Reference genes selection for quantitative real-time PCR using RankAggreg method in different tissues of Capra hircus

Abstract

Identification of reference genes with stable levels of gene expression is an important prerequisite for obtaining reliable results in analysis of gene expression data using quantitative real time PCR (RT-qPCR). Since the underlying assumption of reference genes is that expressed at the exact same level in all sample types, in this study, we evaluated the expression stability of nine most commonly used endogenous controls (GAPDH, ACTB, 18S rRNA, RPS18, HSP-90, ALAS, HMBS, ACAC, and B2M) in four different tissues of the domestic goat, Capra hircus, including liver, visceral, subcutaneous fat and longissimus muscles, across different experimental treatments (a standard diet prepared using the NRC computer software as control and the same diet plus one mg chromium/day). We used six different software programs for ranking of reference genes and found that individual rankings of the genes differed among them. Additionally, there was a significant difference in ranking patterns of the studied genes among different tissues. A rank aggregation method was applied to combine the ranking lists of the six programs to a consensus ranking. Our results revealed that HSP-90 was nearly always among the two most stable genes in all studied tissues. Therefore, it is recommended for accurate normalization of RT-qPCR data in goats, while GAPDH, ACTB, and RPS18 showed the most varied expressions and should be avoided as reference genes.

Figure 1. The distribution of gene expression levels of nine candidate reference genes analyzed for four different tissues (liver (a), visceral fat (b), subcutaneous muscle (c), and longissimus muscle (d)) in pooled C_T value.

Figure 2. The distribution of gene expression levels of nine candidate reference genes in pooled C_T value.

The variations are related to data taken together from four different tissues (liver, visceral fat, subcutaneous muscle, and longissimus muscle).