Polymerase chain reaction-aided analysis of gene expression in frozen tissue sections.
A simple method for the detection and localization of mRNA in single frozen breast biopsy tissue sections is described. Several extraction procedures were compared. Resuspending sections, which could be left at 0 or -70 degrees C for up to 20 min in H2O containing RNAse inhibitor, optimally released RNA with minimal DNA contamination. Reverse transcription followed by polymerase chain reaction amplification using specific primers yielded products visible by ethidium bromide staining (abundant sequences) or after Southern blotting (low copy message). We found that it was possible, by microdissection, to separate stromal and tumor cells and demonstrated differential expression of several genes in the two populations. With 40 cycles of amplification, dissected stromal and tumor tissue both yielded products encoding glyceraldehyde 3'-phosphate dehydrogenase but only the tumor cells gave products with primers specific for either keratin 19, heat shock protein 89 alpha or the fig oncogene, which encodes one of the fibroblast growth factor receptors that we have recently found to be expressed in breast cancers. With refinement of the dissection technique this offers a very sensitive analytical tool for measuring and defining the cellular sites of synthesis of low-abundance message, requiring only single histological sections.
Luqmani
Y A
YA
Department of Medical Oncology, Charing Cross Hospital, London, England.
Smith
J
J
Coombes
R C
RC
D001483:Base Sequence; D001706:Biopsy; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D005260:Female; D005615:Freezing; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics*; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012194:RNA-Directed DNA Polymerase