An Effective Method for Quantifying RNA Expression of IbsC‐SibC, a Type I Toxin‐Antitoxin System in
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Toxin and antitoxin (TA) systems are small genetic modules consisting of a toxin protein and an RNA or protein antitoxin. It is difficult to study their functions in a large part due to the lack of effective methods to study toxin RNAs, which usually exist at exceptionally low levels. Herein, we describe a sensitive reverse transcription quantitative PCR (RT-qPCR) method that is able to quantitate such RNA species. The method was directed at detection of the toxin mRNA of the ibsC-sibC TA pair, and its high specificity was validated by sequencing. The approach was used to determine relative expression of the IbsC and SibC RNAs at different cell-growth phases; this revealed an expression pattern that cannot be explained by the prevailing notion of growth stasis by the toxin and rescue by the antitoxin. The usefulness of the method was further showcased by the determination of average cellular copy numbers of the IbsC-SibC RNAs in wild-type E. coli cells and RNA abundance in E. coli cells engineered with extra copies of the ibsC-sibC genes. With a robust method to quantitate cellular small RNAs at very low concentrations, we are now equipped to study the expression of TA systems under different conditions to gain useful insights about their functions.
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