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Tiling microarray-based identification of small regulatory RNAs in the group A Streptococcus (GAS)

ABSTRACT: Small regulatory RNAs (sRNAs) represent a major mechanism of virulence regulation in several pathogens. To date, only three sRNAs have been described in GAS in any detail. To identify whether sRNAs are common within the GAS genome, we created a custom Affymetrix tiling microarray in which all intergenic regions (>49 bp) of the MGAS2221 genome were tiled at high density. Triplicate cultures of strain MGAS2221 in THY broth were grown to the exponential phase (O.D.600 of 0.5) of growth and RNA was isolated, converted to cDNA, fragmented, labeled, and hybridized to our custom array (one array per RNA sample [ hence three arrays total]). Our data identified the presence of 40 candidate sRNAs within the MGAS2221 genome.

ORGANISM(S): Streptococcus pyogenes

PROVIDER: GSE17789 | GEO | 2009/08/25

SECONDARY ACCESSION(S): PRJNA123287

REPOSITORIES: GEO

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Tiling microarray-based identification of small regulatory RNAs in the group A Streptococcus (GAS)

Project description:Small regulatory RNAs (sRNAs) represent a major mechanism of virulence regulation in several pathogens. To date, only three sRNAs have been described in GAS in any detail. To identify whether sRNAs are common within the GAS genome, we created a custom Affymetrix tiling microarray in which all intergenic regions (>49 bp) of the MGAS2221 genome were tiled at high density. Triplicate cultures of strain MGAS2221 in THY broth were grown to the exponential phase (O.D.600 of 0.5) of growth and RNA was isolated, converted to cDNA, fragmented, labeled, and hybridized to our custom array (one array per RNA sample [ hence three arrays total]). Our data identified the presence of 40 candidate sRNAs within the MGAS2221 genome. Three cultures of MGAS2221 were grown in THY broth to the exponential (O.D. 0.5) phase of growth. Two volumes of RNA protect were added to the samples, the samples incubated at room temperature for 5 minutes, and the bacteria collected through centrifugation. Total RNA was isolated via a mechanical disruption method, converted to cDNA, fragmented, labeled, and hybridized to our Affymetrix microarray. Data was analyzed using tiling analysis software to combine the data from the three arrays and generate signal intensities. The supplementary files 'GSE17789_2221sRNA_signal.bar' and 'GSE17789_2221sRNA_pvalue.bar' contain the signals and p-values, respectively, for Samples GSM444010-GSM444012. The BAR files are generated by TAS software using PM/MM signal intensities. Signal intensity data is reported in a linear scale; the p-value is reported in -10log10 scale. Probe analysis used a bandwidth of 40. Interval analysis used a threshold of 0, a maximum gap of 20, and a minimum run of 60.

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