Home › Biochemistry › Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein
Biochemistry JoVE (Open Access) Citable · DOI

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

DOI: 10.3791/57021-v
What you'll learn
  • ✓ Perform immunoprecipitation of endogenous TERT from cell lysates
  • ✓ Execute RNA-dependent RNA polymerase activity assay on TERT protein
  • ✓ Detect and semi-quantify dsRNA products using RNase treatment and analysis
Protocol

Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.

Difficulty
advanced
Total time
~4–6 hours per sample (cell lysis through final detection)
Model organism
HeLa cells
Biosafety
BSL-1

Steps

1
Prepare cell lysate from cultured cells

Harvest and lyse cultured HeLa cells to extract soluble protein containing endogenous TERT. This lysate serves as the substrate for downstream immunoprecipitation.

▶ 00:29
2
Immunoprecipitate endogenous TERT protein

Incubate cell lysate with antibodies and affinity beads to selectively capture TERT from the crude protein mixture, isolating it for downstream enzyme assay.

▶ 01:34
3
Conduct RNA-dependent RNA polymerase reaction

Incubate immunoprecipitated TERT with RNA substrate and nucleotides to allow TERT-mediated synthesis of double-stranded RNA products under controlled conditions.

▶ 03:54
4
Treat reaction product with RNase

Apply RNase digestion to the RdRP reaction products to selectively degrade single-stranded RNA and enrich double-stranded RNA for downstream detection.

▶ 06:19
5
Analyze RdRP products in mitotic cells

Examine and semi-quantify double-stranded RNA products generated by endogenous TERT activity in synchronized mitotic HeLa cells using the completed assay.

▶ 07:38
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