Department of Agricultural Sciences, University of Bologna, viale Fanin 42, 40127 Bologna, Italy.
Department of Agricultural Sciences, University of Bologna, viale Fanin 42, 40127 Bologna, Italy.
Anaerobe. 2014 Jun;27:34-9. doi: 10.1016/j.anaerobe.2014.03.004. Epub 2014 Mar 19.
Normalization is an essential prerequisite for producing accurate real-time PCR expression analyses. The objective of this study is the selection of a set of optimal reference genes in Bifidobacterium adolescentis gene expression studies under bile exposure. B adolescentis is a particularly abundant species in the human adults gut microbiota, exerting relevant probiotic activities. In the gastrointestinal tract, bile represents a hard challenge for bacterial survival, because of its toxic effect. The natural exposure to bile in the colonic environment induces cells adaptation and tolerance mechanisms in bifidobacteria, which determines changes in gene expression profile, influencing the expression levels of housekeeping genes. In this context, the stability of 9 putative reference genes (cysS, purB, recA, rpoB-L, GADPH-R, 16S rRNA, glnA1, gyrA2, sdhA) was examined in B. adolescentis exposed to bile extract, using two different software (BestKeeper and NormFinder). Both algorithms identified gyrA2 and sdhA as the most stable genes under our experimental conditions, while 16S rRNA is the least reliable HKGs. To our best knowledge, this is the first attempt to validate reference genes in Bifidobacterium spp. and the results offer an appropriate set of reference genes suitable for qRT-PCR studies on B. adolescentis strains under bile stress.
归一化是进行准确实时 PCR 表达分析的基本前提。本研究的目的是在胆汁暴露下选择一组双歧杆菌最佳的内参基因。双歧杆菌是成人肠道微生物群中特别丰富的物种，具有相关的益生菌活性。在胃肠道中，胆汁对细菌的生存具有毒性作用，是一个严峻的挑战。双歧杆菌在结肠环境中自然暴露于胆汁会诱导细胞适应和耐受机制，这决定了基因表达谱的变化，影响管家基因的表达水平。在这种情况下，使用两种不同的软件（BestKeeper 和 NormFinder）检测了双歧杆菌暴露于胆汁提取物时 9 个候选内参基因（cysS、purB、recA、rpoB-L、GADPH-R、16S rRNA、glnA1、gyrA2 和 sdhA）的稳定性。这两种算法都将 gyrA2 和 sdhA 鉴定为我们实验条件下最稳定的基因，而 16S rRNA 是最不可靠的 HKGs。据我们所知，这是首次在双歧杆菌属中验证内参基因，结果提供了一组适合胆汁应激下双歧杆菌菌株 qRT-PCR 研究的合适内参基因。
