Latest developments in in situ PCR.
Since the first publication on the method of in situ polymerase chain reaction (PCR), several thousand research papers have appeared in peer-reviewed journals describing various findings based solely on the application of this method or combined with other more robust methods, including solution-based PCR, immunohistochemistry, Southern blot, etc. A few years after the advent of PCR, several investigators developed in situ PCR methods that differed considerably from each other with regard to tissue preparations, fixation, mounting of slides, reverse transcription technique, primer design, target selection, size, and amplicon size, and thermocycler designs and the use, among many other fine details. This chapter describes the detail procedures that are used in the author's laboratory. It also discusses the variations and modification that can be used for the specific needs of an investigator. This protocol should serve as a primer for the investigators, and each researcher must use his or her variation according to their needs.
Bagasra
Omar
O
Department of Biology, Claflin University, Orangeburg, SC, USA.
Harris
Twaina
T
D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000096:biosynthesis; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D019286:Endopeptidase K / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry; D012313:RNA / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000639:trends*; D012260:Ribonucleases / Q000378:metabolism; D013194:Staining and Labeling; D016707:Tissue Fixation
D017931:DNA Primers; D018076:DNA, Complementary; D009711:Nucleotides; C034477:RNA primers; D012313:RNA; D012260:Ribonucleases; D019286:Endopeptidase K; D004047:Diethyl Pyrocarbonate