8 ml of the culture was mixed with 1/10V 10% phenol:ethanol buffer to stabilize the RNA, and centrifuged at 4 oC, 4300 × g for 30 min to pellet cells. The supernatant was decanted and cell pellets were suspended in 5 ml of buffer (2 mM EDTA, 20 mM NaOAc, pH 5.2) before mixing with equal volume of hot acid-phenol:chloroform (pH 4.5 with Iso Amyl Alcohol (IAA), 125:25:1) (Ambion, Austin, TX) and incubating at 65 oC with periodic shaking for 10 min. The samples were centrifuged at 3220 × g for 20 min and the supernatant was subjected to further extractions with phenol:chloroform and chloroform:IAA (3). RNA was precipitated overnight at -70 oC in 2.5 volume 100% ethanol and 1/10 volume 3 M sodium acetate pH 5.2. RNA purification and DNase treatment of RNA samples were done with the Rneasy kit (Qiagen, Valencia, CA) and RNA quality was assessed on a formaldehyde-agarose gel.
Label
cy3
Label protocol
Reverse transcription reactions contained 6ug RNA, 2ug random primers (Invitrogen, Carlsbad, Calif.), 1x first strand buffer (Invitrogen), 10mM DTT, 400U Superscript II (Invitrogen), 0.5mM each dATP, dCTP, and dGTP, 0.3mM dTTP, and 0.2mM reactions wereamino-allyl dUTP. 30uL C. cDNA was purified using PCR incubated overnight at 42 cleanup columns (Qiagen) with Phosphate wash buffer (5mM K2HPO4, pH 8.0, 80%EtOH) and phosphate elution buffer (4mM K2HPO4, pH 8.5). Amino-allyl labeled cDNA was dried and resuspended in 0.1M sodium carbonate pH 9.3 and coupled with either Cy3 or Cy5 (Amersham Biosciences, Piscataway, N.J.). Uncoupled dye was removed by another purfication using the PCR cleanup kit. Concentration of cDNA and amount of incoporated dye was measured for each sample using a Nanodrop spectrophotometer (Ambion).
8 ml of the culture was mixed with 1/10V 10% phenol:ethanol buffer to stabilize the RNA, and centrifuged at 4 oC, 4300 × g for 30 min to pellet cells. The supernatant was decanted and cell pellets were suspended in 5 ml of buffer (2 mM EDTA, 20 mM NaOAc, pH 5.2) before mixing with equal volume of hot acid-phenol:chloroform (pH 4.5 with Iso Amyl Alcohol (IAA), 125:25:1) (Ambion, Austin, TX) and incubating at 65 oC with periodic shaking for 10 min. The samples were centrifuged at 3220 × g for 20 min and the supernatant was subjected to further extractions with phenol:chloroform and chloroform:IAA (3). RNA was precipitated overnight at -70 oC in 2.5 volume 100% ethanol and 1/10 volume 3 M sodium acetate pH 5.2. RNA purification and DNase treatment of RNA samples were done with the Rneasy kit (Qiagen, Valencia, CA) and RNA quality was assessed on a formaldehyde-agarose gel.
Label
cy5
Label protocol
Reverse transcription reactions contained 6ug RNA, 2ug random primers (Invitrogen, Carlsbad, Calif.), 1x first strand buffer (Invitrogen), 10mM DTT, 400U Superscript II (Invitrogen), 0.5mM each dATP, dCTP, and dGTP, 0.3mM dTTP, and 0.2mM reactions wereamino-allyl dUTP. 30uL C. cDNA was purified using PCR incubated overnight at 42 cleanup columns (Qiagen) with Phosphate wash buffer (5mM K2HPO4, pH 8.0, 80%EtOH) and phosphate elution buffer (4mM K2HPO4, pH 8.5). Amino-allyl labeled cDNA was dried and resuspended in 0.1M sodium carbonate pH 9.3 and coupled with either Cy3 or Cy5 (Amersham Biosciences, Piscataway, N.J.). Uncoupled dye was removed by another purfication using the PCR cleanup kit. Concentration of cDNA and amount of incoporated dye was measured for each sample using a Nanodrop spectrophotometer (Ambion).
Hybridization protocol
Arrays were cross-linked by exposure to 600 mJ UV before blocking in 1% SDS, 5× SSC, and 1 mg/mL BSA at 42°C for 1 hour. After blocking, arrays were washed 2× 5 min in 0.1× SSC and 2× 30 s in H2O. Dried arrays were placed into hybridization cassettes (TeleChem International, Sunnyvale, Calif.) and the cDNA samples were suspended in 10 mM EDTA, denatured at 95°C for 5 min and then mixed with 40 uL of SlideHyb buffer 1 (Ambion) and loaded under a coverslip onto the array. Hybridizations were carried out at 47°C for 16–18 h. After hybridization, arrays were washed in 2× SSC, 0.5% SDS 37C for 5 min, followed by 2× 5 min in 0.1× SSC, 0.1% SDS 37°C, and then 2 × 2.5 min in room temperature 0.1× SSC. Arrays were scanned using an Axon 4000b scanner (Molecular Devices, Sunnyvale, Calif) and images were analyzed using GenePix 6.0 (Molecular Devices).
Scan protocol
Arrays were scanned using an Axon 4000b scanner (Molecular Devices, Sunnyvale, Calif) and images were analyzed using GenePix 6.0 (Molecular Devices).
Description
There are 6088 probes, spotted in duplicate, on the array that target three different E. coli genomes: serotype O157:H7 strains Sakai and EDL-933, serotype K12 MG 1655.
Data processing
Thirty-six dye-swap hybridizations were performed between four groups with six strains per group, according to a balanced double loop design. Strains were grouped based on cladestx profiles and each strain was considered an independent biological replicate of its group (n=6). The six strains from each group were randomly hybridized with six strains from every other group; each hybridization compared a pair of strains that differed in either clade or stx profile, or both. Subsequent to local Lowess normalization, averaging of replicate probes and log2 transformation, the microarray data were fitted to a 2-factor mixed ANOVA model (intensity = Array + Dye + Clade + Stx + Clade:Stx + Sample; where the biological replicate (Sample) and array effects were considered random effects, while all other effects were considered fixed effects), using the MAANOVA package (version 0.98-8) in R software (version 2.2.1). This model allows independent consideration of the effect of ‘Clade’ (clade divergence) and ‘Stx’ (stx type variation) parameters on differences in gene expression among O157:H7 strains, as well as their interaction (combined) effect (Clade:Stx). Overall differences in gene expression between groups were determined using the Fs test, followed by pair-wise contrasts to determine significant differential expression between each pair of groups. Subsequently, the Fs statistic was estimated for the ‘Clade’ effect to determine significant differences in gene expression between clades 8 (n=12) and 2 (n=12), regardless of stx profile. Similarly, the Fs statistic was estimated for the ‘Stx’ effect as well as the ‘Clade:Stx’ interaction effect to examine the combined effect of clade and stx type on differences in gene expression among O157:H7 strains. In other words, this analysis will determine whether the expression of any given gene among groups with different stx types is also dependent on clade. An interaction effect would be observed if expression estimates between strain groups clade8stx2 and clade2stx2 are different from expression estimates between strain groups clade8stx2,2c and clade2stx1,2. For every analysis, 1000 permutations of the data were performed to generate P values; estimates were considered significant if the P value was < 0.05 after adjusting for multiple comparisons.
