Lathem et al. 10.1073/pnas.0506840102. |
Fig. 4. Survival of C57BL/6 mice infected intranasally with Y. pestis strain CO92. Groups of four mice were infected as described with successive 10-fold dilutions of Y. pestis (1 × 105 to 1 × 102 bacteria) and monitored twice daily for 7 days. One hundred percent of mice receiving the highest doses (105, 104) succumbed to the infection by 3.5 days, while increasing percentages of mice survived the lower doses. The analysis was done in duplicate in two independent experiments. The LD50 for this strain via the intranasal route is calculated as 2.8 × 102 bacteria.
Fig. 5. Localization of bacteria in infected lungs of C57BL/6 mice. Lungs were inflated and fixed with formalin, and 5-mm sections were stained by the Steiner method. Lungs were examined from mice infected with 1 × 104 colony-forming units (cfu) Y. pestis for 24, 48, or 72 h. Bacteria stain brown with the Steiner stain. (Bar = 50 mm.)
Fig. 6. Comparison of homogenization and BAL of mouse lungs. C57BL/6 mice were infected intranasally with Y. pestis (1 × 104 cfu). After 48 h, lungs were either lavaged with PBS (3 × 1 ml) or homogenized. (A) cfu recovered from BAL fluid or lung homogenates were determined by plating on BHI agar. A solid line indicates the mean of cfu recovered. There was no statistical difference in the number of bacteria recovered by the two methods (P = 0.57, unpaired t test). (B) RNA from BAL fluid and homogenized lungs was extracted as described and examined on an Agilent 2100 Bioanalyzer. Ribosomal RNA subunits are indicated. The ratio of bacterial rRNA (16S + 23S subunits) to eukaryotic rRNA (18S + 28S subunits) fluorescence intensity was »1:5 from homogenized lung-extracted RNA and 10:1 from BAL fluid-extracted RNA.
Fig. 7. Validation of DNA microarray results by quantitative RT-PCR (qRT-PCR). cDNA was synthesized from the same aRNA samples used for hybridization to the Y. pestis DNA microarrays. Twenty-seven ORFs that were differentially regulated by microarray analysis were selected at random for validation by qRT-PCR. Fold change was normalized to the expression of gyrB and calculated using the DDCt method. Of the 27 ORFs tested, 24 (88.9%) showed differential regulation in the same direction (i.e., up-regulated by both qRT-PCR and microarray).
Supporting Text
Microarray fabrication.
To construct a gene-specific microarray of the Yersinia pestis genome, we designed 70-mer oligonucleotide array elements. Predicted Y. pestis genes, encoded on the chromosome and the three plasmids of strain CO92, were obtained from the Wellcome Trust Sanger Institute (www.sanger.ac.uk/Projects/Y_pestis). Oligonucleotides were designed from these sequences by using arrayoligoselector (1) and were synthesized by using standard methods by illumina (San Diego). The oligonucleotides were dissolved at a concentration of 50 mM in 3 × SSC with 0.75 M betaine and were printed in duplicate on MWG Epoxy slides (MWG Biotech, Ebersburg, Germany) by a locally constructed linear servo arrayer (after the DeRisi model) (2).RNA isolation, T7-based linear amplification, and microarray hybridization.
Two groups of 10 mice each were independently infected with Y. pestis strain CO92, as described in the main text. After 48 h, mice were killed and their tracheas cannulated and lungs lavaged with 3 × 1 ml of PBS. After each wash, lavage fluid was immediately mixed with an excess of RNAlater RNA stabilization solution (Ambion, Austin, TX) to inhibit additional transcription and prevent RNA degradation. The elapsed time between the addition of PBS to the lungs and the subsequent mixing of the extracted lavage fluid to the stabilization solution was <30 seconds. The lavage fluid/RNAlater mixture from the 10 animals was centrifuged for 15 min at 27,000 ´ g to pellet the host and bacterial cells, and the supernatant was discarded. The pelleted cells were resuspended in a host cell lysis buffer (0.2% saponin in 50% PBS/50% RNAlater) and mixed at room temperature for 10 min. The mixture was centrifuged again for 2 minutes and washed once with lysis buffer. Control RNA was prepared from two independent cultures of Y. pestis grown at 37°C for 12 h in brain-heart infusion (BHI) broth supplemented with 2.5 mM CaCl2 to late-logarithmic phase in a rotary shaker set at 250 rpm. Total RNA was extracted from cultured and lavage-derived bacteria by using the RiboPure-Bacteria kit (Ambion). Integrity of the RNA samples was confirmed using the Agilent 2100 Bioanalyzer. Total RNA (500 ng) from each extraction was amplified in duplicate using the MessageAmp II-Bacteria amplified antisense RNA (aRNA) kit (Ambion) according to the manufacturer’s instructions. aRNA (5 mg) was labeled by using Micromax ASAP (PerkinElmer) using a variation of the manufacturer’s instructions in which an RNA Clean-up Kit (Zymo Research, Orange, CA) was used rather than the recommended Oligotex beads. The labeled aRNA was resuspended in Formamide-Based Hybridization Buffer (Genisphere, Hatfield, PA), hybridized to the microarrays at 42°C overnight and washed in a series of room temperature baths (2 × SSC, 0.2% SDS; then 2 × SSC, then 0.2 × SSC) for 12 min each.Microarray data processing and analysis
. Slides were scanned immediately after hybridization on a ScanArray Express HT Scanner (PerkinElmer) to detect Cy3 and Cy5 fluorescence. Laser power was kept constant, and photomultiplier tube (PMT) values were set for optimal intensity with minimal background (high-PMT scan). An additional scan was done for each slide with the PMT such that <1% of the elements are saturated (low-PMT scan) to characterize spots which were saturated in the high-PMT scan. Gridding and analysis of images were performed with scanarray software express Ver. 3.0 (PerkinElmer). The resulting median pixel values were imported into genespring 7.1 software (Agilent), and local background intensity were subtracted from individual spot intensities. To account for dye swap, the "signal" channel and "control" channel measurements were reversed in such samples so that signal derived from cultured RNA occupied the control channel and lavage RNA the signal channel. The mean signal and control intensities of the on-slide duplicate spots were calculated, and the control values were Lowess-normalized (3). If the control channel was lower than 10 relative fluorescence units (rfu), then 10 was used instead. Signal-to-Lowess-adjusted controlled ratios were calculated and the cross-chip averages were derived from the antilog of the mean of the natural log ratios across all eight microarray slides. Oligonucleotide elements that received a "present" call (intensity >200 rfu in at least one channel or local signal-to-background >2) by scanarray software in 8 of 16 scans in either the Cy3 or Cy5 were identified, and all others were excluded from the analysis. Each set of eight technical replicates was filtered using the one-sample Student’s t test function in genespring. In addition, the Bonferroni Multiple Testing correction was used to minimize type II error. The test is applied to the natural log of the mean of each normalized value against the baseline value of 0. Genes with differences corresponding to P value <0.05 in either the high- or the low-PMT scans and that had signal-to-control or control-to-signal ratios >2.0 were considered to be significantly regulated. The microarray data discussed in this publication have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO, www.ncbi.nlm.nih.gov/geo) and are accessible through GEO Series accession number GSE3575.Microarray validation.
The same aRNA samples used for microarray hybridization were reverse-transcribed with a set of random primers and the SuperScript II polymerase (Stratagene), as outlined by the manufacturer. cDNAs were then used as templates for amplification and detection with the SYBR green dye (Bio-Rad) in an iCycler thermocycler (Bio-Rad). For each gene, the calculated threshold cycle (Ct) was normalized to the Ct of the gyrB gene from the same cDNA sample before calculating the fold change using the DDCt method (4).(2003) Genome Biol. 4, R9.
2. Lashkari, D. A., DeRisi, J. L., McCusker, J. H., Namath, A. F., Gentile, C., Hwang, S. Y., Brown, P. O. & Davis, R. W. (1997) Proc. Natl. Acad. Sci. USA 94, 13057-13062.
3. Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., Ngai, J. & Speed, T. P. (2002) Nucleic Acids Res. 30, e15.
4. Applied Biosystems (1997) in ABI Prism 7700 Sequence Detection System User Bulletin #2 (Applied Biosystems, Foster City, CA).