Counting small RNA in disease-causing organisms

June 18, 2013
Artist's concept of the fluorescence labeling and detection of small RNA in pathogenic bacteria.

Small molecules of RNA (tens to hundreds of nucleotides in length) play a key regulatory role in bacteria. Due to their small size, directly measuring the number of small RNA (sRNA) present in a single bacterium has proven so far to be an impossible task. Standard methods of measuring the number of specific nucleic acid molecules present in a single cell suffer from too much background and false positives when scientists attempt to image short targets. In research featured on the cover of the journal Analytical Chemistry, Los Alamos researchers demonstrated improved technical methods capable of directly counting small RNA molecules in pathogenic (disease-causing) bacteria.

The ability to quantify the expression level changes of sRNA in single cells as a function of provides important information to understand the role of sRNA in cellular regulatory networks and disease. The scientists found that only a small fraction of either (causative agent for the plague) or Yersinia pseudotuberculosis bacteria express two sRNAs thought to be important in pathogenesis, with the copy number typically between 0-10 transcripts per bacteria. The relatively low sRNA copy number in the bacterial populations raises the question of how many sRNAs are required for regulatory control over (mRNA) and protein populations.

The team learned that the change in the distribution of sRNAs per bacterium for a situation that mimics infection of a warm-blooded host could be described through a bursting model of gene transcription, where sRNA are produced for a discrete period of time. The numbers of these sRNAs increased upon a temperature shift from 25 to 37 °C, corresponding to a change from Y. pestis infection conditions of the flea host to a human host during infection. These results suggest that the sRNAs play a role in pathogenesis. The team's modeling suggests that sRNA copy numbers are controlled by a change in the on-rate of the gene under the human host infection conditions.

Counting small RNA in disease-causing organisms
Fluorescence microscope image of small RNA detected inside individual bacteria.

Research achievements

The team directly visualized single short (approximately 200 nucleotide) nucleic acid targets in bacteria by neutralizing the fluorescence of unbound dye probes. They used an automated, multi-color wide-field microscope and data analysis package to analyze the statistics of sRNA expression in thousands of individual bacteria. The new technique reduced the number of false positives, which improved the accuracy of the count statistics, and it significantly reduced the image processing time. The team performed modeling of the sRNA increase under simulated conditions of infection in a human host, finding a bursting model of well described the observed sRNA upregulation.

Explore further: MIT reels in RNA surprise with microbial ocean catch

Related Stories

MIT reels in RNA surprise with microbial ocean catch

May 13, 2009

An ingenious new method of obtaining marine microbe samples while preserving the microbes' natural gene expression has yielded an unexpected boon: the presence of many varieties of small RNAs — snippets of RNA that act ...

An sRNA controls a bacterium's social life

May 20, 2010

For the first time, biologists have directly shown how spontaneous mutation of a small RNA (sRNA) regulatory molecule can provide an evolutionary advantage. Reporting in this week's Science, Indiana University Bloomington ...

Bacterial gene 'therapy' to combat cholera

July 9, 2012

Cholera is an extremely virulent intestinal infection caused by ingestion of the bacterium Vibrio cholerae (V. cholerae). EU researchers elucidated the molecular mechanisms behind expression of virulence genes with important ...

Recommended for you

New method facilitates research on fuel cell catalysts

October 8, 2015

While the cleaning of car exhausts is among the best known applications of catalytic processes, it is only the tip of the iceberg. Practically the entire chemical industry relies on catalytic reactions. Therefore, catalyst ...

Better fluorescent lighting through physics

October 8, 2015

General Electric (GE), Lawrence Livermore National Laboratory (LLNL) and Oak Ridge National Laboratory (ORNL) have created new kinds of fluorescent lighting phosphors that use far less rare-earth elements than current technology.

Porous material holds promise for prosthetics, robots

October 8, 2015

Cornell researchers have developed a new lightweight and stretchable material with the consistency of memory foam that has potential for use in prosthetic body parts, artificial organs and soft robotics. The foam is unique ...


Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.