Selection and Validation of Reference Genes for qRT-PCR in Lentinula edodes under Different Experimental Conditions

Yi Luo,Yan Zhou,Yuhua Gong,Chen Wang,Gangzheng Wang,Yinbing Bian

doi:10.3390/genes10090647

Yi Luo, Yan Zhou + Show 4 more

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https://doi.org/10.3390/genes10090647

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Abstract

Lentinula edodes is the most consumed mushroom in Asia due to its nutritional and medicinal values, and the optimal reference gene is crucial for normalization of its gene expression analysis. Here, the expression stability of 18 candidate reference genes (CRGs) in L. edodes was analyzed by three statistical algorithms (geNorm, NormFinder and BestKeeper) under different stresses (heat, cadmium excess and Trichoderma atroviride infection), different substrates (straw, sawdust and corn stalk) and different development stages (mycelia, primordia and fruit bodies). Among the 18 CRGs, 28S, Actin and α-tub exhibited the highest expression stability in L. edodes under all conditions, while GPD, SPRYP and MSF showed the least stable expression. The best reference gene in different conditions was different. The pairwise variation values showed that two genes would be sufficient for accurate normalization under different conditions of L. edodes. This study will contribute to more accurate estimation of the gene relative expression levels under different conditions using the optimal reference gene in qRT-PCR (quantitative reverse transcription polymerase chain reaction) analysis.

Highlights

With the advantages of high sensitivity and repeatability as well as dynamic quantification range, quantitative reverse transcription polymerase chain reaction is widely used to analyze and validate the expression of target genes [1,2,3]
For abiotic and biotic stress treatment group, mycelia of L. edodes strain W1 cultured on sawdust medium were treated under 40 ◦ C heat stress for 24 h, and mycelia growing on the PDA medium were subjected to Trichoderma atroviride infection for 24h
BLAST analysis revealed that the primers of every candidate reference gene were only matched to the target gene sequence in the L. edodes genome database

Summary

Introduction

With the advantages of high sensitivity and repeatability as well as dynamic quantification range, quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used to analyze and validate the expression of target genes [1,2,3]. The results of qRT-PCR were usually not accurate because of errors caused by many factors such as the efficiency of complementary DNA (cDNA) synthesis and amplification, gene expression normalization, and so on [4,5]. In order to minimize those errors, one or more genes which are usually stably expressed in any experimental conditions are applied in qRT-PCR analysis [5,6]. The application of unstable internal reference genes will result in significant variations or differences in the qRT-PCR results [10]. It is necessary to identify and analyze the stability of the reference genes under different conditions to guarantee the accuracy and reliability of qRT-PCR results

Objectives

Methods

Results