Real time PCR and importance of housekeepings genes for normalization and quantification of mRNA expression in different tissues

• Published in: Brazilian archives of biology and technology Vol. 56; no. 1; pp. 143 - 154
• Main Authors: Rebouças, Emanuela de Lima, Costa, José Jackson do Nascimento, Passos, Maria Juliane, Passos, José Renato de Sousa, Hurk, Robert van den, Silva, José Roberto Viana
• Format: Journal Article
• Language: English
• Published: Instituto de Tecnologia do Paraná (Tecpar) 01.02.2013
• Subjects: gene expression; mRNA; normalization; PCR; reference genes
• ISSN: 1516-8913; 1516-8913; 1678-4324
• DOI: 10.1590/S1516-89132013000100019

The aim of this review was to evaluate the importance of the real-time PCR (qRT-PCR) as a technique for mRNA expression analysis in different tissues. Real-time PCR is widely used for quantification of mRNA levels and is a fundamental tool for basic research, molecular medicine and biotechnology. Genes of references are expressed in a wide variety of tissues and cells with minimal variations in their expression levels, and thus are used to normalize data of mRNA quantification. Software programs, such as geNorm, BestKeeper and NormFinder, have been developed to perform the normalization of data, which help to choose the most stable reference gene. Several genes, such as GAPDH, beta -actin, beta -tubulin, PGK, UBQ, RPL-19 and 18S rRNA have been suggested as standards in PCR studies, but these genes can have variation in their expression in different tissues, reinforcing the idea that there is no ideal reference gene.