(2004) have shown that some reference genes are highly specific for a particular model and that experimental validation for each situation is a crucial requirement. Although the products of these genes are constitutively expressed, the variation in the levels of their messenger RNA in certain experimental conditions shows genetic instability, which makes these genes inappropriate in certain situations. The best known housekeeping genes in literature are glyceraldehyde-3-phosphate desidrogenase (GAPDH), β-actin, ribosomal proteins (RPL), ubiquitin (UBQ), β-tubulin, 18S ribosomal protein (18S rRNA) and phosphoglycerate kinase (PGK). Reference or housekeeping genes are expressed constitutively by different cell types (Nelson and Cox 2004) and are used to normalize the data (Karge et al. This technique has been successfully used to evaluate the levels of mRNA in a given cell type.
A variant of this method is real-time quantitative PCR (qRT-PCR), which allows quantification of a specific region of DNA. The polymerase chain reaction (PCR) is a sensitive technique by which a single DNA molecule can serve as a template for amplification (Azevedo et al. Key words: PCR, normalization, reference genes, gene expression, mRNA
Several genes, such as GAPDH, β-actin, β-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.
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. 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. 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. IIDepartment of Pathobiology Faculty of Veterinary Medicine Utrecht University Utrecht - The Netherlands Real time PCR and importance of housekeepings genes for normalization and quantification of mRNA expression in different tissuesĮmanuela de Lima Rebouças I José Jackson do Nascimento Costa I Maria Juliane Passos I José Renato de Sousa Passos I Robert van den Hurk II José Roberto Viana SilvaI, * *Īuthor for de Biotecnologia de Sobral Universidade Federal do Ceara 62042-280 Sobral - CE - Brasil