Quantification of transcript-to-template ratios as a measure of gene expression using RT-PCR.
A new protocol was established for the quantitative analysis of gene expression in small muscle biopsies. Reverse transcription-PCR was performed with a preparation of total nucleic acids (DNA+RNA), amplifying the sequences of interest (targets; mitochondrial transcripts: 12S rRNA, cytochrome-c-oxidase [COX I] mRNA) together with an endogenous, non-transcribed reference sequence (template: D-loop region of mtDNA). Synthesis of PCR products at consecutive cycles within the exponential phase was quantified by measuring incorporation of radioactivity. Product accumulation was determined by regression analysis of these data. Gene expression could then be quantified as a ratio of target transcripts to reference DNA. The results revealed a ratio of 12S rRNA:mtDNA and COX I mRNA:mtDNA of 14 and 2, respectively, or a ratio of 12 S rRNA:COX I mRNA of 7 in human left ventricle and are in good agreement with previously published values for rat liver and muscle. In addition to the investigation of mitochondrial gene expression in the steady state and during mitochondrial proliferation, this newly developed method will easily be applicable to expression analysis of any nuclear gene using an intron sequence as endogenous reference.
Harting
I
I
University of Heidelberg, Germany.
Wiesner
R J
RJ
D000818:Animals; D004272:DNA, Mitochondrial / Q000032:analysis*; D003576:Electron Transport Complex IV / Q000235:genetics; D015870:Gene Expression; D006352:Heart Ventricles / Q000648:ultrastructure; D006801:Humans; D008928:Mitochondria / Q000737:chemistry; D009132:Muscles / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D012335:RNA, Ribosomal / Q000032:analysis; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D012044:Regression Analysis; D013698:Templates, Genetic
D004272:DNA, Mitochondrial; D012333:RNA, Messenger; D012335:RNA, Ribosomal; C075526:RNA, ribosomal, 12S; D003576:Electron Transport Complex IV; D012194:RNA-Directed DNA Polymerase