In situ localization of PCR-amplified human and viral cDNAs.
We describe a technique, called reverse transcriptase (RT) in situ PCR, whereby RNA may be nonisotopically detected in fixed cells when amplified by PCR after cDNA synthesis by RT. RT in situ PCR using primers specific for the measles virus generated an intense signal in most measles-infected HeLa cells, as compared to the weak signal generated in few cells using standard in situ hybridization analysis. The viral RNA that localized to the nucleus spared the nucleoli, was most evident when the RT step used the primer complementary to the negative genomic strand, and was demonstrated in all multinucleated cells and the majority of uninucleate cells. A hybridization signal was evident with standard RNA in situ hybridization using the human megakaryocyte cell line Dami and a probe for glycoprotein IIB (GIIB) mRNA but not a probe for amyloid precursor protein (APP) or gelsolin (GEL) mRNA. After RT in situ PCR, signals were evident for each target localizing to the nucleolus for APP and to perinucleolar and cytoplasmic locations for GEL and GIIB. The latter findings suggest that mRNAs may follow different geographic pathways as they progress from premessage to transcriptionally active message.
D001483:Base Sequence; D003588:Cytopathogenic Effect, Viral; D004247:DNA / Q000235:genetics*; D006367:HeLa Cells / Q000737:chemistry* / Q000382:microbiology; D006801:Humans; D017403:In Situ Hybridization; D008459:Measles virus / Q000302:isolation & purification*; D008533:Megakaryocytes / Q000737:chemistry* / Q000382:microbiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity
D012367:RNA, Viral; D004247:DNA; D012194:RNA-Directed DNA Polymerase