In situ localization of PCR-amplified DNA and cDNA.
Combining the high sensitivity of PCR with the cell localizing ability of in situ hybridization allows for the reproducible detection of low copy targets in intact cells. This article describes several key variables that include fixation, protease digestion, the hot start maneuver, stringency, and, for RNA analysis, DNase digestion that are important to successful in situ PCR. Also stressed is the importance of performing and interpreting controls with each experiment. Important controls include omission of key components, use of samples known either to contain or lack the target of interest and, most importantly, the in-built controls invariably present in the heterogeneous component of any given tissue type.
MGN Medical Consultants, Inc., Setauket, NY 11733, USA. gnuovomd@pol.net
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D001710:Biotin; D018076:DNA, Complementary / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis*; D004076:Digoxigenin; D019286:Endopeptidase K; D005260:Female; D018628:Gene Dosage; D006367:HeLa Cells / Q000821:virology; D006526:Hepatitis C / Q000235:genetics; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008099:Liver / Q000821:virology; D008457:Measles / Q000235:genetics; D027383:Papillomaviridae / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000821:virology
D018076:DNA, Complementary; D004279:DNA, Viral; D001710:Biotin; D019286:Endopeptidase K; D004076:Digoxigenin