BACKGROUND: Optimization of in situ hybridization protocols is of real interest when trying to detect small amounts of mRNA or when using low concentrations of probes. To enhance the hybridization signal, we have developed a modification of an in situ hybridization (ISH) protocol with radiolabeled cRNA probes. The detailed protocol of ISH used for paraffin sections is also described. EXPERIMENTAL DESIGN: In microwave (MW) heating, the tissue sections are heated in a sodium citrate buffer (0.01 M, pH 6). The effects of the pretreatment with MW were studied on kidneys and adrenals of young rats and on human pathologic samples using [35S]-RNA probes complementary to the mRNAs of some components of the renin angiotensin system. RESULTS: The heating pretreatment with the MW permitted us to obtain an enhancement of the hybridization signal, especially when using low doses of radioactive probes. This enhancement could be evaluated to 60 to 120% by computer-assisted quantification of the signal. Furthermore, the histologic structures and the staining with toluidine blue were not impaired by the heating treatment. CONCLUSIONS: The enhancing effect of the hybridization signal obtained using MW allows shorter autoradiographic exposure times and/or the use of lower concentrations of radioactive probes for the detection of mRNA or the detection of mRNA expressed at the threshold of detection with usual protocols.
BACKGROUND: Optimization of in situ hybridization protocols is of real interest when trying to detect small amounts of mRNA or when using low concentrations of probes. To enhance the hybridization signal, we have developed a modification of an in situ hybridization (ISH) protocol with radiolabeled cRNA probes. The detailed protocol of ISH used for paraffin sections is also described. EXPERIMENTAL DESIGN: In microwave (MW) heating, the tissue sections are heated in a sodium citrate buffer (0.01 M, pH 6). The effects of the pretreatment with MW were studied on kidneys and adrenals of young rats and on human pathologic samples using [35S]-RNA probes complementary to the mRNAs of some components of the renin angiotensin system. RESULTS: The heating pretreatment with the MW permitted us to obtain an enhancement of the hybridization signal, especially when using low doses of radioactive probes. This enhancement could be evaluated to 60 to 120% by computer-assisted quantification of the signal. Furthermore, the histologic structures and the staining with toluidine blue were not impaired by the heating treatment. CONCLUSIONS: The enhancing effect of the hybridization signal obtained using MW allows shorter autoradiographic exposure times and/or the use of lower concentrations of radioactive probes for the detection of mRNA or the detection of mRNA expressed at the threshold of detection with usual protocols.
Authors: I Arrighi; M Bloch-Faure; F Grahammer; M Bleich; R Warth; R Mengual; M D Drici; J Barhanin; P Meneton Journal: Proc Natl Acad Sci U S A Date: 2001-07-03 Impact factor: 11.205
Authors: J R Hagaman; J S Moyer; E S Bachman; M Sibony; P L Magyar; J E Welch; O Smithies; J H Krege; D A O'Brien Journal: Proc Natl Acad Sci U S A Date: 1998-03-03 Impact factor: 11.205
Authors: A P Gimenez-Roqueplo; J Favier; P Rustin; J J Mourad; P F Plouin; P Corvol; A Rötig; X Jeunemaitre Journal: Am J Hum Genet Date: 2001-10-16 Impact factor: 11.025
Authors: Giovana S Di Marco; Antoine Alam; Frédéric Dol; Pierre Corvol; Jean-Marie Gasc; Etienne Larger Journal: Mol Med Date: 2008-07-27 Impact factor: 6.354