Modified PAXgene method allows for isolation of high-integrity total RNA from microlitre volumes of mouse whole blood.
Analysis of gene expression is often used to evaluate the effects of experimental manipulations in laboratory animals. Blood is a rich source of potential biomarkers, including gene expression information, which may be obtained from whole blood. When compared with the end of a study, when whole blood samples can be easily obtained for gene expression measurements, the limiting volumes of whole blood obtainable from animals during the course of an experiment requires a method for RNA isolation from a minimal volume of whole blood. The PAXgene Blood RNA Extraction System originally designed for isolation of total RNA from 2.5 mL of human whole blood, was modified and successfully used to isolate high-integrity total RNA from as little as 50 microL of mouse whole blood. Fifty microlitres of mouse whole blood yielded an average of 2.3 microg highly intact total RNA, of sufficient quality and quantity allowing for multiple gene expression determinations. The utility of this method was demonstrated by confirming the time- and dose-dependent upregulation of haem oxygenase-1 (Hmox1) mRNA in response to a single injection of cobalt protoporphyrin. The successful isolation of total RNA from small volumes of mouse whole blood can allow for serial sampling on the same animals, thereby reducing the number of animals required for experimentation.
Krawiec
J A
JA
Cardiovascular Center for Excellence in Drug Discovery, Department of Vascular Inflammatory Diseases, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, USA. John.A.Krawiec@gsk.com
Chen
H
H
Alom-Ruiz
S
S
Jaye
M
M
D000818:Animals; D001800:Blood Specimen Collection / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D051547:Heme Oxygenase-1 / Q000097:blood / Q000235:genetics; D007756:Laboratory Animal Science / Q000379:methods; D008565:Membrane Proteins / Q000097:blood / Q000235:genetics; D051379:Mice; D011524:Protoporphyrins / Q000494:pharmacology; D012313:RNA / Q000097:blood*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation / Q000187:drug effects
D008565:Membrane Proteins; D011524:Protoporphyrins; D012333:RNA, Messenger; D012313:RNA; C007095:cobaltiprotoporphyrin; D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse