Isolation and selection of reference genes for Ganoderma boninense gene expression study using quantitative real-time PCR (qPCR)

Abstract

Quan­ti­ta­tive real-time PCR (qPCR) has become a favourite method for quan­tifi­ca­tion of mRNA tran­scripts. How­ever, sev­eral opti­mi­sa­tion steps must be per­formed to avoid mis­lead­ing qPCR results. One of the steps is selec­tion of ref­er­ence genes for nor­mal­i­sa­tion pur­pose and these genes should be sta­bly expressed across the sam­ples. In this study, iso­la­tion of partial-length cDNA encod­ing seven poten­tial ref­er­ence genes from Gan­o­derma boni­nense has been per­formed. These poten­tial ref­er­ence genes are α-tubulin, β-tubulin, β-actin, elon­ga­tion fac­tor 2 (eef2), glyc­er­alde­hyde 3-phosphate dehy­dro­ge­nase (gapdh), 40S ribo­so­mal (r40s) and ubiq­ui­tin C (ubc). The expres­sion of these ref­er­ence genes was stud­ied in mycelia, white but­ton and fruit­ing body tis­sues of G. boni­nense. The qPCR data were analysed using Best­Keeper and geNorm algo­rithms and both soft­wares have iden­ti­fied β-tubulin, eEF2 and α-tubulin as the most sta­ble ref­er­ence genes and r40s and ubc as the least sta­ble ref­er­ence genes. Three ref­er­ence genes with the low­est M value (eEF2, β-tubulin and α-tubulin) were rec­om­mended by the geNorm soft­ware to be used in the qPCR analy­sis for more accu­rate normalisation.