Quantitation of ERCC-2 gene expression in human tumor cell lines by reverse transcription-polymerase chain reaction in comparison to northern blot analysis

Anal Biochem. 1997 Jan 1;244(1):50-4. doi: 10.1006/abio.1996.9825.


Excision repair cross-complementing rodent repair deficiency genes (ERCC) are human genes implicated in nucleotide excision repair. ERCC-2 has been implicated in the repair of DNA damaged by chemotherapeutic agents, and may thus play an important role in anticancer drug resistance. ERCC-2 gene expression is low in primary tumor samples rendering it difficult to quantitate. We have developed a semiquantitative method to measure ERCC-2 gene expression utilizing reverse transcription-polymerase chain reaction (RT-PCR). Total RNA extracted from established human tumor cell lines was reverse-transcribed to obtain cDNA. Serially diluted reference ERCC-2 DNA fragment was amplified by PCR to obtain a 617-bp fragment. A standard curve was then created using densitometry readings of the 617-bp bands on agarose gel. A fixed amount of sample cDNA from each cell line was amplified at the same time and the resultant PCR product was read by densitometer. Using the standard curve, ERCC-2 gene expression in a given amount of total RNA was quantitated and normalized to beta-actin expression. There was minimal variation in three repeated experiments with PCR amplification. ERCC-2 gene expression determined by this semiquantitative PCR was also correlated to ERCC-2 quantitation by Northern blot analysis, with a significant concordance (r = 0.912, P = 0.0002). We also successfully applied this sensitive method to quantify five clinical glioma samples.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / analysis
  • Blotting, Northern
  • DNA Helicases*
  • DNA Repair
  • DNA-Binding Proteins*
  • Gene Expression
  • Glioma / genetics
  • Humans
  • Polymerase Chain Reaction
  • Proteins / analysis*
  • Proteins / genetics
  • Reproducibility of Results
  • Transcription Factors*
  • Tumor Cells, Cultured / metabolism
  • Xeroderma Pigmentosum Group D Protein


  • Actins
  • DNA-Binding Proteins
  • Proteins
  • Transcription Factors
  • DNA Helicases
  • Xeroderma Pigmentosum Group D Protein
  • ERCC2 protein, human