Genome-Wide Screening and Stability Verification of the Robust Internal Control Genes for RT-qPCR in Filamentous Fungi

Abstract

In real-time quantitative PCR (RT-qPCR), internal control genes (ICGs) are crucial for normalization. This study screened 6 novel ICGs: Pre-mRNA-splicing factor cwc15 (Cwf15); ER associated DnaJ chaperone (DnaJ); E3 ubiquitin-protein ligase NEDD4 (HUL4); ATP-binding cassette, subfamily B (MDR/TAP), member 1 (VAMP); Exosome complex exonuclease DIS3/RRP44 (RNB); V-type H+-transporting ATPase sub-unit A (V-ATP) from the 22-transcriptome data of 8 filamentous fungi. The six novel ICGs are all involved in the basic biological process of cells and share the different transcription levels from high to low. In order to further verify the stability of ICGs candidates, the six novel ICGs as well as three traditional housekeeping genes: β-actin (ACTB); β-tubulin (β-TUB); glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and the previously screened reference genes: SPRY-domain-containing protein (SPRYp); Ras-2 protein (Ras); Vacuolar protein sorting protein 26 (Vps26) were evaluated by geNorm and NormFinder statistical algorithms. RT-qPCR of 12 ICGs were performed at different developmental stages in Flammulina filiformis and under different treatment conditions in Neurospora crassa. The consistent results of the two algorithms suggested that the novel genes, RNB, V-ATP, and VAMP, showed the highest stability in F. filiformis and N. crassa. RNB, V-ATP, and VAMP have high expression stability and universal applicability and therefore have great potential as ICGs for standardized calculation in filamentous fungi. The results also provide a novel guidance for the screening stable reference genes in RT-qPCR and a wide application in gene expression analysis of filamentous fungi.

Evaluation and validation of suitable reference genes for quantitative real-time PCR analysis in lotus (Nelumbo nucifera Gaertn.)

“…As an example, Jain’s research highlighted the high stability of the UBQ and EF-1α genes in Oryza sativa, emphasizing the need for species-specific gene selection 26 . The Coffea arabica GAPDH gene exhibits high stability, contrasting its low stability in peach, as previously reported 27 , 28 . Our study employed a combined approach of software analysis and experimentation to identify the optimal reference genes.…”

Bioprospecting of endophytic fungi from semidesert candelilla (Euphorbia antisyphiliticaZucc): Potential for extracellular enzyme production

“…These genes contain conserved sequences common to all fungi; however, for some genera, this target does not allow the identification of all the species that compose it, making it necessary to search for other targets with greater resolving power. Different gene regions, such as beta-tubulin, have been used that show potential for the identification of fungi such as N. crassa and other genera such as Colletotrichum and Aspergillus with DNA sequence analysis [42][43][44][45][46].…”

Transcriptome analysis reveals the effect of cold storage time on the expression of genes related to oxidative metabolism in Chinese black truffle

“…A total of 20 DEGs were randomly selected for quantitative real-time PCR (qRT-PCR) validation, which was performed on the CFX96 Real-Time System (BIO-RAD) with SYBR green used as the fluorescent dye. Each experiment was repeated with three biological replicates, using 18S rRNA as an internal reference gene ( 34 ). The relative expression of genes was calculated using the 2 –ΔΔCt method ( 35 ).…”