Quantitative RT-PCR Protocol for Gene Expression

RT-qPCR was conducted as described (23 (link)). One microgram of total RNA from all experimental conditions were treated with DNase I (Thermo Fisher Scientific) to digest DNA, according to the manufacturer's protocol. Samples were then reverse transcribed into cDNA with SuperScript™ IV One-Step RT-PCR System (Invitrogen) and random primer hexamers (Thermo Fischer Scientific). After reverse transcription, cDNA samples were treated with RNase H (NEB) to hydrolyze leftover RNA. qPCR was done using GoTaq^® qPCR Master Mix (Promega), 50-fold diluted cDNA, and a final concentration of 250 nM to 1 M of oligonucleotides. All primer pairs used for qPCR analysis were optimized using a standard curve. qPCR reactions were prepared by the CAS-1200 Corbett robot (Corbett Robotics) and analyzed using the Rotor Gene 6000, with suggested standard cycling conditions. recA was used as an internal control for the normalization of gene expression. All three biological replicates used for this analysis were run in duplicates. The 2^−ΔΔCT method was used to calculate the fold-change relative to the control (45 ). The mean log₂ fold-change and standard error of the mean were computed.