Simultaneous amplification of four DNA repair genes and beta-actin in human lymphocytes by multiplex reverse transcriptase-PCR.
We describe here the development, optimization, and use of a non-radioactive, quantitative, multiplex reverse transcriptase-PCR technique to measure, in a single reaction, the relative levels of the transcripts of four DNA repair genes (XPCC, hMSH2, XRCC1, and ERCC1) and the beta-actin gene in lymphoblastoid cell lines and frozen peripheral blood lymphocytes. Expression of defective DNA repair genes was not detected in DNA repair-deficient human cell lines, whereas the intact genes were detected in repair-proficient cell lines and in lymphocytes from a normal donor. The assay was reproducible, and repeated determinations of the same samples generated highly consistent results for each target gene. This approach should facilitate molecular epidemiological studies that incorporate screening for germline alterations that may affect gene expression and for changes in the levels of gene expression.
Wei
Q
Q
Department of Epidemiology, University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
Xu
X
X
Cheng
L
L
Legerski
R J
RJ
Ali-Osman
F
F
D000199:Actins / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D004260:DNA Repair / Q000235:genetics*; D005784:Gene Amplification; D015870:Gene Expression; D006801:Humans; D008214:Lymphocytes / Q000502:physiology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012194:RNA-Directed DNA Polymerase; D015203:Reproducibility of Results
D000199:Actins; D012194:RNA-Directed DNA Polymerase