Guideline to reference gene selection for quantitative real-time PCR.
Today, quantitative real-time PCR is the method of choice for rapid and reliable quantification of mRNA transcription. However, for an exact comparison of mRNA transcription in different samples or tissues it is crucial to choose the appropriate reference gene. Recently glyceraldehyde 3-phosphate dehydrogenase and beta-actin have been used for that purpose. However, it has been reported that these genes as well as alternatives, like rRNA genes, are unsuitable references, because their transcription is significantly regulated in various experimental settings and variable in different tissues. Therefore, quantitative real-time PCR was used to determine the mRNA transcription profiles of 13 putative reference genes, comparing their transcription in 16 different tissues and in CCRF-HSB-2 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate and ionomycin. Our results show that "Classical" reference genes are indeed unsuitable, whereas the RNA polymerase II gene was the gene with the most constant expression in different tissues and following stimulation in CCRF-HSB-2 cells.
Radonić
Aleksandar
A
Charité-Campus Charité Mitte, II. Medizinische Klinik mit Schwerpunkt Onkologie und Hämatologie, Humboldt Universität, Berlin, Germany.
Thulke
Stefanie
S
Mackay
Ian M
IM
Landt
Olfert
O
Siegert
Wolfgang
W
Nitsche
Andreas
A
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic
D017931:DNA Primers; D015342:DNA Probes; D012333:RNA, Messenger