In situ cDNA polymerase chain reaction. A novel technique for detecting mRNA expression.
We report a novel method for detecting intracellular messenger RNA by combining the techniques of in situ hybridization and polymerase chain reaction (PCR) (in situ cDNA PCR). The technique could detect low abundancy signals and distinguish different levels of gene expression. We examined the expression of the functional markers of activated cytotoxic T lymphocytes, granzyme A, and perforin in human lymphocytes from in vitro cultures. The amplification products were found in the cells and the supernatants, with the distribution critically affected by the protease digestion conditions. The specificity of amplification was confirmed by electrophoretic analysis and Southern blotting. We conclude that the in situ cDNA PCR technique offers a sensitive method of measuring the frequency of signal-expressing cells and has significant research and clinical applications.
Chen
R H
RH
Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, United Kingdom.
Fuggle
S V
SV
D001706:Biopsy; D015139:Blotting, Southern; D002478:Cells, Cultured; D018076:DNA, Complementary; D010450:Endopeptidases / Q000494:pharmacology; D005784:Gene Amplification; D053804:Granzymes; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D007668:Kidney / Q000737:chemistry / Q000473:pathology; D016030:Kidney Transplantation; D008562:Membrane Glycoproteins / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D054353:Perforin; D016133:Polymerase Chain Reaction / Q000379:methods*; D052899:Pore Forming Cytotoxic Proteins; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012697:Serine Endopeptidases / Q000032:analysis / Q000235:genetics; D013601:T-Lymphocytes / Q000737:chemistry / Q000166:cytology; D013602:T-Lymphocytes, Cytotoxic / Q000737:chemistry / Q000201:enzymology
D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D012333:RNA, Messenger; D054353:Perforin; D010450:Endopeptidases; D053804:Granzymes; D012697:Serine Endopeptidases; C509212:GZMA protein, human