RNA was isolated from NHA, A172, GBM28, and GBM43 using Trizol extraction with chloroform as previously described44 (link). RNA was purified using DNase and quantified using NanoDrop 2000 (ThermoFisher Scientific) for a 260/280 value ~ 2.0, and reverse transcribed using the SuperScript First-Strand Synthesis RT-PCR kit (Thermo Fisher Scientific). Polymerase chain reactions were then used to amplify HML-6 transcripts on the cDNA using primers (10 uM) which target the protein-coding region of HML-6 (ERVK3-1). Fast SybR green master mix was used for the PCR with the following cycling conditions: 95 °C for 20 s, [95 °C for 3 s, 60 °C for 30 s] repeated for 40 cycles followed by 95 °C for 20 s [95 °C for 1 s, 60 °C for 20 s] for 40 cycles. Both primers targeted the env region of the HERVK3-1 downstream of LTR13. Ct values were normalized to expression of NHA using the delta-delta Ct method45 (link). Reverse transcriptase negative controls were included and did not amplify.
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