Reference genes for normalization: A study of rat brain tissue

Abstract

Quantitative real-time polymerase chain reaction (qPCR) has become a widely used tool in the search for disease genes. When examining gene expression with qPCR in psychiatric diseases, endogenous reference gene(s) must be used for normalization. Traditionally, genes such as beta-actin (ActB), Gapd, and 18s rRNA, assumed to be stably expressed, have been used for normalization. However, it has become clear that expression of these genes is influenced by different experimental paradigms. Since stable gene expression of houskeeping genes (HKGs), therefore, cannot be expected, alternative strategies are warranted. With the overall aim to inspect the gene expression of three target genes, NMDAR1, SORT, and CREB, in rat hippocampus, we tested a panel of eight HKGs, 18s rRNA, ActB, CycA, Gapd, Hmbs, Hprt1, Rpl13A, and Ywhaz in order to select the most stably expressed gene, using the NormFinder and geNorm software applets. Additionally, we have applied four different normalization approaches for normalization of the three target genes. We found using the NormFinder software that Ywhaz is the most stably expressed gene among the eight tested HKGs. However, the results of the analysis of the target genes are highly dependent on the choice of normalization approach. Moreover, the number of HKGs, used for selection of the most stable HKG, also influences on the result of the gene expression analysis of target genes. These results underline the importance of choosing a proper normalization strategy when analyzing gene expression with qPCR. The strategy should be unbiased and carried out in every specific experimental setting.

Selection and validation of reference house­keeping genes in the J774A1 macrophage cell line for quantitative real-time PCR

“…This indicated that these HKGs are not good macrophage reference genes; these results also indicated that the expression of these genes could be reshaped by various ECM components present in culture, or be sensitive to adhesion-specific signaling. Previous studies have also reported divergent qPCR results for ACTB in rat oligodendrocytes and rat brain tissue (Bonefeld et al, 2008;Nelissen et al, 2010) and B2M in human heart tissues (Molina et al, 2014). These findings emphasize the importance of evaluating all genes used for normalization, even if they are traditionally used in other studies (Nelissen et al, 2010).…”

Genomewide Analysis of Rat Barrel Cortex Reveals Time- and Layer-Specific mRNA Expression Changes Related to Experience-Dependent Plasticity

“…Relative expression of the genes of interest was calculated after obtaining the corresponding Ct values and correcting for unequal sample input using geNorm (Vandesompele et al, 2002), which identifies the two most stably expressed housekeeping genes (Ywhaz and CycA) from a set of three tested candidate genes reported previously to be stably expressed in the brain (Bonefeld et al, 2008) to calculate a normalization factor for each sample. This normalization factor was then used to obtain the relative differences between the samples for each primer pair.…”

Different modes of GH administration influence gene expression in the male rat brain

“…The amount of each transcript was normalized to the amount of Gapdh expressed in the same sample. For stability comparison of candidate reference genes, we applied the NormFinder Software (http://www.mdl.dk; Andersen et al 2004, Bonefeld et al 2008. All samples were analyzed in duplicates.…”