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

Abstract

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, 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.

Real Time PCR: the Use of Reference Genes and Essential Rules Required to Obtain Normalisation Data Reliable to Quantitative Gene Expression

“…The increase in fluorescent signal is directly proportional to the number of PCR product molecules generated in the reaction. (Reid et al, 2006), showing none or only minimal changes in the expression levels between individual samples and experimental conditions (Rebouças et al, 2013). These genes are largely used as internal controls for normalisation in gene expression studies in different tissues and/or condition as in plants and animals (Wong et al, 2005;Kumar et al, 2013;Sara et al, 2013;Rocha et al, 2013;Nakayama et al, 2004).…”

In vitro culture of secondary follicles and prematuration of cumulus–oocyte complexes from antral follicles increase the levels of maturation‐related transcripts in bovine oocytes

“…The primers designed to perform amplification of mRNA for CCNB1 , H1FOO , GDF9, cMOS , PARN , and eIF4E are shown in Table . This table also shows glyceraldehyde3‐phosphate dehydrogenase ( GAPDH ), which was used as endogenous controls for normalization of mRNA expression, according to previous studies (Bezerra et al, ; Rebouças et al, ; Rossi et al, ; Rossi et al, ; Silva et al, ). The specificity of each primer pair was confirmed by melting curve analysis of PCR products.…”

Isolation of Partial Housekeeping Genes on Tuntun Angin (Elaeocarpus floribundu)

“…In plants, the genes encoding actin and tubulin proteins have many variants and are called gene family. They participate in movement of chromosomes during mitosis and meiosis, movement of organells, and intracellular transport (McDowell et al, 1996;Dominguez & Holmes, 2011;Kandasamy et al, 2012;Rebouças et al, 2013). Arabidopsis thaliana has ten actin genes (McDowell et al, 1996) while Pinus taeda (Schwarzerova et al, 2010) and Physcomitrella patens usually have seven to eleven actin variants (Zhang et al, 2010).…”

“…Previously, a housekeeping gene was isolated from E. floribundus such as partial actin gene (Roslim & Herman, 2017). However, for the purposes of gene expression studies, more than one reference gene are needed as internal control (Rebouças et al, 2013;Wang et al, 2017;Bao et al, 2016). Moreover, the most widely used housekeeping genes as internal control are 18S rRNA, 26S rRNA, EF1α, GAPDH, actin, and beta tubulin (βTUB) in plants like tea (Camellia sinensis), jute (Corchorus capsularis), and olive (Olea europaea) (Kozera & Rapacz, 2013;Ray & Johnson, 2014;Niu et al, 2015;Wang et al, 2017).…”