IN SITU LOCALIZATION OF mRNA USING THYMINE-THYMINE DIMERIZED cDNA

概要

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DNA labeled with non-radioactive markers have been used for hybridizatiori with specific DNA or RNA either on filter or in cells and tissues. The presence of protruding markers on the probe DNA has been attributed to the cause for the loss of sensitivity and specificity of hybridization. In search of a non-protruding marker, we investigated the possibility of use of T-T dimer which can be generated easily in DNA and is a potent hapten as a marker for DNA. T-T dimer in DNA was generated by UV irradiation (254 μm) for total of 4,000-5,000 joules/m^2. For in situ hybridization; cells or tissues were first fixed either with Carnoy's fixative or formaldehyde and were usually treated with 0.2 N HC1, then hybridized with the T-T dimerized DNA (T-T DNA). The hybridized T-T DNA was detected immunohistochemically using rabbit anti-T-T DNA and peroxidase-labeled goat anti-rabbit IgG. With the Southern dot hybridization, the presence of 1 or less pg of complementary DNA can be detected. In cells and tissues, mRNA such as c-myc mRNA and growth hormone mRNA, and various viral DNA mRNA could be localized. The use of T-T as marker offers several advantages over other markers, it appears not interfere with the hybridization efficiency, simple to make and can be detected with high sensitivity.
日本組織細胞化学会の論文