In Situ Hybridization for RNA: Radioactive RNA Probe — A Protocol for Practice With a Kit

2000 ◽  
pp. 115-127
Author(s):  
Hidefumi Yoshioka ◽  
Sumihare Noji
Keyword(s):  
In Situ Hybridization ◽  
Rna Probe

scholarly journals PCR-derived ssDNA Probes for Fluorescent In Situ Hybridization to HIV-1 RNA

2000 ◽  
Vol 48 (2) ◽  
pp. 285-293 ◽  
Author(s):  
Marlyse C. Knuchel ◽  
Brigit Graf ◽  
Erika Schlaepfer ◽  
Herbert Kuster ◽  
Marek Fischer ◽  
...  
Keyword(s):  
T Cells ◽  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Uracil Dna Glycosylase ◽  
Rna Probes ◽  
Immunodeficiency Virus ◽  
Polymerase Chain ◽  
Rna Probe ◽  
Hiv 1

We developed a simple and rapid technique to synthesize single-stranded DNA (ssDNA) probes for fluorescent in situ hybridization (ISH) to human immunodeficiency virus 1 (HIV-1) RNA. The target HIV-1 regions were amplified by the polymerase chain reaction (PCR) and were simultaneously labeled with dUTP. This product served as template for an optimized asymmetric PCR (one-primer PCR) that incorporated digoxigenin (dig)-labeled dUTP. The input DNA was subsequently digested by uracil DNA glycosylase, leaving intact, single-stranded, digoxigenin-labeled DNA probe. A cocktail of ssDNA probes representing 55% of the HIV-1 genome was hybridized to HIV-1-infected 8E5 T-cells and uninfected H9 T-cells. For comparison, parallel hybridizations were done with a plasmid-derived RNA probe mix covering 85% of the genome and a PCR-derived RNA probe mix covering 63% of the genome. All three probe types produced bright signals, but the best signal-to-noise ratios and the highest sensitivities were obtained with the ssDNA probe. In addition, the ssDNA probe syntheses generated large amounts of probe (0.5 to 1 μg ssDNA probe per synthesis) and were easier to perform than the RNA probe syntheses. These results suggest that ssDNA probes may be preferable to RNA probes for fluorescent ISH.

scholarly journals In situ hybridization of a radioactive RNA probe on resin-embedded legume root-nodule sections: a tool for observing gene expression in the rhizosphere?

Agronomie ◽  
2003 ◽  
Vol 23 (5-6) ◽  
pp. 489-493 ◽  
Author(s):  
Olivier Schumpp ◽  
Hassen Gherbi ◽  
Jacques Escoute ◽  
Hélène Payre ◽  
Jean-Jacques Drevon
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Root Nodule ◽  
Rna Probe

scholarly journals Localization of neuropeptide Y mRNA in neurons of human cerebral cortex by means of in situ hybridization with a complementary RNA probe.

1987 ◽  
Vol 84 (20) ◽  
pp. 7315-7318 ◽  
Author(s):  
G. Terenghi ◽  
J. M. Polak ◽  
Q. Hamid ◽  
E. O'Brien ◽  
P. Denny ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
In Situ Hybridization ◽  
Neuropeptide Y ◽  
Human Cerebral Cortex ◽  
Rna Probe

Flow cytometric detection of yeast by in situ hybridization with a fluorescent ribosomal RNA probe

1990 ◽  
Vol 12 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Brigitte Bertin ◽  
Odile Broux ◽  
Michel Van Hoegaerden
Keyword(s):  
In Situ Hybridization ◽  
Ribosomal Rna ◽  
Flow Cytometric ◽  
Rna Probe

scholarly journals In situ hybridization and immunocytochemistry in serial sections of rabbit skeletal muscle to detect myosin expression.

1988 ◽  
Vol 36 (12) ◽  
pp. 1519-1526 ◽  
Author(s):  
D J Dix ◽  
B R Eisenberg
Keyword(s):  
Skeletal Muscle ◽  
In Situ Hybridization ◽  
Medial Gastrocnemius ◽  
Distinct Advantage ◽  
Rabbit Muscle ◽  
Serial Sections ◽  
Slow Twitch ◽  
Rna Probe ◽  
Slow Twitch Fibers

We performed in situ hybridization of myosin heavy-chain (MHC) mRNA on rabbit muscle using a biotin-labeled complementary RNA probe. An 1107-nucleotide fragment from an alpha-cardiac MHC cDNA was used to transcribe an RNA probe 97% similar to slow-twitch and 75% similar to fast-twitch sequences. Serial sections were used to identify slow-twitch fibers in medial gastrocnemius, soleus, and tibialis anterior by immunofluorescence of slow MHC and oxidative capacity by histochemistry. Slow-twitch fibers hybridized by the RNA probe stained heavily after detection with streptavidin-alkaline phosphatase (89% dark and 11% medium density). Fast-oxidative fibers stained intermediately (26% dark, 58% medium, and 16% light) and fast-glycolytic fibers stained lightly (12% medium and 88% light). Biotin-labeled probe and enzymatic detection allowed greater resolution of the subcellular location of the MHC mRNA, a distinct advantage over isotope labeling and autoradiography. A non-uniform distribution of MHC mRNA was recognized within an adult skeletal muscle fiber. High concentrations of MHC mRNA were found under the sarcolemma and between the myofibrils, suggesting the existence of a distribution mechanism. The combination of in situ hybridization and immunocytochemistry allows rapid subcellular localization of both MHC mRNA and its translated protein.

Pro-opiomelanocortin (POMC) gene expression, as identified by in situ hybridization, in purified populations of interstitial macrophages and Leydig cells of the adult rat testis

1993 ◽  
Vol 5 (5) ◽  
pp. 545 ◽  
Author(s):  
H Li ◽  
GP Risbridger ◽  
JA Clements
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Leydig Cells ◽  
Cell Types ◽  
Rat Testis ◽  
Adult Rat ◽  
Pomc Gene ◽  
Rna Probe ◽  
Silver Grains

The presence of testicular pro-opiomelanocortin (POMC) mRNA and POMC-derived peptides has recently been demonstrated in purified preparations of interstitial macrophages and in Leydig cells of the adult rat testis by Northern blot analysis and immunocytochemistry. In the present study, in situ hybridization provided further evidence that the POMC gene is expressed by both purified interstitial macrophages and Leydig cells. The cellular localization of the POMC transcripts was similar for both cell types, silver grains being predominantly located in the cytoplasm. The specificity of the labelling was demonstrated by the lack of silver grains in the preparations pretreated with RNAase or hybridized with an insulin cDNA probe, a gene known not to be expressed in these cell types. An additional control was provided by hybridization with a sense POMC RNA probe, which gave a less intense signal when compared with the antisense RNA probe under the same experimental conditions. The results confirm POMC gene expression in both macrophages and Leydig cells in the adult rat testis.

scholarly journals Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 524-527 ◽  
Author(s):  
ST Koury ◽  
MC Bondurant ◽  
MJ Koury
Keyword(s):  
Endothelial Cells ◽  
In Situ Hybridization ◽  
Basement Membrane ◽  
Interstitial Cells ◽  
Tubular Basement Membrane ◽  
Paraffin Embedding ◽  
Capillary Endothelial Cells ◽  
Rna Probe ◽  
Labeled Rna

Abstract In situ hybridization was used to localize the cells that produce erythropoietin (EP) in anemic murine kidneys. Kidneys from anemic and nonanemic mice were fixed and processed for paraffin embedding. Sections were hybridized with a 35S-labeled RNA probe complementary to mRNA coding for EP. An uncommon, but specific type of cell was intensely labeled in the cortices of anemic kidneys. The labeled cells were clearly nonglomerular and nontubular. Their location outside of the tubular basement membrane was consistent with that of a subset of interstitial cells or capillary endothelial cells.

scholarly journals Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 524-527 ◽  
Author(s):  
ST Koury ◽  
MC Bondurant ◽  
MJ Koury
Keyword(s):  
Endothelial Cells ◽  
In Situ Hybridization ◽  
Basement Membrane ◽  
Interstitial Cells ◽  
Tubular Basement Membrane ◽  
Paraffin Embedding ◽  
Capillary Endothelial Cells ◽  
Rna Probe ◽  
Labeled Rna

In situ hybridization was used to localize the cells that produce erythropoietin (EP) in anemic murine kidneys. Kidneys from anemic and nonanemic mice were fixed and processed for paraffin embedding. Sections were hybridized with a 35S-labeled RNA probe complementary to mRNA coding for EP. An uncommon, but specific type of cell was intensely labeled in the cortices of anemic kidneys. The labeled cells were clearly nonglomerular and nontubular. Their location outside of the tubular basement membrane was consistent with that of a subset of interstitial cells or capillary endothelial cells.

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