Device and method for high-throughput quantification of mRNA from whole blood
View Patent ↗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.
1. A method of high throughput quantification of a specific mRNA in whole blood, comprising the following steps in order:
(a) obtaining a blood sample by a method consisting of:
(i) collecting whole blood in a container consisting of a blood collection tube, wherein, prior to the collecting step, the content of the blood collection tube consists of an anticoagulant;
(ii) freezing the whole blood; and
(iii) thawing the whole blood;
(b) lysing leukocytes of the whole blood thereby producing a lysate comprising mRNA;
wherein said lysing step is performed with a lysis buffer which inhibits RNases; and
(c) quantifying the specific mRNA in the lysate.
2. The method of claim 1 , wherein erythrocytes and blood components other than leukocytes are removed from the whole blood before the lysing step.
3. The method of claim 2 , wherein
the erythrocytes and blood components other than the leukocytes are removed by filtration and the leukocytes are captured on a filter membrane; and
the leukocytes are lysed on the filter membrane.
4. The method of claim 1 , wherein, after the lysing step, the lysate is transferred to an oligo(dT)-immobilized plate to capture the mRNA.
5. A method of high throughput quantification of a specific mRNA in whole blood, comprising the following steps in order:
(a) obtaining a blood sample by a method consisting of:
(i) collecting whole blood in a container consisting of a blood collection tube, wherein, prior to the collecting step, the content of the blood collection tube consists of an anticoagulant;
(ii) stimulating the whole blood with a bioactive agent;
(iii) freezing the whole blood; and
(iv) thawing the whole blood;
(b) lysing leukocytes of the whole blood thereby producing a lysate comprising mRNA;
wherein said lysing is performed with a lysis buffer which inhibits RNases; and
(c) quantifying the specific mRNA in the lysate.