Revertaid mmlv reverse transcriptase

Real time PCR was performed to quantify the expression of selected DEGs (Table S5) obtained from Illumina sequencing. The cDNAs, used as template, were generated from mRNA using the REVERTAID MMLV reverse transcriptase (Fermentas). Primers (Table S6) were designed using the primer designing tool at IDT (https://eu.idtdna.com/site) to amplify an amplicon of 80–150 nucleotides with Tm around 60 °C. Reactions were run in triplicates (technical and biological) for each sample using Power-Up SYBR Green on an ABI StepOnePlus real time PCR machine (Applied Biosystems Inc, USA) and the data analyzed was the mean of biological triplicates. The reaction was set up in 20 µl as follows: 1 µl of cDNA, 10 µl SYBR Green Dye master mix (2X), 5 pmol each of forward and reverse primers and water up to 20 µl. The general steps performed during real-time PCR experiment were as follows: step 1, 50 °C, 2 min, step 2, 95 °C, 10 min, step 3 (95 °C 15 s, 60 °C 1 min) × 40 cycles. The specificity of the amplicon was analyzed by a melt curve analysis. The relative mRNA level of the gene in different RNA samples was normalized with respect to ACTIN as the internal control gene^{60 (link)} and analyzed by 2^−∆CT method^{133 (link)}.

Total RNA was isolated using the total RNA kit peqGOLD (#12-6834-02, PeqLab, Erlangen, Germany) and subjected to reverse transcription using Oligo dT Primer (# SO-132), Ribolock RNA Inhibitor (#EO-0382), dNTP-Mix (#RO-193) and Revert Aid M-MLV Reverse Transcriptase (#EP-0442) (all from Fermentas via Thermo Fisher Scientific, Darmstadt, Germany) according to manufacturer’s instructions. Quantitative Real-Time PCR analysis was performed as duplicate analysis on a LightCycler 480 (Roche Diagnostics, Mannheim, Germany) including melting curve analysis as quality control. Primers, primer sequences and annealing temperatures are listed in Table 3.