Evaluation of candidate reference genes stability for gene expression analysis by reverse transcription qPCR in Clostridium perfringens

Abstract

Identification of stable reference genes for normalization purposes is necessary for obtaining reliable and accurate results of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses. To our knowledge, no reference gene(s) have been validated for this purpose in Clostridium perfringens. In this study, the expression profile of ten candidate reference genes from three strains of C. perfringens were assessed for stability under various experimental conditions using geNorm in qbase + . These stability rankings were then compared to stability assessments evaluated by BestKeeper, NormFinder, delta Ct, and RefFinder algorithms. When comparing all the analyses; gyrA, ftsZ, and recA were identified within the most stable genes under the different experimental conditions and were further tested as a set of reference genes for normalization of alpha toxin gene expression over a 22-h period. Depending on the condition, rpoA and rho might also be suitable to include as part of the reference set. Although commonly used for the purpose of normalizing RT-qPCR data, the 16S rRNA gene (rrs) was found to be an unsuitable gene to be used as a reference. This work provides a framework for the selection of a suitable stable reference gene set for data normalization of C. perfringens gene expression.

Study of an Enterococcus faecium strain isolated from an artisanal Mexican cheese, whole-genome sequencing, comparative genomics, and bacteriocin expression

“…The housekeeping gene rpoA, which encodes the α-subunit of RNA polymerase, was selected as the normalization gene because its expression is fairly constant. It has proven reliable in RT-qPCR procedures in Clostridium perfringens and Campylobacter jejuni (Williams and Ghanem 2022 ; Ritz et al 2009 ). The expression of ORF 621 was also considered as a baseline.…”

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

“…It is recognized that these genes may exhibit species-specific regulation, with differential expression patterns observed. 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 .…”

The Potential Regulatory Role of miR-378 in the Expression of Bone Morphogenetic Protein-15 in Infertile Women with Hyperprolactinemia (HPL)

“…The expression of the BMP15 gene is determined via total RNA extraction followed by the reverse transcription quantitative polymerase chain reaction (RTqPCR) technique. The relative mRNA levels were determined by reverse transcriptionquantitative PCR (RT-qPCR), as previously described [18]. Total RNA was extracted using the TRIzol reagent (TransZol Up Plus, TransGen, biotech.)…”

“…(Trizo, Trans; India), and the cDNA was synthesized with a real-time (RT) reagent SuperMix Kit and quantified using TransStart® Top Green qPCR Super Mix (TransGen, biotech. AQ131-01) by quantitative PCR (RT-qPCR) with specific primer pairs (Table 1) according to the previously described protocol [18]. The forward and reverse primers (oligonucleotide) of the BMP15 gene were designed.…”