Validation of extraction methods for total RNA and miRNA from bovine blood prior to quantitative gene expression analyses.
The benefit and precision of blood diagnosis by quantitative real-time PCR (qPCR) is limited by sampling procedures and RNA extraction methods. We have compared five different RNA extraction protocols from bovine blood regarding RNA and miRNA yield, quality, and most reproducible data in the qRT-PCR with the lowest point of quantification. Convincing results in terms of highest quantity, quality, and best performance for mRNA qPCR were obtained by leukocyte extraction following blood lysis as well as extraction of PAXgene stabilized blood. The best microRNA qPCR results were obtained for samples extracted by the leukocyte extraction method.
Physiology Weihenstephan, Technische Universitaet Muenchen, Weihenstephaner Berg 3, 85354, Freising, Germany.
Andrea
A
Hammerle-Fickinger
Irmgard
I
Riedmaier
Christiane
C
Becker
Heinrich H D
HH
Meyer
Michael W
MW
Pfaffl
Susanne E
SE
Ulbrich
D000818:Animals; D002417:Cattle; D007962:Leukocytes / Q000378:metabolism; D035683:MicroRNAs / Q000097:blood* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D025341:Principal Component Analysis; D012313:RNA / Q000097:blood* / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000097:blood / Q000302:isolation & purification
D035683:MicroRNAs; D012333:RNA, Messenger; D012313:RNA