AUTHOR=Jureckova Katerina , Raschmanova Hana , Kolek Jan , Vasylkivska Maryna , Branska Barbora , Patakova Petra , Provaznik Ivo , Sedlar Karel 

TITLE=Identification and Validation of Reference Genes in Clostridium beijerinckii NRRL B-598 for RT-qPCR Using RNA-Seq Data

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.640054

DOI=10.3389/fmicb.2021.640054

ISSN=1664-302X

ABSTRACT=<p>Gene expression analysis through reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) depends on correct data normalization by reference genes with stable expression. Although <italic>Clostridium beijerinckii</italic> NRRL B-598 is a promising Gram-positive bacterium for the industrial production of biobutanol, validated reference genes have not yet been reported. In this study, we selected 160 genes with stable expression based on an RNA sequencing (RNA-Seq) data analysis, and among them, seven genes (<italic>zmp</italic>, <italic>rpoB1</italic>, <italic>rsmB</italic>, <italic>greA</italic>, <italic>rpoB2</italic>, <italic>topB2</italic>, and <italic>rimO</italic>) were selected for experimental validation by RT-qPCR and gene ontology (GO) enrichment analysis. According to statistical analyses, <italic>zmp</italic> and <italic>greA</italic> were the most stable and suitable reference genes for RT-qPCR normalization. Furthermore, our methodology can be useful for selection of the reference genes in other strains of <italic>C. beijerinckii</italic> and it also suggests that the RNA-Seq data can be used for the initial selection of novel reference genes, however, their validation is required.</p>