Identification and evaluation of reference genes for qRT-PCR studies in Lentinula edodes

Abstract

Lentinula edodes (shiitake mushroom) is a common edible mushroom with a number of potential therapeutic and nutritional applications. It contains various medically important molecules, such as polysaccharides, terpenoids, sterols, and lipids, were contained in this mushroom. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to analyze the mechanisms underlying the biosynthetic pathways of these substances. qRT-PCR is used for accurate analyses of transcript levels owing to its rapidity, sensitivity, and reliability. However, its accuracy and reliability for the quantification of transcripts rely on the expression stability of the reference genes used for data normalization. To ensure the reliability of gene expression analyses using qRT-PCR in L. edodes molecular biology research, it is necessary to systematically evaluate reference genes. In the current study, ten potential reference genes were selected from L. edodes genomic data and their expression levels were measured by qRT-PCR using various samples. The expression stability of each candidate gene was analyzed by three commonly used software packages: geNorm, NormFinder, and BestKeeper. Base on the results, Rpl4 was the most stable reference gene across all experimental conditions, and Atu was the most stable gene among strains. 18S was found to be the best reference gene for different development stages, and Rpl4 was the most stably expressed gene under various nutrient conditions. The present work will contribute to qRT-PCR studies in L. edodes.

Fig 1. The amplicon length and specificity of candidate reference genes.

A: Amplified fragments of candidate reference genes shown by agarose gel electrophoresis with ethidium bromide staining. B: Melting curves generated by qRT-PCR. For each sub-graph, temperature is displayed in the x axis, the derivative reporter signal is displayed in the y axis.

Fig 2. Distribution overview of expression profiles of different reference genes.

Boxplot representation of raw Ct values obtained from amplification curves. The box indicates the 25^th and 75^th percentiles. Whiskers represent the maximum and minimum values, the thin line within the box marks the median and mean (rhombus).

Fig 3

Ranking of the candidate reference genes for all samples according to geNorm (A), BestKeeper (B), and NormFinder.

Fig 4. Pairwise variation calculated by geNorm between Vn and Vn + 1to determine the minimum number of reference genes required for accurate normalization in four different samples.

The cut off value is 0.150, below which the inclusion of an additional reference gene is not required.

Fig 5

Relative expression levels of UGPase (A), PGI (B) and PGM(C) among various developmental stages of L. edodes. M: mycelium; P: primordial; F: fruiting bodies. The most stable genes recommended for Development sample (18S and Btu) and the least stable gene F-actin were used for normalization. For 18S and Btu geometric mean was calculated and used for normalization of expression. Error bars show standard deviation calculated from three biological replicates. The relative expression levels of the three genes were indicated as percentage to the expression of candidate reference genes. Different letters above histograms indicate statistical difference highlighted using ANOVA (p-value < 0.05).