Quantitative RT-PCR for Gene Expression Analysis

Approximately 10 ng of RNA was converted to cDNA in a 30 μL reaction using qScript XLT cDNA Supermix Kit (Quantabio, Beverly, MA, USA) in a thermocycler. Single-stranded cDNA of 1 ng/μL was used for qRT-PCR. The SYBR Premix Ex (TaKaRa, Shiga, Japan, SYBR Premix Ex Taq) was used to prepare reactions in 20 μL volumes, and dispensed into LightCycler 480 Multiwell Plate 96-well plates. Each sample was run in triplicate as technical replicates for every experiment. The reference genes chosen for this study were RPL32 and SRP14. These reference genes were selected based on previous data from our lab group studying EBF3 expression in melanoma cells [10 (link)]. Real-time PCR reactions were run on the LightCycler 480 (Roche, Vienna, Austria). After acquiring the C_t values for each replicate, the values were normalised to the reference genes, RPL32 and SRP14, using the 2^−ΔΔCt method [35 (link)]. Subsequently, the results were analysed and presented graphically with GraphPad Prism 9 (v.9.0.1). This data was analysed using an unpaired t-test to determine the mean difference in relation to the standard error between the two groups.