Quantitative detection of reverse transcriptase-PCR products by means of a novel and sensitive DNA stain.
We constructed a plasmid for the in vitro synthesis of a competitor RNA for use as an internal exogenous control during reverse transcriptase--PCR (RT-PCR) detection of epidermal growth factor receptor (EGFR) expression. The competitor RNA harbors a 32-base deletion compared with wild-type EGFR mRNA and generates a PCR product that is easily distinguished from the wild-type PCR product by agarose gel electrophoresis. We encountered the problem of heteroduplex formation during later stages of PCR, which could be solved by decreasing the PCR cycle number. This was accompanied by a significant loss of sensitivity. Sensitivity could be restored by using a novel and extremely sensitive DNA stain (SYBR Green I) instead of ethidium bromide.
Schneeberger
C
C
Department of Obstetrics and Gynecology, University of Vienna, Austria.
Speiser
P
P
Kury
F
F
Zeillinger
R
R
D001483:Base Sequence; D052160:Benzothiazoles; D002460:Cell Line; D004396:Coloring Agents; D004247:DNA / Q000032:analysis* / Q000235:genetics; D017931:DNA Primers; D003959:Diamines; D004587:Electrophoresis, Agar Gel / Q000379:methods; D066246:ErbB Receptors / Q000096:biosynthesis*; D004996:Ethidium; D005456:Fluorescent Dyes; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis*; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D052160:Benzothiazoles; D004396:Coloring Agents; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; D009692:Nucleic Acid Heteroduplexes; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; C098022:SYBR Green I; D004247:DNA; D066246:ErbB Receptors; D012194:RNA-Directed DNA Polymerase; D004996:Ethidium