Prolonged hybridization with a cRNA probe improves the signal to noise ratio for in-tube in situ hybridization for quantification of mRNA after fluorescence-activated cell sorting

2012 ◽  
Vol 87 (5) ◽  
pp. 366-371 ◽  
Author(s):  
H Yamada ◽  
N Yamakawa ◽  
M Watanabe ◽  
Y Hidaka ◽  
Y Iwatani ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Cell Sorting ◽  
Signal To Noise Ratio ◽  
Fluorescence Activated Cell Sorting ◽  
Signal To Noise ◽  
Crna Probe ◽  
Noise Ratio

scholarly journals N-Ethylmaleimide (NEM) Can Significantly Improve In Situ Hybridization Results Using 35S-labeled Oligodeoxynucleotide or Complementary RNA Probes

1997 ◽  
Vol 45 (7) ◽  
pp. 1035-1041 ◽  
Author(s):  
R. Thomas Zoeller ◽  
Donald L. Fletcher ◽  
Olimpia Butnariu ◽  
Christopher A. Lowry ◽  
Frank L. Moore
Keyword(s):  
In Situ Hybridization ◽  
Chemical Reaction ◽  
Signal To Noise Ratio ◽  
Significant Source ◽  
Signal To Noise ◽  
Additional Element ◽  
Covalent Bonds ◽  
Rna Probes ◽  
Noise Ratio

We predicted that a significant source of background labeling after in situ hybridization (ISH) using 35S-labeled probes is attributable to a chemical reaction between the phosphorothioate moiety of the probe [O3 P=S] and disulfides in tissue. These covalent bonds would immobilize probe in the tissue, thereby increasing background labeling. On the basis of this view, we have explored the use of N-ethylmaleimide (NEM) to irreversibly alkylate the phosphorothioate moiety of the probe and/or to alkylate free sulfhydryls in tissue to block the formation of disulfides as a method of reducing background labeling. We report that NEM can significantly decrease background labeling of 35S-labeled oligodeoxy-nucleotide or cRNA probes but does not affect specific labeling. We conclude that the use of NEM in ISH protocols, as outlined here, may be an additional element researchers may consider to improve the 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 Messenger RNA quantification after fluorescence-activated cell sorting using in situ hybridization

2010 ◽  
Vol 77A (11) ◽  
pp. 1032-1037 ◽  
Author(s):  
Hiroya Yamada ◽  
Rie Maruo ◽  
Mikio Watanabe ◽  
Yoh Hidaka ◽  
Yoshinori Iwatani ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Cell Sorting ◽  
Messenger Rna ◽  
Fluorescence Activated Cell Sorting ◽  
Rna Quantification
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