Highly fluorochrome labeled gene probes for quantitative tracing of RNA in individual cells by in situ hybridization.
A new method is presented for preparing highly fluorochrome labeled gene probes suitable for in situ hybridization. For this purpose fluorochromes were attached to a synthetic polypeptide, which was then coupled covalently to various gene probes. The advantage of the reported method is its high labeling efficiency and the easy coupling procedure. The method allows rapid and quantitative detection of homologous RNA at the single cell level. Optimal conditions for the hybridization of fluorochrome-labeled gene probes were established microfluorimetrically, and the specificity and sensitivity of the method were tested. Quantitation of the RNA with a fluorochrome-labeled gene probe in situ in individual cells allows determination of the degree of gene activation in individual cells and may thus provide a new tool for investigation of normal and malignant cells with respect to activation of genes controlling differentiation and proliferation.
Pachmann
K
K
Medizinische Klinik Innenstadt, University of München, Germany.
Reinecke
K
K
Emmerich
B
B
Thiel
E
E
D002478:Cells, Cultured; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005452:Fluoresceins; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000502:physiology; D009693:Nucleic Acid Hybridization; D011094:Polyethyleneimine; D012313:RNA / Q000032:analysis*; D015347:RNA Probes / Q000138:chemical synthesis*; D012333:RNA, Messenger / Q000032:analysis; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D015347:RNA Probes; D012333:RNA, Messenger; D013861:Thiocyanates; C069224:fluorescein isothiocyanate-polyethylenimine; D012313:RNA; D011094:Polyethyleneimine; D016650:Fluorescein-5-isothiocyanate