Total RNA was extracted from four independent cell cultures. Before bacterial harvest, double-volume of RNAprotect Bacteria Reagent (Qiagen) was added immediately to each cell culture. Total RNA extraction was performed by using MasterPureTM RNA Purification kits (Epicenter). RNA quality was monitored by agarose gel electrophoresis and RNA quantity was measured by spectrophotometer.
Label
Cy3
Label protocol
Genome directed primers (GDPs) were designed by using FindGDPs software (University of Texas Southwestern Medical Center at Dallas). The desired length of GDPs was set as hexamers, and 50% of the 3' end of each ORF was defined as target region to scan for GDPs. Finally, 42 GDPs were picked out for annealing to all the 4005 genes of Y. pestis represented on the microarray. Three separated labeled probes for each RNA preparation were made as technical replicates. For the total of six 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 rRNasin 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, followed by the addition of a 200 U of Superscript II reverse transcriptase. The mixture was 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 the addition of 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 (pH 9.3) and incubated for 2h 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.
Total RNA was extracted from four independent cell cultures. Before bacterial harvest, double-volume of RNAprotect Bacteria Reagent (Qiagen) was added immediately to each cell culture. Total RNA extraction was performed by using MasterPureTM RNA Purification kits (Epicenter). RNA quality was monitored by agarose gel electrophoresis and RNA quantity was measured by spectrophotometer.
Label
Cy5
Label protocol
Genome directed primers (GDPs) were designed by using FindGDPs software (University of Texas Southwestern Medical Center at Dallas). The desired length of GDPs was set as hexamers, and 50% of the 3' end of each ORF was defined as target region to scan for GDPs. Finally, 42 GDPs were picked out for annealing to all the 4005 genes of Y. pestis represented on the microarray. Three separated labeled probes for each RNA preparation were made as technical replicates. For the total of six 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 rRNasin 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, followed by the addition of a 200 U of Superscript II reverse transcriptase. The mixture was 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 the addition of 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 (pH 9.3) and incubated for 2h 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 xSSC, 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 dried 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 0.5µg/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 Scientific).
Scan protocol
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 resulting slides were dried and then scanned at a resolution of 10 µm by using a GenePix Personal 4100A Microarray Scanner (Axon Instruments).
Description
Slide C
Data processing
For microarray hybridization, four independent bacterial cultures from each condition were prepared as biological replicates for RNA isolation. Accordingly, for each time point, four dual-fluorescence-labeled cDNA probes were prepared to hybridize with four slides, respectively. Pairwise comparisons were made using dye swaps to avoid labeling bias. 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 fold 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 ΔompR /WT signal for each spot was recorded. Genes with less than six 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. After the SAM analysis, only genes with at least two-fold changes in expression were collected for further analysis.
Raw data are unavailable due to computer disk failure.
