title: Direct in situ reverse transcriptase-polymerase chain reaction.
abstract: In situ hybridization has been used for localization of specific nucleic acid sequences at the cellular level despite providing relatively low-detection sensitivity. In situ reverse transcriptase-polymerase chain reactions (RT-PCR) enhance sensitivity and thus enable localization of low-abundance mRNA in a cell. However, the available methods are fraught with problems of nonspecific amplifications as a result of mispriming and/or amplification from partially digested residual genomic DNA in tissue. Herein, we demonstrate that nonspecific background amplification can be eliminated by pretreatment of samples with restriction enzymes before DNase I digestion. Primers tagged with a far-red shifted fluorescent dye such as Cy5 in PCR reactions allow identification of target mRNA by fluorescence microscopy. These novel modifications lead to increased specificity and rapid in situ detection of cellular mRNA and thus may be used for pathological diagnosis.
authors.affiliation: Division of Nephrology and Hypertension, Richard Roudebusch Veterans Affairs Medical Center, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
authors.forename: R
authors.initials: R
authors.lastname: Kher
authors.forename: R
authors.initials: R
authors.lastname: Bacallao
mesh_terms: D000818:Animals; D002232:Carbocyanines; D003593:Cytoplasm / Q000378:metabolism; D004262:DNA Restriction Enzymes; D003850:Deoxyribonuclease I; D004622:Embryo, Mammalian / Q000378:metabolism; D005456:Fluorescent Dyes; D007668:Kidney / Q000196:embryology / Q000378:metabolism*; D007686:Kidney Tubules, Distal / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000378:metabolism; D008722:Methods; D051379:Mice; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; C085321:cyanine dye 5; D020794:Receptor Protein-Tyrosine Kinases; C084944:TYRO3 protein, human; D004262:DNA Restriction Enzymes; D003850:Deoxyribonuclease I