Validation of candidate reference genes in Bifidobacterium adolescentis for gene expression normalization

Area of Science:

• Microbiology
• Molecular Biology
• Gut Microbiome Research

Background:

• Bifidobacterium adolescentis is a key gut bacterium with probiotic properties.
• Bile exposure in the gastrointestinal tract poses a survival challenge, inducing adaptive gene expression changes.
• Accurate gene expression analysis requires stable reference genes for normalization.

Purpose of the Study:

• To identify optimal reference genes for quantitative real-time PCR (qRT-PCR) studies in B. adolescentis under bile exposure.
• To evaluate the stability of nine candidate reference genes in B. adolescentis exposed to bile extract.

Main Methods:

• Examined the stability of nine putative reference genes (cysS, purB, recA, rpoB-L, GADPH-R, 16S rRNA, glnA1, gyrA2, sdhA).
• Utilized BestKeeper and NormFinder software for stability analysis.
• Tested gene expression in B. adolescentis samples exposed to bile extract.

Main Results:

• Identified gyrA2 and sdhA as the most stable reference genes under bile exposure conditions.
• Determined 16S rRNA to be the least reliable reference gene (HKG).
• Provided validated reference genes for B. adolescentis gene expression studies.

Conclusions:

• gyrA2 and sdhA are recommended as optimal reference genes for qRT-PCR in B. adolescentis under bile stress.
• This study represents the first validation of reference genes in Bifidobacterium spp. under bile exposure.
• The findings support accurate gene expression profiling in B. adolescentis adapting to gastrointestinal conditions.