title: Immunofluorescence protects RNA signals in simultaneous RNA-DNA FISH.
abstract: 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.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore.
authors.forename: Lan-Tian
authors.initials: LT
authors.lastname: Lai
authors.forename: Pin Jie
authors.initials: PJ
authors.lastname: Lee
authors.forename: Li-Feng
authors.initials: LF
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002232:Carbocyanines / Q000737:chemistry / Q000494:pharmacology; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D051379:Mice; D008954:Models, Biological; D011309:Preservation, Biological / Q000379:methods; D012313:RNA / Q000032:analysis* / Q000378:metabolism; D062085:RNA, Long Noncoding / Q000737:chemistry / Q000378:metabolism; D012815:Signal Processing, Computer-Assisted; D013048:Specimen Handling
chemical_list: D002232:Carbocyanines; D062085:RNA, Long Noncoding; C077885:XIST non-coding RNA; C089187:cyanine dye 3; D012313:RNA; D001710:Biotin; D004247:DNA