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.

Validation of candidate reference genes for qRT-PCR studies in symbiotic and non-symbiotic Casuarina glauca Sieb. ex Spreng. under salinity conditions

“…The most stable genes were Ap47, PP2A and S23 in branchlets; S23, GAPDH and PP2A in roots; and S23, Ap47 and PP2A in nodules (Online Resource 6). Altogether, these results are in agreement with recent studies, where it was reported that the expression of reference genes may vary with the experimental conditions as well as with cell/tissue type (Kozera and Rapacz 2013;Mehta et al 2010;Ling and Salvaterra 2011;Najafpanah et al 2013). On the other hand, the results support the findings of Klie and Debener (2011), that a new generation of reference genes will replace classical reference genes such as Ubi and Tub.…”

Selection of Reference Genes for Gene Expression Studies Related to Intramuscular Fat Deposition in Capra hircus Skeletal Muscle

“…Given the differences observed in our comparison, we conclude that normalization against a single “commonly used reference gene” may lead to serious discrepancies in data interpretation. GAPDH was found to be unsuitable for normalization in skeletal muscle development in pig [ 48 ]; similar results were reported in goat [ 16 ] and cattle [ 51 ]. In addition, GAPDH was observed to be up-regulated and down-regulated under various conditions [ 7 , 9 , 52 , 53 ].…”

“…More recently, Najafpanah MJ et.al. [ 16 ] carried out an analysis of nine candidate reference genes in goat; HSP-90 was almost always the most stable reference gene in longissimus muscle, liver and visceral and subcutaneous fat.…”

Validation of reference genes for cryopreservation studies with the gorgonian coral endosymbiont Symbiodinium

“…Previous studies indicate that pcna, gapdh, 18S rRNA, hsp90 , and rbcl have been selected as potential reference genes for determining gene expression in different tissues from various species, including plants1531, goat32, and fish33. In the present study, the three algorithms provided slightly different stability rankings, which was not unexpected because these algorithms use different approaches.…”