Yersinia pestis strain 201 treated with treatment of 80 μg/ml polymyxin B for 30 min.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from two independent batches of cultures. To minimize bacterial RNA degradation and preserve gene transcription profile, double-volume of RNAprotect Bacteria Reagent (Qiagen) was added immediately to each aliquot, mixed thoroughly and placed at room temperature for 5 min. Total RNA extraction was performed by using MasterPureTM RNA Purification kits (Epicenter), exactly following the kit’s manual. Briefly, cell pellets were collected from the bacteria cultures by centrifugation. The lysis of cell samples was performed by Protease K digestion at 65°C for 15min, and protein was then removed from the lysed sample with the protein precipitation reagent provided by the kit. Isopropanol was added to precipitate nucleic acid and the residue DNA was removed with RNase-free DNase I. The RNA was then recovered with isopropanol. The RNAs from each four aliquots were pooled together. RNA quality was monitored by agarose gel electrophoresis and RNA quantity was measured by spectrophotometer.
Label
Cy5 or Cy3
Label protocol
Each RNA preparation was used to prepare fluorescence-labeled probes for three separate slides. For the total of eight replicate slides, the incorporated dye was reversed to make up three flip dye pairs. 20 µg of total RNA was mixed with 5 µg of GDPs and 5 µg random hexamer primers, heated to 70°C for 10 min, and cooled quickly on ice. 1 µl of RNasin RNase inhibitor (Promega), 8 µl of 5×reverse transcription buffer, 4 µl of 0.1 M DDT, 1.6 µl of unmodified dNTP mixture (12.5 mM of each dATP, dGTP, and dCTP, plus 7.5 mM dTTP), 2 µl of 4 mM aminoallyl-dUTP (Sigma) and 200 U of SuperScript II reverse transcriptase (Invitrogen) were added to a final volume of 40 µl. The reaction occurred at 42°C for 1 hr, after which an additional 200 U of Superscript II reverse transcriptase was added. The mixture was then incubated at 42°C for an additional hour. The reaction was stopped by the addition of 20 mM EDTA, and the RNA was degraded by the addition of NaOH to a final concentration of 30 mM, followed by incubation at 65°C for 20 min. The mixture was neutralized by adding HCl to a final concentration of 30 mM. Free amines were removed by using Qiaquick PCR purification columns (Qiagen). The purified cDNA was air dried at 50°C, and then was labeled by the addition of 1/10 of one reaction vial of Cy5 or Cy3 monofunctional dye (Amersham) in 0.05 M sodium bicarbonate (pH9.3) and incubated for 2 hr at room temperature in the dark. The Cy3 and Cy5 reaction mixtures were combined, and the unincorporated dye was removed by using MinElute reaction cleanup columns (Qiagen). The purified probe was air dried at 50°C, and was stored at -20°C until use.
Channel 2
Source name
Yersinia pestis_without treatment of antibacterial peptide
Yersinia pestis strain 201 without treatment of 80 μg/ml polymyxin B for 30 min.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from two independent batches of cultures. To minimize bacterial RNA degradation and preserve gene transcription profile, double-volume of RNAprotect Bacteria Reagent (Qiagen) was added immediately to each aliquot, mixed thoroughly and placed at room temperature for 5 min. Total RNA extraction was performed by using MasterPureTM RNA Purification kits (Epicenter), exactly following the kit’s manual. Briefly, cell pellets were collected from the bacteria cultures by centrifugation. The lysis of cell samples was performed by Protease K digestion at 65°C for 15min, and protein was then removed from the lysed sample with the protein precipitation reagent provided by the kit. Isopropanol was added to precipitate nucleic acid and the residue DNA was removed with RNase-free DNase I. The RNA was then recovered with isopropanol. The RNAs from each four aliquots were pooled together. RNA quality was monitored by agarose gel electrophoresis and RNA quantity was measured by spectrophotometer.
Label
Cy3 or Cy5
Label protocol
Each RNA preparation was used to prepare fluorescence-labeled probes for three separate slides. For the total of eight replicate slides, the incorporated dye was reversed to make up three flip dye pairs. 20 µg of total RNA was mixed with 5 µg of GDPs and 5 µg random hexamer primers, heated to 70°C for 10 min, and cooled quickly on ice. 1 µl of RNasin RNase inhibitor (Promega), 8 µl of 5×reverse transcription buffer, 4 µl of 0.1 M DDT, 1.6 µl of unmodified dNTP mixture (12.5 mM of each dATP, dGTP, and dCTP, plus 7.5 mM dTTP), 2 µl of 4 mM aminoallyl-dUTP (Sigma) and 200 U of SuperScript II reverse transcriptase (Invitrogen) were added to a final volume of 40 µl. The reaction occurred at 42°C for 1 hr, after which an additional 200 U of Superscript II reverse transcriptase was added. The mixture was then incubated at 42°C for an additional hour. The reaction was stopped by the addition of 20 mM EDTA, and the RNA was degraded by the addition of NaOH to a final concentration of 30 mM, followed by incubation at 65°C for 20 min. The mixture was neutralized by adding HCl to a final concentration of 30 mM. Free amines were removed by using Qiaquick PCR purification columns (Qiagen). The purified cDNA was air dried at 50°C, and then was labeled by the addition of 1/10 of one reaction vial of Cy3 or Cy5 monofunctional dye (Amersham) in 0.05 M sodium bicarbonate (pH9.3) and incubated for 2 hr at room temperature in the dark. The Cy3 and Cy5 reaction mixtures were combined, and the unincorporated dye was removed by using MinElute reaction cleanup columns (Qiagen). The purified probe was air dried at 50°C, and was stored at -20°C until use.
Hybridization protocol
For hybridization, the slides were incubated in 50 µl of prehybridization buffer (5×SSC, 0.1% SDS and 0.1% BSA) at 42°C for at least 1 hr. The slides were washed twice in water for 1 min and once in 95% ethanol for 1 min, and then blown to dry with hot air. Dual-fluorescence labeled dried probes were resuspended in 35 µl of hybridization solution (50% deionized formamide, 5×SSC, 0.1% SDS, 5×Denhardt’s solution, and 0.5 µg/µl of sheared salmon sperm DNA). The mixture was denatured at 98°C for 5 min, and then was applied to the microarray slide followed by covering a 24×50 mm glass coverslip. The slides were hybridized in a moisture chamber at 42°C for 18 to 20 hr in a hybridization incubator (Robbins Scintific). After hybridization, the slides were washed in 1×SSC with 0.06% SDS for 2 min, then in 0.06×SSC for 2 min and finally in ethanol for 1 min. The slides were blown dry for scanning.
Scan protocol
The slides were scanned at a resolution of 10 µm by using a GenePix Personal 4100A Microarray Scanner (Axon Instruments).
Description
Y. pestis strain 201 (F1+,VW+, Pgm+, and Pst+) avirulent to humans, belonging to a newly established biovar, Microtus was grown at 26 °C in a chemically defined TMH medium to the middle exponential growth phase (A620nm approximately 0.8). Cell cultures were diluted 1:20 in fresh TMH medium, and the cells were grown for at least 10 generations at 26 °C until reaching A620nm of approximately 0.6. Bacterial cells were then exposed to different in vitro stresses.
Data processing
The scanning images were processed and the data were further analyzed by using GenePix Pro 4.1 software (Axon Instruments) combined with Microsoft Excel software. Spots were analyzed by adaptive quantitation, and the local background was subsequently subtracted. Spots with background-corrected signal intensity (median) in both channels less than two folds of background intensity (median) were rejected from further analysis. Data normalization was performed on the remaining spots by total intensity normalization methods. The normalized log2 ratio of test/reference signal for each spot was recorded. Genes with less than three data points were considered unreliable, and their data points were discarded as well. The averaged log2 ratio for each remaining gene on the four replicate slides was ultimately calculated. Significant changes of gene transcription level were identified with SAM software using one class mode.
Comparative transcriptomics in Yersinia pestis: a global view of environmental modulation of gene expression
Data table header descriptions
ID_REF
VALUE
Data normalization was performed on all the spots by total intensity normalization methods. The normalized log2 ratio of test/reference signal for each spot was recorded.
