Total RNA (200 ng) was reverse-transcribed using the SensiFAST cDNA synthesis kit (Bioline) as per the manufacturer's instructions. Complementary DNA (cDNA) samples then were diluted 1:12 with nuclease-free water. Oligonucleotide primers (
Table) were designed using Primer-BLAST to span the exon-exon junction.
All RT-qPCR reactions were performed using the SensiFAST SYBR No-ROX kit (Bioline). Reactions (10 μl) were set up in a LightCycler 480, 384-well plate (Roche Diagnostics Ltd., Forrenstrasse, Switzerland) using a Freedom EV075 robotic station with Freedom EVOware Standard 3.2 software (Tecan, Port Melbourne, VIC, Australia) consisting of 4 μl cDNA, 5 μl SYBR, and 300 nM forward and reverse primers. Reverse transcriptase qPCR analysis was done using the Roche LightCycler 480 (Roche Diagnostics Ltd.) under the following thermal cycling conditions: 95°C for 2 minutes followed by 45 cycles consisting of denaturation (95°C, 5 seconds), annealing (60°C, 10 seconds), and extension (72°C, 15 seconds). At the end of each run, melting curve profiles (95°C for 5 minutes, 60°C for 1 minute, and then slowly heating at 0.11°C/s up to 98°C with continuous measurement of fluorescence per 5°C) to confirm amplification of specific transcripts. Standard curves were generated by serially diluting (1:2), covering an appropriate concentration range. Relative gene expression was determined as a ratio of the gene of interest and GAPDH using the second derivative maximum method of the LightCycler software (Roche Diagnostics Ltd.). All reactions, including no-template controls and minus RT controls, were run in duplicate.