Selection and validation of reference genes for RT-qPCR in ophiocordyceps sinensis under different experimental conditions

Abstract

The Chinese caterpillar mushroom, Ophiocordyceps sinensis (O. sinensis), is a rarely medicinal fungus in traditional chinese herbal medicine due to its unique medicinal values, and the expression stability of reference genes is essential to normalize its gene expression analysis. In this study, BestKeeper, NormFinder and geNorm, three authoritative statistical arithmetics, were applied to evaluate the expression stability of sixteen candidate reference genes (CRGs) in O. sinensis under different stress [low temperature (4°C), light treatment (300 lx), NaCl (3.8%)] and different development stages (mycelia, primordia and fruit bodies) and formation of morphologic mycelium (aeriasubstrate, hyphae knot mycelium). The paired variation values indicated that two genes could be enough to accurate standardization exposed to different conditions of O.sinensis. Among these sixteen CRGs, 18S ribosomal RNA (18S rRNA) and beta-Tubulin (β-TUB) showed the topmost expression stability in O.sinensis exposed to all conditions, while glutathione hydrolase proenzym (GGT) and Phosphoglucose isomerase (PGI) showed the least expression stability. The optimal reference gene in different conditions was various. β-TUB and Ubiquitin (UBQ) were identified as the two most stable genes in different primordia developmental stage, while phosphoglucomutase (PGM) with elongation factor 1-alpha (EF1-α) and 18S rRNA with UBQ were the most stably expressed for differentially morphologic mycelium stages and different stresses, respectively. These results will contribute to more accurate evaluation of the gene relative expression levels in O.sinensis under different conditions using the optimal reference gene in real-time quantitative PCR (RT-qPCR) analysis.

Fig 1. Expression stability analysis of sixteen candidate reference genes in O. sinensis.

(A) variation in RT-qPCR values of nineteen candidate reference genes (CRGs) in all samples, (B-E) expression stability ranking of sixteen CRGs using geNorm under different development stages (mycelia, primordia, young and mature fruiting bodies), total samples, different temperature stresses (heat and cold) and all samples, respectively. (F) pairwise variation (Vn/Vn+1) exhibiting the optional number of reference genes required in an accurate normalization. DDS, differentially developmental stages; Total,total samples;DM, Differentially Morphologic Mycelium Stages; DS, Different Stresses.

Fig 2. The relative expression levels of nine genes normalized by the selected reference genes under different testing conditions.

(A) RT-qPCR analysis of three genes normalized using the most stable reference genes (β-TUB or UBQ) or a combination (β-TUB and UBQ) and the least stable genes (GGT) during different development stages. (B) RT-qPCR analysis of three genes normalized using the most stable reference genes (PGM or EF1-α) or a combination (PGM and EF1-α) and the least stable genes (TPI) under differentially morphologic mycelium stages. (C) RT-qPCR analysis of three genes normalized using the most stable reference genes (18S rRNA or UBQ) or a combination (18S rRNA and UBQ) and the least stable genes (H⁺-ATPase) under cold stress (-2°C). Error bars indicate the mean standard error calculated from three biological replicates. The statistical level was according to * P < 0.05, **P < 0.01.