Immunofluorescence protects RNA signals in simultaneous RNA–DNA FISH

Cell research often requires combinational detection of RNA and DNA by fluorescence in situ hybridization (RNA–DNA FISH). However, it is difficult to preserve the fragile RNA signals through the harsh conditions used to denature the DNA template in DNA FISH. The current protocols of RNA –DNA FISH st...

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Main Authors: Lai, Lan-Tian, Lee, Pin Jie, Zhang, Li-Feng
Other Authors: School of Biological Sciences
Format: Journal Article
Language:English
Published: 2015
Subjects:
Online Access:https://hdl.handle.net/10356/80848
http://hdl.handle.net/10220/38893
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author Lai, Lan-Tian
Lee, Pin Jie
Zhang, Li-Feng
author2 School of Biological Sciences
author_facet School of Biological Sciences
Lai, Lan-Tian
Lee, Pin Jie
Zhang, Li-Feng
author_sort Lai, Lan-Tian
collection NTU
description Cell research often requires combinational detection of RNA and DNA by fluorescence in situ hybridization (RNA–DNA FISH). However, it is difficult to preserve the fragile RNA signals through the harsh conditions used to denature the DNA template in DNA FISH. The current protocols of RNA –DNA FISH still cannot work robustly in all experiments. RNA–DNA FISH remains as a technically challenging and tedious experiment. By incorporating protein components into the signal detection steps of RNA FISH, which is then followed by a post-fixation step, we established an improved protocol of RNA–DNA FISH. The established method worked satisfyingly and robustly in our studies on Xist (inactivated X chromosome specific transcript) RNA and Terra (telomeric repeat-containing RNA). Our results provided the direct evidence to show that, not all the telomeres are associated with Terra, and a significant fraction of Terra foci do not overlap with telomere DNA in interphase cell nuclei. The improved method of simultaneous RNA–DNA FISH is reliable and time-efficient. It can be used in a variety of biological studies.
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spelling ntu-10356/808482023-02-28T17:00:06Z Immunofluorescence protects RNA signals in simultaneous RNA–DNA FISH Lai, Lan-Tian Lee, Pin Jie Zhang, Li-Feng School of Biological Sciences Simultaneous RNA–DNA FISH X chromosome inactivation (XCI) Terra Fluorescence in situ hybridization (FISH) Xist Cell research often requires combinational detection of RNA and DNA by fluorescence in situ hybridization (RNA–DNA FISH). However, it is difficult to preserve the fragile RNA signals through the harsh conditions used to denature the DNA template in DNA FISH. The current protocols of RNA –DNA FISH still cannot work robustly in all experiments. RNA–DNA FISH remains as a technically challenging and tedious experiment. By incorporating protein components into the signal detection steps of RNA FISH, which is then followed by a post-fixation step, we established an improved protocol of RNA–DNA FISH. The established method worked satisfyingly and robustly in our studies on Xist (inactivated X chromosome specific transcript) RNA and Terra (telomeric repeat-containing RNA). Our results provided the direct evidence to show that, not all the telomeres are associated with Terra, and a significant fraction of Terra foci do not overlap with telomere DNA in interphase cell nuclei. The improved method of simultaneous RNA–DNA FISH is reliable and time-efficient. It can be used in a variety of biological studies. NMRC (Natl Medical Research Council, S’pore) Accepted version 2015-12-02T05:27:25Z 2019-12-06T14:00:16Z 2015-12-02T05:27:25Z 2019-12-06T14:00:16Z 2013 Journal Article Lai, L.-T., Lee, P. J., & Zhang, L.-F. (2013). Immunofluorescence protects RNA signals in simultaneous RNA–DNA FISH. Experimental Cell Research, 319(3), 46-55. 0014-4827 https://hdl.handle.net/10356/80848 http://hdl.handle.net/10220/38893 10.1016/j.yexcr.2012.11.009 en Experimental Cell Research © 2012 Elsevier Inc. All rights reserved. This is the author created version of a work that has been peer reviewed and accepted for publication by Experimental Cell Research, Elsevier Inc. It incorporates referee’s comments but changes resulting from the publishing process, such as copyediting, structural formatting, may not be reflected in this document. The published version is available at: [http://dx.doi.org/10.1016/j.yexcr.2012.11.009] 22 p. application/pdf
spellingShingle Simultaneous RNA–DNA FISH
X chromosome inactivation (XCI)
Terra
Fluorescence in situ hybridization (FISH)
Xist
Lai, Lan-Tian
Lee, Pin Jie
Zhang, Li-Feng
Immunofluorescence protects RNA signals in simultaneous RNA–DNA FISH
title Immunofluorescence protects RNA signals in simultaneous RNA–DNA FISH
title_full Immunofluorescence protects RNA signals in simultaneous RNA–DNA FISH
title_fullStr Immunofluorescence protects RNA signals in simultaneous RNA–DNA FISH
title_full_unstemmed Immunofluorescence protects RNA signals in simultaneous RNA–DNA FISH
title_short Immunofluorescence protects RNA signals in simultaneous RNA–DNA FISH
title_sort immunofluorescence protects rna signals in simultaneous rna dna fish
topic Simultaneous RNA–DNA FISH
X chromosome inactivation (XCI)
Terra
Fluorescence in situ hybridization (FISH)
Xist
url https://hdl.handle.net/10356/80848
http://hdl.handle.net/10220/38893
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