TY - JOUR
T1 - Counting small RNA in pathogenic bacteria
AU - Shepherd, Douglas P.
AU - Li, Nan
AU - Micheva-Viteva, Sofiya N.
AU - Munsky, Brian
AU - Hong-Geller, Elizabeth
AU - Werner, James H.
PY - 2013/5/21
Y1 - 2013/5/21
N2 - Here, we present a modification to single-molecule fluorescence in situ hybridization that enables quantitative detection and analysis of small RNA (sRNA) expressed in bacteria. We show that short (∼200 nucleotide) nucleic acid targets can be detected when the background of unbound singly dye-labeled DNA oligomers is reduced through hybridization with a set of complementary DNA oligomers labeled with a fluorescence quencher. By neutralizing the fluorescence from unbound probes, we were able to significantly reduce the number of false positives, allowing for accurate quantification of sRNA levels. Exploiting an automated, mutli-color wide-field microscope and data analysis package, we analyzed the statistics of sRNA expression in thousands of individual bacteria. We found that only a small fraction of either Yersinia pseudotuberculosis or Yersinia pestis bacteria express the small RNAs YSR35 or YSP8, with the copy number typically between 0 and 10 transcripts. The numbers of these RNA are both increased (by a factor of 2.5× for YSR35 and 3.5× for YSP8) upon a temperature shift from 25 to 37 C, suggesting they play a role in pathogenesis. The copy number distribution of sRNAs from bacteria-to-bacteria are well-fit with a bursting model of gene transcription. The ability to directly quantify expression level changes of sRNA in single cells as a function of external stimuli provides key information on the role of sRNA in cellular regulatory networks.
AB - Here, we present a modification to single-molecule fluorescence in situ hybridization that enables quantitative detection and analysis of small RNA (sRNA) expressed in bacteria. We show that short (∼200 nucleotide) nucleic acid targets can be detected when the background of unbound singly dye-labeled DNA oligomers is reduced through hybridization with a set of complementary DNA oligomers labeled with a fluorescence quencher. By neutralizing the fluorescence from unbound probes, we were able to significantly reduce the number of false positives, allowing for accurate quantification of sRNA levels. Exploiting an automated, mutli-color wide-field microscope and data analysis package, we analyzed the statistics of sRNA expression in thousands of individual bacteria. We found that only a small fraction of either Yersinia pseudotuberculosis or Yersinia pestis bacteria express the small RNAs YSR35 or YSP8, with the copy number typically between 0 and 10 transcripts. The numbers of these RNA are both increased (by a factor of 2.5× for YSR35 and 3.5× for YSP8) upon a temperature shift from 25 to 37 C, suggesting they play a role in pathogenesis. The copy number distribution of sRNAs from bacteria-to-bacteria are well-fit with a bursting model of gene transcription. The ability to directly quantify expression level changes of sRNA in single cells as a function of external stimuli provides key information on the role of sRNA in cellular regulatory networks.
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U2 - 10.1021/ac303792p
DO - 10.1021/ac303792p
M3 - Article
C2 - 23577771
AN - SCOPUS:84878319768
SN - 0003-2700
VL - 85
SP - 4938
EP - 4943
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 10
ER -