Using amino-labeled nucleotide probes for simultaneous single molecule RNA-DNA FISH.

Reelina Basu,Jun Wu,Li-Feng Zhang,Zhenyu Meng,Lan-Tian Lai,Fangwei Shao,Stefan Maas

doi:10.1371/journal.pone.0107425

Reelina Basu, Jun Wu + Show 5 more

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https://doi.org/10.1371/journal.pone.0107425

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Journal: PloS one	Publication Date: Sep 16, 2014
Citations: 25	License type: CC BY 4.0

Affiliation: Nanyang Technological University

Abstract

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.

Highlights

A growing list of long non-coding RNAs are being discovered in the current biological research [1]
The fixation step is used to crosslink the RNA signal with the cellular proteins in its close proximity, such that the RNA targets or the RNA Fluorescence in situ hybridization (FISH) probes may still be damaged in the subsequent DNA FISH, but the RNA signals survive
We detected HOTAIR and nuclear-enriched abundant transcript 2 (NEAT2) RNAs simultaneously with the telomeric DNA regions in human breast cancer cells (Figure 3). These results show that an amino-labeled nucleotide probe set can be generated by nick translation to detect various non-repetitive RNA targets in RNA-DNA FISH

Summary

Introduction

A growing list of long non-coding RNAs (lncRNAs) are being discovered in the current biological research [1]. For the fixation step to work, it is critical to include immunostain into RNA FISH, because formaldehyde-mediated crosslinking (Figure 1A) can only be efficiently applied to proteins, which contain lysine residues to offer the amino groups as crosslinking sites 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. The immunostain unnecessarily amplifies the RNA signals and raises the background noise To circumvent these disadvantages, in the current method, we introduced amino labels directly to the oligonucleotide probes for formaldehyde fixation between the probe and cellular proteins in RNA FISH (Figure 1B). We successfully applied the method to detect a variety of RNA targets including single RNA molecules

Methods

Results

Conclusion