Selection and validation of endogenous reference genes for RT-qPCR normalization in different stresses and tissues of the tiger milk mushroom, Pleurotus tuber-regium.

Abstract

Pleurotus tuber-regium is a sclerotium-producing mushroom with pharmacological and nutritional value. Many researches applied molecular biological strategies that allow analyzing numerous genes expressed in response to different growth stresses or tissues. In order to capture accurate and reliable results in gene expression studies, it is necessary to select appropriate internal reference genes. They must have relatively uniform expression in the majority of tested samples, thereby reducing the impact of the sample itself on the RT-qPCR results. The selection and validation of 14 candidate reference genes, including α-tub, β-tub, γ-tub, GAPDH, Tif-5a, Tef-1α, ATPase, acyl-CoA, U2ribo, S/G, Wlp, VtpAsE, E3upl, and Sdh, were carried out for gene expression analysis in P. tuber-regium mycelia in response to different temperatures, drought levels, and salinity shifts, fruitbody, and sclerotium. Four statistical algorithms, NormFinder, geNorm, BestKeeper, and RefFinder, were recruited to evaluate the transcription stability of candidate reference genes. The RT-qPCR expression stability analysis indicated that the E3upl and Tif-5a were the most stable expressed genes among all experimental samples, so the combination of these two reference genes is suitable for the analysis of gene expression patterns in P. tuber-regium.