Retrotranscription was carried out with 400 ng of RNA and the RevertAid First Strand cDNA Synthesis Kit (Life Technologies), using the specific minigene primer RTPSPL3-RV (5′-TGAGGAGTGAATTGGTCGAA-3′). Samples were incubated at 42°C for 1 h, followed by 5 min at 70°C. Transcripts were amplified with Platinum Taq DNA polymerase (Life Technologies) using 40 ng of cDNA and the primers pMAD_607FW (Patent P201231427, CSIC) and RTBR2_ex17RV2 (5′-GGCTTAGGCATCTATTAGCA-3′). PCR consisted of: denaturation step at 94°C for 2 min, followed by 35 cycles 94°C-30 s, 60°C-30 s and 72°C-1 min/kb, and a final extension step at 72°C for 5 min. Transcripts were sequenced at the Macrogen Spain facility.
In order to relatively quantify all transcripts, semi-quantitative fluorescent RT-PCRs were undertaken in triplicate with the primers pMAD_607FW (FAM-labeled) and RTBR2_ex17RV2 and Platinum Taq DNA polymerase (Life Technologies) under standard conditions except that 26 cycles were herein applied (Acedo et al., 2015 (link)). FAM-labeled products were run with LIZ-1200 Size Standard at the Macrogen facility and analyzed with the Peak Scanner software V1.0 (Life Technologies). Only peak heights ≥ 50 RFU (Relative Fluorescence Units) were considered.
In order to relatively quantify all transcripts, semi-quantitative fluorescent RT-PCRs were undertaken in triplicate with the primers pMAD_607FW (FAM-labeled) and RTBR2_ex17RV2 and Platinum Taq DNA polymerase (Life Technologies) under standard conditions except that 26 cycles were herein applied (Acedo et al., 2015 (link)). FAM-labeled products were run with LIZ-1200 Size Standard at the Macrogen facility and analyzed with the Peak Scanner software V1.0 (Life Technologies). Only peak heights ≥ 50 RFU (Relative Fluorescence Units) were considered.
Free full text: Click here
