Reference gene selection for real-time quantitative PCR normalization in enterotoxigenic Escherichia coli exposed to baicalin-aluminum complexes

Abstract:

[Background] Diarrhea due to enterotoxigenic Escherichia coli (ETEC) is one of the most frequent diseases in piglets. Baicalin-aluminum has a good therapeutic effect on ETEC-induced intestinal injury in piglets, but the mechanism of action remains to be clarified. [Objective] Screening out the most stable internal reference genes in ETEC under the stress of baicalin-aluminum is the basis for studying the effect of baicalin-aluminum on the expression of ETEC target genes. [Methods] In this study, 12 internal reference genes 16S rRNA, mdh, recA, gapA, gyrA, gyrB, rpoA, rpoB, mdoG, dnaG, secA and fusA were determined at different concentrations (250, 500 and 1 000 μg/mL) baicalin-aluminum using qPCR technology. The expression level after different culture time (4.5 h and 6 h) under baicalin-aluminum stress. Four methods including comparative Ct value method, geNorm, BestKeeper and NormFinder software were used to evaluate the stability of the expression of 12 candidate internal reference genes. [Results] The results show that rpoA expression is moderately abundant and the most stable expression among multiple analysis methods. [Conclusion] It was determined that rpoA is the best internal reference gene for ETEC qPCR. This study laid the foundation for the subsequent use of qPCR to study the expression of target gene function after baicalin-aluminum stress ETEC.