Detection of mRNA by non-radioactive direct primed in situ reverse transcription.
There are various techniques to detect mRNA in tissue specimens. Among these in situ hybridization is widely applied, and for the detection of small quantities of RNA in situ reverse transcriptase polymerase chain reaction (in situ RT-PCR) has been applied. Furthermore in situ transcription, where signal is produced by direct incorporation of labeled nucleotides during production of a cDNA by reverse transcription, has been shown by a few investigators. We present a non-radioactive in situ reverse transcriptase (in situ RT) protocol which is at least as sensitive as in situ hybridization but avoids probe production and long procedures of preincubation, incubation, and washing. Digoxigenin-labeled UTP is incorporated into a cDNA produced by in situ reverse transcription of mRNA. This method is combined with the fast and sensitive immunogold-silver detection system allowing demonstration of the mRNA within 7 h compared to days in the case of in situ hybridization. Contrary to in situ RT-PCR this new method of in situ RT has no background problems due to non-specific amplification or diffusion of the reaction product.
Kriegsmann
J
J
Institute of Pathology, Johannes Gutenberg University, Langenbeckstrasse 1, 55101 Mainz, Germany. f0x113@web.de
Müller-Ladner
U
U
Sprott
H
H
Bräuer
R
R
Petrow
P K
PK
Otto
M
M
Hansen
T
T
Gay
R E
RE
Gay
S
S
D000818:Animals; D001172:Arthritis, Rheumatoid / Q000378:metabolism; D053804:Granzymes; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000235:genetics; D013583:Synovial Membrane / Q000378:metabolism / Q000473:pathology; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics
D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D019010:Vascular Cell Adhesion Molecule-1; D053804:Granzymes; D012697:Serine Endopeptidases; C509212:GZMA protein, human