Transcription of VEGFA RNA Fragments

The templates for transcription reactions for VEGFA RNA fragments derived from pre-mRNAs were obtained in PCR using specific oligonucleotide sets (_TF/TR) having a promoter for polymerase T7. The PCR products were then purified prior to transcription reaction according to the manufacturer's protocol (A&A Biotechnology). The transcription reaction and 5′-end radiolabeling were performed as previously described with slight changes (Sznajder et al. 2016 (link)). Briefly, transcription reaction was performed in 40 µL composed of 10 µL of DNA template, 1 mM NTPs (Invitrogen), 3 mM guanosine (Sigma-Aldrich), 20 U T7 RNA Polymerase (Promega), 1× T7 transcription buffer (Promega), 40 U Rnasin Plus RNase Inhibitor (Promega). Purification of transcript was conducted on a denaturing 6% polyacrylamide gel (19:1 acrylamid:bisacrylamid) and through ethanol precipitation. For radio-labeling, 2 pmol of transcript was incubated with 2 pmol of [γ-³²P] ATP, 1 U Rnasin Plus, 10 U OptiKinase (Affymetrix), 1× reaction buffer (Affymetrix) and ddH₂0 up to 10 µL, at 37°C for 30 min. Labeled RNA was subsequently run on a denaturing 8% polyacrylamide gel (19:1) in 0.5× TBE, at 100 V for 1 h. A band of RNA was visualized on IP through FLA-5100 (FujiFilm), cut out followed by ethanol precipitation and resuspended in 20 µL ddH₂O. (CUG)₂₀ was a kind gift from Włodzimierz J. Krzyżosiak.