Model system for optimising mRNA non-isotopic in situ hybridisation: riboprobe detection of lysozyme mRNA in archival gut biopsy specimens.
The aim of this study was to optimise conditions for mRNA detection by nonisotopic in situ hybridisation (NISH) using biotinylated and digoxigenin labelled riboprobes. Because lysozyme gene transcripts are present at high concentrations in Paneth and other alimentary cells, archival gut biopsy specimens were chosen as a model system for these experiments. Most of the variables in NISH, from unmasking of mRNA, to its ultimate detection by peroxidase or alkaline phosphatase based detection systems, were examined in detail. The most important findings were that simultaneous heating of tissue targets and riboprobes at 95 degrees C for 15 minutes before hybridisation at 50 degrees C for two hours gave the most intense signal for lysozyme mRNA in Paneth cells, Brunner's glands, and lamina propria macrophages; digoxigenin labelled riboprobes gave a higher signal to noise ratio than their biotinylated counterparts, and probes 600 base pairs long were superior to shorter probes. It is concluded that the mRNA NISH method may be generally useful for detecting gene transcription in archival clinical biopsy specimens.
Martinez-Montero
J C
JC
University of Oxford, Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital, Headington.
Herrington
C S
CS
Stickland
J
J
Sawyer
H
H
Evans
M
M
Flannery
D M
DM
McGee
J O
JO
D001109:Archives; D001706:Biopsy; D006801:Humans; D006868:Hydrolysis; D007422:Intestines / Q000737:chemistry*; D009113:Muramidase / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis*; D012260:Ribonucleases / Q000037:antagonists & inhibitors; D013048:Specimen Handling / Q000379:methods; D013696:Temperature
D015347:RNA Probes; D012333:RNA, Messenger; D012260:Ribonucleases; D009113:Muramidase