Quantifying HERVK3-1 Expression in Glioma Cells
Corresponding Organization : National Institutes of Health
Other organizations : University of Miami, Center for Cancer Research, National Cancer Institute
Variable analysis
- RNA isolation method (Trizol extraction with chloroform)
- Cell lines (NHA, A172, GBM28, GBM43)
- Quantification of HML-6 transcripts using qPCR
- RNA purity (260/280 value ~2.0)
- RNA purification (DNase treatment)
- Reverse transcription (SuperScript First-Strand Synthesis RT-PCR kit)
- PCR 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)
- PCR primers (targeting the env region of the HERVK3-1 downstream of LTR13)
- PCR master mix (Fast SybR green master mix)
- Normalization (delta-delta Ct method using NHA as reference)
- Reverse transcriptase positive control
- Reverse transcriptase negative control (did not amplify)
Annotations
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