scholarly journals Detection of Single Copy Genes by Two-Pass Tyramide Signal Amplification Fluorescence in situ Hybridization (Two-Pass TSA-FISH) with Single Oligonucleotide Probes

2010 ◽  
Vol 25 (1) ◽  
pp. 15-21 ◽  
Author(s):  
Shuji Kawakami ◽  
Kengo Kubota ◽  
Hiroyuki Imachi ◽  
Takashi Yamaguchi ◽  
Hideki Harada ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Signal Amplification ◽  
Single Copy ◽  
Oligonucleotide Probes ◽  
Tyramide Signal Amplification

scholarly journals Quantitative Assessment of Picoeukaryotes in the Natural Environment by Using Taxon-Specific Oligonucleotide Probes in Association with Tyramide Signal Amplification-Fluorescence In Situ Hybridization and Flow Cytometry

2003 ◽  
Vol 69 (9) ◽  
pp. 5519-5529 ◽  
Author(s):  
Isabelle C. Biegala ◽  
Fabrice Not ◽  
Daniel Vaulot ◽  
Nathalie Simon
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Natural Environment ◽  
Signal Amplification ◽  
Cell Counting ◽  
Oligonucleotide Probes ◽  
Tyramide Signal Amplification ◽  
Routine Application

ABSTRACT Picoeukaryotes (cells of <3 μm in diameter) contribute significantly to marine plankton biomass and productivity, and recently molecular studies have brought to light their wide diversity. Among the methods that have been used so far to quantify aquatic microorganisms, fluorescence in situ hybridization of oligonucleotide probes combined with flow cytometry offers the advantages of both high resolution for taxonomic identification and automated cell counting. However, cell losses, cell clumps, and low signal-to-background ratio have often been mentioned as major problems for routine application of this combination of techniques. We developed a new protocol associating tyramide signal amplification-fluorescence in situ hybridization and flow cytometry, which allows the detection of picoeukaryotes in cultures during both the exponential and stationary phases. The use of surfactant and sonication proved to be essential for the detection and quantification of picoeukaryotes from the natural environment, with as little as a few tenths of a milliliter of 3-μm-pore-size prefiltered sea water. The routine application of the technique was tested along a coastal transect off Brittany (France), where the different groups of picoeukaryotes were investigated using already published specific probes and a newly designed probe that targets the order Mamiellales (Prasinophyceae, Chlorophyta). Among the picoeukaryotes, Mamiellales outnumbered by 1 order of magnitude both the cyanobacteria and the non-Chlorophyta, which were represented mainly by the Pelagophyceae class. Picoeukaryote abundance increased from open toward more estuarine water, probably following changes in water temperature and stability.

Horse Radish Peroxidase Labelled Oligonucleotides and Tyramide Signal Amplification for Fluorescence in Situ Hybridization to Low Abundant Cytokine Mrnas

1997 ◽  
Vol 3 (S2) ◽  
pp. 203-204
Author(s):  
Mariette van de Corput ◽  
Rob van Gijlswijk ◽  
Mark Bobrow ◽  
Tom Erickson ◽  
Roel Dirks ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Signal Amplification ◽  
Signal To Noise Ratio ◽  
High Specificity ◽  
Tyramide Signal Amplification ◽  
Horse Radish Peroxidase ◽  
Metaphase Chromosomes ◽  
Generation Capacity

In recent years, Tyramide Signal Amplification (TSA) has gained acclaim as a very sensitive detection method for immunocytochemsitry and fluorescence in situ hybridization (FISH). To maximally exploit the great signal generation capacity of TSA in mRNA-FISH, minimizing signals emanating from non-specifically bound nucleic acid probe becomes of prime importance, because a specificity check of the signals observed in the cytoplasm is virtually impossible. We reasoned that utilization of synthetic oligonucleotides (ONTs) in stead of commonly used cDNAs or cRNAs would diminish non-specific probe binding and that direct Horse Radish Peroxidase (HRP) labelling of ONTs and TSA would enable their in situ detection.This approach was first tested in metaphase DNA-FISH using chromosome-specific repeats as targets. Using bifunctional crosslinking chemistry and HPLC, 5’-hexylamino oligonucleotides for chromosome specific simple satellite and alphoid sequences were conjugated to HRP and purified. Following 15 - 20 min of situ hybridization of a single HRP-ONT probe to metaphase chromosomes and a direct flurochrome-tyramide detection step, such repeat targets could be visualized with high specificity and excellent signal-to noise ratio.

scholarly journals Sensitive mRNA Detection by Fluorescence In Situ Hybridization Using Horseradish Peroxidase-labeled Oligodeoxynucleotides and Tyramide Signal Amplification

1998 ◽  
Vol 46 (11) ◽  
pp. 1249-1259 ◽  
Author(s):  
Mariëtte P.C. van de Corput ◽  
Roeland W. Dirks ◽  
Rob P.M. van Gijlswijk ◽  
Erica van Binnendijk ◽  
Claudia M. Hattinger ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Horseradish Peroxidase ◽  
Fluorescence In Situ Hybridization ◽  
Signal Amplification ◽  
Tyramide Signal Amplification ◽  
Mrna Detection
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