title: Per-unit-living tissue normalization of real-time RT-PCR data in ischemic rat hearts.
abstract: In studies of gene expression in acute ischemic heart tissue, internal reference genes need to show stable expression per-unit-living tissue to hinder dead cells from biasing real-time RT-PCR data. Until now, this important issue has not been appropriately investigated. We hypothesized that the expression of seven internal reference genes would show stable per-unit-living tissue expression in Langendorff-perfused rat hearts subjected to ischemia-reperfusion. This was found for cyclophilin A, GAPDH, RPL-32, and PolR2A mRNA, with GAPDH showing the highest degree of stability (R = 0.11), suggesting unchanged rates of mRNA transcription in live cells and complete degradation of mRNA from dead cells. The infarct size-dependent degradation of GAPDH was further supported by a close correlation between changes in GAPDH mRNA and changes in RNA quality measured as RNA integrity number (R = 0.90, P < 0.05). In contrast, β-actin and 18S rRNA showed stable expression per-unit-weight tissue and a positive correlation with infarct size (R = 0.61 and R = 0.77, P < 0.05 for both analyses). The amount of total RNA extracted per-unit-weight tissue did not differ between groups despite wide variation in infarct size (7.1-50.1%). When β-actin expression was assessed using four different normalization strategies, GAPDH and geNorm provided appropriate per-unit-living expression, while 18S and total RNA resulted in marked underestimations. In studies of ischemic tissues, we recommend using geometric averaging of carefully selected reference genes for normalization of real-time RT-PCR data. A marked shift in the mRNA/rRNA ratio renders rRNA as useless for normalization purposes.
authors.affiliation: Lillehammer University College, Lillehammer, Norway.
authors.forename: S
authors.initials: S
authors.lastname: Ellefsen
authors.forename: M
authors.initials: M
authors.lastname: Bliksøen
authors.forename: A
authors.initials: A
authors.lastname: Rutkovskiy
authors.forename: I B
authors.initials: IB
authors.lastname: Johansen
authors.forename: M-L
authors.initials: ML
authors.lastname: Kaljusto
authors.forename: G E
authors.initials: GE
authors.lastname: Nilsson
authors.forename: J I
authors.initials: JI
authors.lastname: Vaage
authors.forename: K-O
authors.initials: KO
authors.lastname: Stensløkken
mesh_terms: D000818:Animals; D001483:Base Sequence; D021984:Cyclophilin A / Q000235:genetics; D017931:DNA Primers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D008297:Male; D017202:Myocardial Ischemia / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D021984:Cyclophilin A