Key result
An improved in situ hybridization technique increased sensitivity at least 10-fold, allowing the detection and quantitation of 10-20 copies of visna virus RNA per cell after 2 days of exposure.
Optimized in situ hybridization significantly increases sensitivity, allowing for the quantitative detection of low-copy viral RNA in infected cells.
May advance low-copy viral RNA detection in animal models; leaves open human cardiovascular translation.
The sensitivity of in situ hybridization has been increased at least 10-fold by hybridizing in cDNA excess, by increasing the diffusion of the cDNA through the cells, by hybridizing at optimum temperature, and by stabilizing hybrids during autoradiography. Saturation of intracellular RNA with [3H]cDNA has been achieved. The assay is quantitative. In situ hybridization has been used to detect and quantitate visna virus RNA in infected cells. By using [3H]cDNA with specific activity of 2 X 10(8) dpm/micrograms and conditions that reduce background to negligible levels, 10--20 copies of viral RNA per cell can be detected and quantitated after 2 days of autoradiographic exposure.
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Brahic et al. (1978) studied Visna virus infection (in vitro). Improved in situ hybridization technique vs. Standard in situ hybridization was evaluated on Detection sensitivity of viral RNA copies per cell. An improved in situ hybridization technique increased sensitivity at least 10-fold, allowing the detection and quantitation of 10-20 copies of visna virus RNA per cell after 2 days of exposure.
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