Specific Sorting of Single Bacterial Cells with Microfabricated Fluorescence-Activated Cell Sorting and Tyramide Signal Amplification Fluorescence in Situ Hybridization

2011 ◽  
Vol 83 (19) ◽  
pp. 7269-7275 ◽  
Author(s):  
Chun H. Chen ◽  
Sung H. Cho ◽  
Hsin-I Chiang ◽  
Frank Tsai ◽  
Kun Zhang ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Cell Sorting ◽  
Signal Amplification ◽  
Bacterial Cells ◽  
Tyramide Signal Amplification ◽  
Fluorescence Activated Cell Sorting

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.

Fluorescence In Situ Hybridization of Progenitor Cells Obtained by Fluorescence-Activated Cell Sorting for the Detection of Cells Affected by Chromosome Abnormality Trisomy 8 in Patients With Myelodysplastic Syndromes

Blood ◽  
1998 ◽  
Vol 92 (8) ◽  
pp. 2886-2892 ◽  
Author(s):  
Kohki Saitoh ◽  
Ikuo Miura ◽  
Naoto Takahashi ◽  
Akira B. Miura
Keyword(s):  
Stem Cells ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Progenitor Cells ◽  
Pluripotent Stem Cells ◽  
Cell Sorting ◽  
Chromosome Abnormality ◽  
Fluorescence Activated Cell Sorting ◽  
Trisomy 8

Myelodysplastic syndrome (MDS) is believed to be a stem-cell disorder involving cytopenia and dysplastic changes in three hematopoietic lineages. However, the involvement of pluripotent stem cells and progenitor cells has not been clarified conclusively. To address this issue, we used fluorescence in situ hybridization (FISH) of blood and bone marrow (BM) smears for mature cells and FISH of cells sorted by fluorescence-activated cell sorting for progenitor cells. Seven patients with MDS associated with trisomy 8 were studied. FISH showed +8 in granulocytes, monocytes, and erythroblasts, but not in lymphocytes. Sorted cells of T (CD3+), B (CD19+), and NK cells (CD3−CD56+) from peripheral blood did not contain +8, nor did CD34+ subpopulations from BM including B (CD34+CD19+), T/NK (CD34+CD7+) progenitors, and pluripotent stem cells (CD34+Thy1+). The +8 chromosome abnormality was identified in stem cells only at the level of colony-forming unit of granulocyte-erythrocyte-macrophage-megakaryocyte (CFU-GEMM; CD34+CD33+). It may thus be concluded that cells affected by trisomy 8 in the context of MDS are at the CFU-GEMM level and that cells of lymphoid lineage are not involved. These results provide new insights into the biology of MDS and suggest that intensive chemotherapy and autologous BM transplantation may become important therapeutic strategies. © 1998 by The American Society of Hematology.

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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