Quantifying HERVK3-1 Expression in Glioma Cells

RNA was isolated from NHA, A172, GBM28, and GBM43 using Trizol extraction with chloroform as previously described^{44 (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 method^{45 (link)}. Reverse transcriptase negative controls were included and did not amplify.