on 2)

The method of claim 2, wherein the lysate is produced by lysing said leukocytes on said filter membrane and transferring the lysate to the oligo(dT)-immobilized plate by centrifugation.

### Claim 9 (depends on 2)

The method of claim 2, wherein the lysate is produced by lysing said leukocytes on said filter membrane and transferring the lysate to the oligo(dT)-immobilized plate by vacuum aspiration.

### Claim 10 (depends on 2)

The method of claim 2, wherein the lysate is produced by lysing said leukocytes on said filter membrane and transferring the lysate to the oligo(dT)-immobilized plate by applying positive pressure.

### Claim 11 (depends on 1)

The method of claim 1, wherein the anticoagulant is heparin.

### Claim 12 (depends on 1)

The method of claim 1, wherein the whole blood is frozen and subsequently thawed prior to step (c).

### Claim 13 (depends on 1)

The method of claim 1, wherein the erythrocytes and blood components are removed by filtration to yield the leukocytes on a plurality of filter membranes layered together.

### Claim 14 (depends on 1)

The method of claim 1, wherein the immobilized plate comprises a multi-well oligo(dT)-immobilized plate.

### Claim 15 (depends on 14)

The method of claim 14, additionally comprising application of specific antisense primers to each well of the multi-well oligo(dT)-immobilized plate during said lysate transferring step.

### Claim 16 (depends on 1)

The method of claim 1, wherein the quantification of the target mRNA comprises synthesis of a cDNA from the target mRNA and amplification of the cDNA.

### Claim 17 (depends on 1)

The method of claim 1, wherein the target mRNA is β-actin mRNA.

### Claim 18 (depends on 1)

The method of claim 1, wherein the target mRNA is CD4 mRNA.

### Claim 19 (depends on 1)

The method of claim 1, wherein the target mRNA is the mRNA of a translocation gene involved in leukemia.

### Claim 20 (depends on 1)

The method of claim 1, wherein the target mRNA is an mRNA of cancer-specific genes from micrometastatic cancer.

### Claim 21 (depends on 1)

The method of claim 1, wherein the whole blood comprises white blood cells infected by a virus and the target mRNA is a virus-derived mRNA from the infected white blood cells.

### Claim 22 (depends on 21)

The method of claim 21, wherein the virus is HIV and the virus-derived mRNA is HIV mRNA.

### Claim 23 (depends on 22)

The method of claim 22, wherein said quantifying the target mRNA is the quantification of said HIV mRNA and is used to diagnose HIV.

### Claim 24 (depends on 21)

The method of claim 21, wherein the virus is CMV and the virus-derived mRNA is CMV mRNA.

### Claim 25 (depends on 24)

The method of claim 24, wherein said quantifying the target mRNA is the quantification of said virus-derived mRNA and is used to diagnose CMV.

### Claim 26 (depends on 21)

The method of claim 21, wherein said quantifying the target mRNA is the quantification of said virus-derived mRNA and is used to monitor blood banks for the presence of a viral disease.

### Claim 27 (depends on 21)

The method of claim 21, wherein said quantifying the target mRNA is the quantification of said virus-derived mRNA and is used to study anti-viral drug sensitivity for said virus.

### Claim 28 (depends on 1)

The method of claim 1, wherein the target mRNA is an mRNA of apoptosis genes involved in leukemia.

### Claim 29 (depends on 1)

The method of claim 1, wherein the target mRNA is an mRNA of cytokines.

### Claim 30 (depends on 1)

The method of claim 1, wherein the target mRNA is an mRNA responsible for apoptosis development.

### Claim 31 (depends on 1)

The method of claim 1, wherein the target mRNA is an mRNA of DNA-repair genes.

### Claim 32 (depends on 31)

The method of claim 31, wherein said quantifying the target mRNA is the quantification of said mRNA of DNA-repair genes and is used to test the sensitivity of one of DNA-repair genes that encodes said mRNA to radiation.

### Claim 33 (depends on 1)

The method of claim 1, wherein the target mRNA is an mRNA of allergen response genes.

### Claim 34 (depends on 33)

The method of claim 33, wherein said quantifying the target mRNA is the quantification of said mRNA of allergen response genes and is used to test allergen stimulation.

### Claim 35

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.

### Claim 36 (depends on 35)

The method of claim 35, wherein the blood sample further comprises different target mRNAs and the lysis buffer further comprises a plurality of different antisense primers specific for said different target mRNAs in the blood sample.

### Claim 37 (depends on 36)

The method of claim 36, wherein the antisense primer-derived cDNA is removed from the plate and the plate with the immobilized oligo(dT)-derived cDNA is stored for future use.