Short communication: Genome-wide identification of new reference genes for reverse-transcription quantitative PCR in Streptococcus thermophilus based on RNA-sequencing analysis

Abstract

Streptococcus thermophilus, one of the most important industrial lactic acid bacteria, is widely used for the production of fermented dairy products such as yogurt and cheese. The accuracy of gene expression-based analyses (e.g., reverse-transcription quantitative real-time PCR) relies heavily on the selection of reliable reference genes (RG), which provides the basis for correctly interpreting expression data. However, many traditional RG are not stably expressed in different systems. Here we used RNA-sequencing to systematically investigate gene expression variation at the genome scale and identify more stable RG in S. thermophilus. In total, 21 putative candidate RG were identified with variation coefficient values <10.0 based on the expression of all 1,911 genes under 4 different experimental conditions. We selected and validated 12 RG chosen from transcriptomes by using reverse-transcription quantitative real-time PCR, and ranked their expression stability by statistical algorithms geNorm and NormFinder. Compared with traditional RG 16S rRNA, genes encoding glycine-tRNA ligase subunit β GlyS and fatty acid-binding protein DegV were more stable under all 4 treatments, which have never been used as RG in S. thermophilus. Our finding provides the foundation for more precise analysis of gene expression in S. thermophilus and other lactic acid bacteria species.