Locked Nucleic Acid Flow Cytometry-fluorescence <em>in situ</em> Hybridization (LNA flow-FISH): A Method for Bacterial Small RNA Detection

Abstract

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination 1,2 , cellular identification and gene expression 3,4 , monitoring viral multiplication in infected cells Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (T m ) of these analogs for natural nucleic acids 7,8 . Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence 9,10 . When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes 7,11 and have been successfully used to detect eukaryotic mRNA 12 and viral RNA in mammalian cells 5 .Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes 13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2). Video LinkThe video component of this article can be found at https://www.jove.com/video/3655/ Protocol 1. LNA probes & experimental design 1. Design LNA probes that are the reverse complement of your transcribed sRNA/mRNA or have them custom designed at www.exiqon.com. If using the custom design option, you will know the sequence, but not the LNA spiking pattern. The addition of each LNA residue into a DNA or RNA oligonucleotide ...

Hybridization-Based Detection of Helicobacter pylori at Human Body Temperature Using Advanced Locked Nucleic Acid (LNA) Probes

“…Fluorescent in situ hybridization (FISH) using DNA probes has long been used to rapidly detect and localize microbial cells in human clinical samples [ 4 , 5 ]. Nonetheless, this method was never employed to detect microorganisms within the human body (or other higher-order animals).…”

A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species

“…Formamide lowers the melting temperature of DNAs by 2.4 -2.9°C/ mole of formamide -with an efficiency depending on the properties of the nucleic acid strands themselves, such as their G+C content, the helix topology and the state of hydration (Blake and Delcourt, 1996). The solvent weakens hydrogen bonds, ultimately allowing lower hybridization temperatures, with similar high stringencies (Casey and Davidson, 1977;Sadhu et al, 1984;Robertson and Vora, 2012). Generally, the more concentrated the formamide, the higher is the stringency of reaction, but it was shown that, similarly to urea, an excess of solvent causes a dramatic drop in probe binding and signal detection (Manz et al, 1992;Bond and Banfield, 2001).…”

X-FISH: Analysis of cellular RNA expression patterns using flow cytometry

“…While FISH is conventionally used to study expression in cells or tissues fixed on slides, this technique has also been adapted to study hybridization by flow cytometry (Bauman et al, 1990). A flow cytometry-based technique has been recently used to study gene expression within bacteria and viruses (Robertson et al, 2010;Robertson and Vora, 2012b).…”