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Related Concept Videos

Types of RNA01:23

Types of RNA

Overview
Three main types of RNA are involved in protein synthesis: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). These RNAs perform diverse functions and can be broadly classified as protein-coding or non-coding RNA. Non-coding RNAs play important roles in the regulation of gene expression in response to developmental and environmental changes. Non-coding RNAs in prokaryotes can be manipulated to develop more effective antibacterial drugs for human or animal use.
RNA...
Bacterial RNA Polymerase00:43

Bacterial RNA Polymerase

Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
RNA Interference01:23

RNA Interference

RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
Translational Regulation01:29

Translational Regulation

Translational regulation in prokaryotes ensures efficient protein synthesis by controlling ribosome access to mRNA. This regulation is mediated by secondary RNA structures, including translational riboswitches, RNA thermometers, and small RNAs (sRNAs), which respond to intracellular and environmental signals to modulate gene expression.Translational RiboswitchesRiboswitches in the leader region of mRNAs can regulate translation by altering the accessibility of the Shine-Dalgarno (SD) sequence,...
Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
RNA-seq03:21

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...

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Related Experiment Video

Updated: May 12, 2026

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform
09:13

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

Published on: January 13, 2016

Counting small RNA in pathogenic bacteria.

Douglas P Shepherd1, Nan Li, Sofiya N Micheva-Viteva

  • 1Center for Integrated Nanotechnologies, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, United States.

Analytical Chemistry
|April 13, 2013
PubMed
Summary
This summary is machine-generated.

We developed a new method to accurately count bacterial small RNAs (sRNAs) in single cells. This technique reveals that specific sRNAs are upregulated in pathogenic bacteria upon temperature changes, suggesting a role in disease.

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MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Small RNAs (sRNAs) are crucial regulators in bacterial gene expression.
  • Accurate quantification of sRNAs in individual bacterial cells is challenging.
  • Existing methods often suffer from high background noise and false positives.

Purpose of the Study:

  • To develop a modified single-molecule fluorescence in situ hybridization (smFISH) technique for precise sRNA detection in bacteria.
  • To enable quantitative analysis of sRNA expression at the single-cell level.
  • To investigate the expression patterns of specific sRNAs in pathogenic Yersinia species.

Main Methods:

  • A novel smFISH approach using complementary DNA oligomers with fluorescence quenchers to reduce background noise.
  • Hybridization of dye-labeled DNA oligomers to neutralize unbound probes.
  • Utilizing an automated, multi-color wide-field microscope and data analysis package.
  • Analyzing sRNA expression statistics in thousands of individual Yersinia pseudotuberculosis and Yersinia pestis bacteria.

Main Results:

  • The modified smFISH method significantly reduced false positives, enabling accurate sRNA quantification.
  • Only a small fraction of bacteria expressed the YSR35 and YSP8 sRNAs, with low copy numbers (0-10 transcripts).
  • Expression of YSR35 and YSP8 increased significantly (2.5× and 3.5×, respectively) upon a temperature shift from 25°C to 37°C.
  • Bacterial sRNA copy number distributions fit a bursting model of gene transcription.

Conclusions:

  • The developed smFISH technique provides a powerful tool for quantitative sRNA analysis in single bacterial cells.
  • Temperature-induced upregulation of YSR35 and YSP8 suggests their involvement in bacterial pathogenesis.
  • This method offers key insights into the role of sRNAs in bacterial regulatory networks and responses to environmental stimuli.