Reverse Transcription and Quantitative PCR

SuperScript III (Thermo Fisher Scientific) was used to reverse transcribe 2 μg of RNA into cDNA using the random primers supplied with the SuperScript III kit. Fresh cDNA was diluted 1:10 with ddH₂O and was used immediately. Primers were designed by using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/) and were selected to be located within the first half of the target gene, and to produce a product between 50 and 150 nucleotides in length. Real-time qPCR was performed with Power SYBR Green (Thermo Fisher Scientific). The Applied Biosystems ViiA 7 instrument was used with the following parameters for qPCR: (i) holding stage, 50°C for 2 min and 95°C for 10 min; (ii) PCR stage, 95°C for 15 sec and 60°C for 1 min, repeated 40 times with fluorescence recorded at 60°C; and (iii) melting curve stage, 90°C for 15 sec, 60°C for 1 min, then 95°C for 15 sec with the fluorescence recorded every 0.05 sec. Each reaction produced only one melting curve, indicating only one target was amplified during the qPCR. The threshold cycle (ΔΔC_T) method was used to determine the relative amount of RNA present in the sample tested. The atpI gene was used as an internal reference to normalize the CsrA-RNA coimmunoprecipitation because we have previously shown that CsrA does not bind to the mRNA transcript of this gene (17 (link)).