A method using flow cytometry and fluorescent in situ hybridization (ISH) to detect RNA in cells is described. Ll210 murine leukemia cells were fixed with 1% formaldehyde in HEPES buffered Hank's balanced salt solution (HH) followed by 70% ethanol. Endogenous RNAses were blocked by diethylpyrocarbonate treatment. Single-stranded sense and antisense RNA probes, labeled with biotin-11-UTP, were transcribed from a 2.1 kb 28s ribosomal RNA (rRNA) gene fragment subcloned into the pGEMZ plasmid. For good results, it was essential that the probes were degraded to 100-150 nucleotides before use. Hybridization was performed at 45°C in 50% fomamide, 5 x SSC, 0.5% SDS. Hybrids were detected with streptavidin-FITC by flow cytometry. Antisense rRNA probe signal was Several in situ hybridization (ISH) methods have been described that allow for the detection of specific nuclei acid sequences with nonradioactively labeled probes. Probes have been labeled directly with fluorochromes (3) or cytochemically detectable enzymes (29). Post hybridization immunocytochemical development steps are not needed for such directly labeled probes. Indirect methods employ probes that are labeled by a hapten or affinity label, which is detected by (immuno-) cytochemical means subsequent to the hybridization reaction. Examples are the use of biotinylated probes (15,22,23), probes labeled with N-Acetoxy-N-Acetylaminofluorene (AAF) (2 1,33), or mercurated probes, which are haptenized after the hybridization reaction with a sulfhydryl-TPN (5,17). Probe labeling is not needed when antibodies specific for RNA.DNA hybrids are used to detect hybrids in situ by immunofluorescence (27,28,31).The availability of a fluorescent ISH method would allow its application to flow cytometry (2). The advantages of such flow cytometric ISH would be the simple quantification of hybridization signals and the rapid analysis of many cells, so that heterogeneous populations or aberrant expression levels in small subpopulations of cells can be analyzed. However, hybridization 100 times higher than the background. The hybrids were largely resistant to RNAse and melted at high temperature, The sense probe also gave a signal (5 times background), which was not RNAse resistant and was attributed to the presence of internal inverted repeats in the ribosomal RNA. When sufficient background reduction can be achieved, it is expected that as few as ten mRNA molecules per cell can be detected with the fluorescent in situ hybridization method.Key terms: Suspension hybridizaton, RNA detection, ribosomal RNA, Ll210 cells, biotinylated probes, single-stranded RNA probes, antisense RNA, nonisotopic nucleic acid probes conditions may seem incompatible with keeping cells, chromosomes, or nuclei in suspension, as is required for flow cytometry. Several papers have been published describing the use of fluorescent ISH to detect DNA targets in isolated interphase nuclei or chromosomes in suspension (12,26,34,35).To our knowledge the present paper is the first to describe the application of fluo...
Abstract. Electron microscopy, immunofluorescence, and bioassay demonstrated the presence of a mammary tumor inciting virus in untreated mice of three different inbred strains, and in irradiated or urethan-treated mice of two other mouse strains, indicating the ubiquitous nature of this group of viruses. In general these viruses are transmitted vertically by the gametes of the mouse strain in which they naturally occur. The virus is present in every cell, although often in an incomplete form. If a mammary tumor inciting virus is introduced into a different mouse strain, only milkborne transmission will take place, after which the virus is found in a limited number of tissues.It has been speculated that mammary tumor inciting viruses are transmitted as genetic factors of the host strain to which they belong. There is some evidence that a repressor, produced by a regulator gene, controls the rate of release of such a genetically transferred virus. Repression can be abrogated by a carcinogenic treatment. The repressor would also cause resistance to a superinfecting mammary tumor inciting virus by interference with its replication.
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