Quantitation of ERCC-2 gene expression in human tumor cell lines by reverse transcription-polymerase chain reaction in comparison to northern blot analysis.
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.
Chen
Z P
ZP
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montreal, Quebec, Canada.
Malapetsa
A
A
Mohr
G
G
Brien
S
S
Panasci
L C
LC
D000199:Actins / Q000032:analysis; D015152:Blotting, Northern; D004265:DNA Helicases; D004260:DNA Repair; D004268:DNA-Binding Proteins; D015870:Gene Expression; D005910:Glioma / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D014157:Transcription Factors; D014407:Tumor Cells, Cultured / Q000378:metabolism; D051759:Xeroderma Pigmentosum Group D Protein
D000199:Actins; D004268:DNA-Binding Proteins; D011506:Proteins; D014157:Transcription Factors; D004265:DNA Helicases; D051759:Xeroderma Pigmentosum Group D Protein; C497292:ERCC2 protein, human