Selection of reference genes from Shiraia bambusicola for RT-qPCR analysis under different culturing conditions

Abstract

Stable reference genes are necessary to analyse quantitative real-time reverse transcription PCR (qRT-PCR) data and determine the reliability of the final results. For further studies of the valuable fungus Shiraia bambusicola, the identification of suitable reference genes has become increasingly urgent. In this study, three conventional reference genes and nine novel candidates were evaluated under different light conditions (all-dark, all-light and 12-h light/dark) and in different media (rice medium, PD medium, and Czapek–Dox medium). Three popular software programs (geNorm, NormFinder and BestKeeper) were used to analyse these genes, and the final ranking was determined using RefFinder. SbLAlv9, SbJsn1, SbSAS1 and SbVAC55 displayed the best stability among the genes, while SbFYVE and SbPKI showed the worst. These emerging genes exhibited significantly better properties than the three existing genes under almost all conditions. Furthermore, the most reliable reference genes were identified separately under different nutrient and light conditions, which would help accessible to make the most of the existing data. In summary, a group of novel housekeeping genes from S. bambusicola with more stable properties than before was explored, and these results could also provide a practical approach for other filamentous fungi.

Selection and Evaluation of Appropriate Reference Genes for RT-qPCR Normalization of Volvariella volvacea Gene Expression under Different Conditions

“…The expression levels of the selected genes were also investigated and the mean Cps are listed in Figure 1 . The average expression levels ranged from 9.37 to 29.56, consistent with previous studies [ 29 , 38 ]. Because moderate expression levels (e.g., Cp of 15 to 30) yield accurate normalization [ 39 ], the genes selected in this study were found to be sufficient for experimental needs.…”

“…Utilizing suitable reference genes is necessary to ensure the reliability and accuracy of the resulting data, as the use of unstable reference genes could yield inaccurate results. Therefore, numerous studies have been conducted to investigate reference gene stability under different conditions [ 29 , 30 , 37 , 38 ]. In this present study, the stability of expression of 12 candidate V. volvacea reference genes was systematically analyzed using geNorm, NormFinder, and BestKeeper in the presence of salt (NaCl), oxidative (H 2 O 2 ), metal (CuSO 4 ), acid (pH 4), alkali (pH 9), cold (4°C), and heat stress (42°C), and during different developmental stages.…”

Identification of reference genes for qRT-PCR in granulosa cells of healthy women and polycystic ovarian syndrome patients

“…To the best of our knowledge, there is no gold standard for the optimal number of the candidate reference genes required for the expression stability study. In general, six to twelve candidate genes are commonly included for the analysis 59 – 76 . The supply of cDNA samples and the lab resources dedicated for the characterization should also be considered to make the final decision on the number of candidate genes used for the analysis.…”

Selection of reference genes for quantitative real‐time PCR normalisation in largemouth bass Micropterus salmoides fed on alternative diets

“…The comprehensive ranking was given by RefFinder software and eef1a1 and tuba1 genes were recommended as the two most stable genes for different tissues. Recently, the website tool refFinder software has been used widely to assess suitable reference genes for various species (Auler et al, ; Kałużna et al, ; Sarker et al, ; Zhang et al, ) and human cell lines (Jacob et al, ; Kaszubowska et al, ) in many reports. By the analysis of four major algorithms, the results indicated that although an extent deviation existed in the tissue examples, eef1a1 and tuba1 could be considered the reliable reference genes for target gene expression in diet experiment.…”