Detection of viral sequences of low reiteration frequency by in situ hybridization.

Abstract

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 l cDNA has been achieved. The assay is quantitative. In situ ybridization has been used to detect and quantitate visna virus RNA in infected cells. By using [3HlcDNA with specific activity of 2 X 108 dpm/,ug 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.In situ hybridization is potentially a very powerful technique for localizing specific genes on chromosomes, following their expression in cells, and detecting and quantitating viral genes in infected cells. Unfortunately, until now the lack of sensitivity of the technique has restricted its use to the study of highly repeated genetic sequences such as the tandemly repeated genes coding for rRNA (1). Visna disease is a slow persistent infection of sheep caused by a retrovirus (2, 3). During our investigation of the molecular pathogenesis of this disease, we showed that virus persistence is a consequence of restricted gene expression in vivo (4). Although proviral DNA is present in the tissues of infected animals, no viral proteins can be detected and the amount of infectious virus synthesized is minimal. In order to analyze this phenomenon further and, in particular, to determine the level at which the restriction is imposed, we have improved the sensitivity of in situ hybridization approximately 10-fold. This enables us to detect 1-10 copies of viral genome per cell with radioautographic exposures of a few days. It should now be possible, with this technique, to address a number of important issues in gene regulation by using radioactive cDNA transcribed in vitro from purified mRNA. MATERIALS AND METHODSPreparation of [3HlcDNA.[3H]DNA complementary to the visna virus genome was prepared in an endogenous reaction in which purified visna virus was used as a source of both RNA-dependent DNA polymerase and viral 70S RNA. The reaction mixture contained 50 mM Tris-HCI (pH 7.4), 8 mM MgCI2, 1 mM dATP, dGTP, and dCTP, 0.1 mM [3H]dTTP at a specific activity of 90 Ci/mmol or greater, 10 mM dithiothreitol, 100,ug of actinomycin D per ml, 0.02% (vol/vol) Triton X-100, and 250 ;g of purified visna virus per ml. The total reaction volume was usually 4 ml. The reaction mixture was incubated at 450C for 2 hr. and the reaction was terminated by addition of EDTA and Sarkosyl to final concentrations of The publication costs of this article were defrayed in part by page charge payment. This article must therefore by hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 6125 10 mM and 0.5% (wt/vol), respectively. The mixture was incubated for 10 min at 370C; nonradioactive dTTP (in 20-to 100-fold excess) and 200,gg of ye...

An ovarian follicular epithelium protein of the silkworm (Bombyx mori) that associates with the vitelline membrane and contributes to the structural integrity of the follicle

“…After synthesis, the sizes of the riboprobes were reduced to approximately 100 nt by alkaline hydrolysis [19].…”

Susceptibility of human glial cells to infection with human immunodeficiency virus (HIV)

“…CEM/LAV-NlT [14] producer cells were included as positive controls. Smears of fixed cells were taken through a modified prehybridisation procedure [15] prior to overnight incubation with a PhotobiotinTM [16]-labelled antisense single-stranded RNA probe. The probe consisted of pGAP, pGgag and pGenv combined in equal amounts in hybridisation cocktail as described [ 161.…”

Rapid Fluorescence Detection of in Situ Hybridization with Biotinylated Bovine Herpesvirus-1 DNA Probes

“…Hybridization probes can be used effectively to detect viral sequences in situ at levels as low as 10-20 viral genome copies per ce11. 3,7,8 In situ-only detection is approximately l00-fold less sensitive than nucleic acid extraction of specific tissues and detection on nitrocellulose filters. 8 In situ hybridization allows the localization of viral sequences within the cells of specific tissues and the monitoring of viral mRNA expression, which can be applied to investigations into the pathogenesis of viral pathogens.…”

“…l, l3 In situ hybridization has been used to detect 1 infected cell in 50 uninfected cells and is useful to localize viral genetic material within a ce11. 5,7 The diagnosis and pathogenesis studies of several viral diseases have been documented using gene p r o b e s . 4,8,9,19,28 Most of the early applications using DNA hybridization probes required the use of radionucleotides as reporter groups.…”