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...
| Main Authors: | , , |
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| Format: | Journal Article |
| Language: | English |
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2015
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| Online Access: | https://hdl.handle.net/10356/80848 http://hdl.handle.net/10220/38893 |
| _version_ | 1826111000284233728 |
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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. |
| first_indexed | 2024-10-01T02:43:25Z |
| format | Journal Article |
| id | ntu-10356/80848 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T02:43:25Z |
| publishDate | 2015 |
| record_format | dspace |
| 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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