Quantitative Real-Time PCR Protocol for Gene Expression Analysis

qRT-PCR was performed on the BioRad CFX96 Real-Time PCR System, using the Power SYBR Green PCR Master Mix (Applied Biosystems), according to manufacturer’s instructions. Primers (S3 Table) were obtained from PrimerDesign Ltd, Integrated DNA Technologies (IDT), or Sigma-Aldrich. Relative quantities of target mRNA were determined using the relative standard curve method [27 (link)]. Standard curves were prepared for each primer pair, from serial dilutions of pooled sample cDNA. Universal cycling conditions were used (95°C for 10min, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1min). PCRs were performed in technical triplicate, on at least two biological replicates, and results were analysed using the BioRad CFX Manager software (v3.1). Gene of interest expression was normalised to β Actin (ACTB) expression in each case. The design and reporting of qRT-PCR experiments aimed to comply with the Minimum Information for publication of Quantitative real-time PCR Experiments guidelines [28 (link), 29 (link)] wherever possible. Statistical analysis was performed using the unpaired Students’ t-test. Significance was defined as p < 0.05.