Comparison of 11 endogenous control genes for normalization of mRNA obtained from paraffin-embedded tissues.
Real-time reverse transcriptase PCR (RT-PCR) based assays are being increasingly used in characterization of gene expression. Good quality mRNA is an essential prerequisite for such assays. While fresh tissues provide quality mRNA, the same may not be true of tissues which are formalin-fixed and paraffin-embedded (FFPE). This emphasizes the need to identify a good endogenous control gene to normalize for differences in quality and RNA recovery. We attempted to characterize gene expression patterns of 11 commonly used endogenous control genes among 20 FFPE tissues (both neoplastic and normal). Pearson's coefficient of correlation was determined by comparing the expression of each gene against the mean expression of all other genes. beta2 microglobulin (beta2M) and beta-actin (betaA) (r = 0.95 and 0.94, respectively) were found to be stably expressed across all tissues. However, betaA had greater accuracy (2 x SD) than beta2M and therefore may be a better choice of an endogenous control for experiments that require normalization while using FFPE tissues.
Pai
Rekha
R
Department of Pathology, Christian Medical College, Vellore 632004, Tamil Nadu, India.
Samuel
Prasanna
P
Nehru
Arun G
AG
Manipadam
Marie Therese
MT
Thomas
Susanna N
SN
D000199:Actins / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D052138:Genes, Neoplasm; D006801:Humans; D009369:Neoplasms / Q000235:genetics*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000592:standards; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012015:Reference Standards; D001613:beta 2-Microglobulin / Q000235:genetics*
D000199:Actins; D012333:RNA, Messenger; D001613:beta 2-Microglobulin