RT-qPCR Validation of RNA-seq Transcripts

The RNA material remaining after RNAseq analysis was subjected to reverse transcription using Transcriptor First Strand cDNA Synthesis Kit (Roche Life Sciences, Basel, Switzerland) and the Eppendorf Mastercycler ^® nexus (Eppendorf AG, Hamburg, Germany), according to the manufacturer protocols. The RT-qPCR validation was then performed on a Lightcycler 96 (Roche Life Sciences, Basel, Switzerland) using Eva Green (Syngen Biotech, Wrocław, Poland) as a detection dye. The final reaction mix consisted of 1 μL of cDNA, 1 μL of forward+reverse primer mix, 2 μL of Eva Green and 6 μL of PCR-grade water. The specific primers were designed based on Ensembl transcript sequences [87 (link)], using the Primer3 software (Table 3) [88 (link)]. The design process was based on the exon–exon method to avoid potential remnant genomic DNA amplification. Furthermore, the primers were designed to match all of the known protein-coding transcript variants. The results of the analysis were calculated based on the 2-ΔΔCT method, with ACTB and HPRT used as housekeeping genes [89 (link)].