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Comparison of 11 endogenous control genes for normalization of mRNA obtained from paraffin-embedded tissues

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Pai R, Samuel P, Nehru AG, Manipadam MT, Thomas SN. Comparison of 11 endogenous control genes for normalization of mRNA obtained from paraffin-embedded tissues. APMIS 2009; 117: 886–92.

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. β2 microglobulin (β2M) and β-actin (βA) (r = 0.95 and 0.94, respectively) were found to be stably expressed across all tissues. However, βA had greater accuracy (2 × SD) than β2M and therefore may be a better choice of an endogenous control for experiments that require normalization while using FFPE tissues.

Keywords: Normalization; endogenous control; mRNA; paraffin-embedded

Document Type: Research Article


Affiliations: 1: Departments of Pathology 2: Biostatistics, Christian Medical College, Vellore, Tamil Nadu, India

Publication date: 2009-12-01

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