A rapid and highly sensitive method of non radioactive colorimetric in situ hybridization for the detection of mRNA on tissue sections.

<h4>Background</h4>Non Radioactive colorimetric In Situ Hybridization (NoRISH) with hapten labeled probes has been widely used for the study of gene expression in development, homeostasis and disease. However, improvement in the sensitivity of the method is still needed to allow for the...

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Autores principales:	Electra Stylianopoulou, Dimitrios Lykidis, Petros Ypsilantis, Constantinos Simopoulos, George Skavdis, Maria Grigoriou
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/41f60c2d02e34facba99362bc609b18b
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spelling	oai:doaj.org-article:41f60c2d02e34facba99362bc609b18b2021-11-18T07:23:40ZA rapid and highly sensitive method of non radioactive colorimetric in situ hybridization for the detection of mRNA on tissue sections.1932-620310.1371/journal.pone.0033898https://doaj.org/article/41f60c2d02e34facba99362bc609b18b2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22479470/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Non Radioactive colorimetric In Situ Hybridization (NoRISH) with hapten labeled probes has been widely used for the study of gene expression in development, homeostasis and disease. However, improvement in the sensitivity of the method is still needed to allow for the analysis of genes expressed at low levels.<h4>Methodology/principal findings</h4>A stable, non-toxic, zinc-based fixative was tested in NoRISH experiments on sections of mouse embryos using four probes (Lhx6, Lhx7, ncapg and ret) that have different spatial patterns and expression levels. We showed that Z7 can successfully replace paraformaldehyde used so far for tissue fixation in NoRISH; the morphology of the cryosections of Z7-fixed tissues was excellent, and the fixation time required for tissues sized 1 cm was 1 hr instead of 24 hr for paraformaldehyde. The hybridization signal on the sections of the Z7-treated embryos always appeared earlier than that of the PFA-fixed embryos. In addition, a 50-60% shorter detection time was observed in specimen of Z7-treated embryos, reducing significantly the time required to complete the method. Finally and most importantly, the strength of the hybridization signal on the sections of the Z7-treated embryos always compared favorably to that of the sections of PFA-fixed embryos; these data demonstrate a significant improvement of the sensitivity the method that allows for the analysis of mRNAs that are barely or not detected by the standard colorimetric NoRISH method.<h4>Conclusions/significance</h4>Our NoRISH method provides excellent preservation of tissue morphology, is rapid, highly sensitive, and especially suitable to implement in the study of genes expressed at low levels and/or in sparse cells within a structure.Electra StylianopoulouDimitrios LykidisPetros YpsilantisConstantinos SimopoulosGeorge SkavdisMaria GrigoriouPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 3, p e33898 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Electra Stylianopoulou Dimitrios Lykidis Petros Ypsilantis Constantinos Simopoulos George Skavdis Maria Grigoriou A rapid and highly sensitive method of non radioactive colorimetric in situ hybridization for the detection of mRNA on tissue sections.
description	<h4>Background</h4>Non Radioactive colorimetric In Situ Hybridization (NoRISH) with hapten labeled probes has been widely used for the study of gene expression in development, homeostasis and disease. However, improvement in the sensitivity of the method is still needed to allow for the analysis of genes expressed at low levels.<h4>Methodology/principal findings</h4>A stable, non-toxic, zinc-based fixative was tested in NoRISH experiments on sections of mouse embryos using four probes (Lhx6, Lhx7, ncapg and ret) that have different spatial patterns and expression levels. We showed that Z7 can successfully replace paraformaldehyde used so far for tissue fixation in NoRISH; the morphology of the cryosections of Z7-fixed tissues was excellent, and the fixation time required for tissues sized 1 cm was 1 hr instead of 24 hr for paraformaldehyde. The hybridization signal on the sections of the Z7-treated embryos always appeared earlier than that of the PFA-fixed embryos. In addition, a 50-60% shorter detection time was observed in specimen of Z7-treated embryos, reducing significantly the time required to complete the method. Finally and most importantly, the strength of the hybridization signal on the sections of the Z7-treated embryos always compared favorably to that of the sections of PFA-fixed embryos; these data demonstrate a significant improvement of the sensitivity the method that allows for the analysis of mRNAs that are barely or not detected by the standard colorimetric NoRISH method.<h4>Conclusions/significance</h4>Our NoRISH method provides excellent preservation of tissue morphology, is rapid, highly sensitive, and especially suitable to implement in the study of genes expressed at low levels and/or in sparse cells within a structure.
format	article
author	Electra Stylianopoulou Dimitrios Lykidis Petros Ypsilantis Constantinos Simopoulos George Skavdis Maria Grigoriou
author_facet	Electra Stylianopoulou Dimitrios Lykidis Petros Ypsilantis Constantinos Simopoulos George Skavdis Maria Grigoriou
author_sort	Electra Stylianopoulou
title	A rapid and highly sensitive method of non radioactive colorimetric in situ hybridization for the detection of mRNA on tissue sections.
title_short	A rapid and highly sensitive method of non radioactive colorimetric in situ hybridization for the detection of mRNA on tissue sections.
title_full	A rapid and highly sensitive method of non radioactive colorimetric in situ hybridization for the detection of mRNA on tissue sections.
title_fullStr	A rapid and highly sensitive method of non radioactive colorimetric in situ hybridization for the detection of mRNA on tissue sections.
title_full_unstemmed	A rapid and highly sensitive method of non radioactive colorimetric in situ hybridization for the detection of mRNA on tissue sections.
title_sort	rapid and highly sensitive method of non radioactive colorimetric in situ hybridization for the detection of mrna on tissue sections.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/41f60c2d02e34facba99362bc609b18b
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A rapid and highly sensitive method of non radioactive colorimetric in situ hybridization for the detection of mRNA on tissue sections.

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