Immunofluorescence protects RNA signals in simultaneous RNA–DNA FISH

Lai, Lan-Tian; Lee, Pin Jie; Zhang, Li Feng

doi:10.1016/j.yexcr.2012.11.009

Cited by 17 publications

(25 citation statements)

References 16 publications

(25 reference statements)

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“…The Terra RNA transcripts locate in close proximity to telomeres, but some of the Terra signals do not overlap with telomere DNA. All of these characteristics of Terra are consistent with previous observations [2], [5]. It shows that the RNA-DNA FISH experiment worked well with the amino-labeled oligonucleotide probe.…”

Section: Resultssupporting

confidence: 90%

“…Cells were fixed and permeabilized before RNA or DNA FISH was carried out. The detailed experimental procedures have been described [2], [3]. In brief, for RNA FISH, slides were dehydrated, dried and hybridized with probes for 3 hours at 42°C in a dark and humid environment.…”

Section: Methodsmentioning

confidence: 99%

“…Simultaneous RNA-DNA fluorescence in situ hybridization (RNA-DNA FISH) is a highly useful tool for this research topic. However, an RNA-DNA FISH experiment is often difficult because the fragile RNA FISH signals or the RNA targets cannot survive the harsh treatments (high temperature, low pH) used in DNA FISH to denature the target DNA [2], [3].…”

Section: Introductionmentioning

confidence: 99%

“…In our previous study [2], we developed a method to overcome this difficulty by introducing immunostain into RNA FISH at signal detection steps. This, followed by a fixation step using formaldehyde (post-fixation), can efficiently protect the RNA signals from being damaged by the subsequent DNA FISH.…”

Section: Introductionmentioning

confidence: 99%

“…Although this method provides robust protection for the RNA signals to survive the DNA FISH, the multiple steps of immunostain, which have to be included to preserve the RNA signals, make the method complicated, time-consuming and expensive. It also generates additional difficulties for the subsequent DNA FISH [2]. Furthermore, the immunostain unnecessarily amplifies the RNA signals and raises the background noise.…”

Section: Introductionmentioning

confidence: 99%

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Using Amino-Labeled Nucleotide Probes for Simultaneous Single Molecule RNA-DNA FISH

et al. 2014

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Using amino-labeled oligonucleotide probes, we established a simple, robust and low-noise method for simultaneous detection of RNA and DNA by fluorescence in situ hybridization, a highly useful tool to study the large pool of long non-coding RNAs being identified in the current research. With probes either chemically or biologically synthesized, we demonstrate that the method can be applied to study a wide range of RNA and DNA targets at the single-cell and single-molecule level in cellular contexts.

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Section: Resultssupporting

confidence: 90%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Using Amino-Labeled Nucleotide Probes for Simultaneous Single Molecule RNA-DNA FISH

et al. 2014

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Telomeric noncoding RNA: telomeric repeat‐containing RNA in telomere biology

Cusanelli

Chartrand

2014

WIREs RNA

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Telomeres are nucleoprotein structures that cap the ends of eukaryotic chromosomes, protecting them from degradation and activation of DNA damage response. For this reason, functional telomeres are vital to genome stability. For years, telomeres were assumed to be transcriptionally silent, because of their heterochromatic state. It was only recently shown that, in several organisms, telomeres are transcribed, giving rise to a long noncoding RNA (lncRNA) called telomeric repeat-containing RNA (TERRA). Several lines of evidence now indicate that TERRA molecules play crucial roles in telomere homeostasis and telomere functions. Recent studies have shown that the expression and regulation of TERRA are dynamically controlled by each chromosome end. TERRA has been involved in the regulation of telomere length, telomerase activity, and heterochromatin formation at telomeres. The correct regulation of the telomeric transcripts may be essential to genome stability, and altered TERRA levels associate with tumorigenesis and cellular senescence. Thus, the study of the molecular mechanisms of TERRA biogenesis and function may advance the understanding of telomere-related diseases, including cancer and aging.

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