RNA was isolated from samples using the RNeasy Mini Kit (Qiagen). The lysis and digestion protocol was followed with two 50 μl ddH20 elutions. Each sample was treated with 2 μl of DNase (Ambion, Austin, TX) at 37°C for 30 min. Samples were purified and concentrated using Microcon YM-30 columns (Millipore, Billerica, MA), and the quantity and purity were determined using an ND-1000 spectrophotometer (Nanodrop, Wilmington, DE). Samples were determined to be free of contaminating genomic DNA by absence of a band on a DNA electrophoresis gel after 30 rounds of PCR.
Label
Cy 3
Label protocol
Targets were generated from 10 µg of DNase treated total RNA as follows. Ten µg of total RNA was combined with 1 μl of 10 µg/μl random hexamers (Integrated DNA Technologies) and ddH20 was added to bring the total reaction volume to 15.5 μl. Samples were incubated at 70°C for 10 min followed by 10 min incubation on ice. After incubation, 14.5 μl of a reaction mix (6 μl of 5x First Strand Buffer, 1.2 μl of 25x aa-dUTP/dNTP mix (Fermentas, Glen Burnie, MD), 3 μl 0.1M DTT, 2 μl Superscript III 200U/ μl (Invitrogen Corp., Carlsbad, CA), 2.4 μl RNase-free water) was added and reactions were incubated at 42°C for at least 2 hrs. After incubation, RNA was immediately hydrolyzed by incubation with 3 μl 0.1 M ETDA and 3.5 μl 0.1 M NaOH at 65°C for 10 min. The pH was neutralized with the addition of 36.5 μl of 500 mM HEPES (pH 7.0). Reactions were purified using the Mo Bio UltraClean PCR Clean-Up Kit (MO BIO Laboratories, Inc., Carlsbad, CA), quantified using the ND-1000 spectrophotometer, dried-down, and resuspended in 10 μl RNase-free water and 1.5 μl sodium bicarbonate (pH 8.7). After 10 min, 6 μl of Cy 3 (catalog number PA25001) or Cy 5 (catalog number PA23001) (GE Healthcare, Piscataway, NJ) were added according the microarray experimental design, and samples were incubated at room temperature for 3 h. Dyes were prepared by resuspending a single tube of dye in 72 μl of DMSO and storing in 6 μl aliquots at -70°C until use. Dye coupled reactions were purified in the same manner as cDNA synthesis reactions. Frequency of dye incorporation and yield was determined using the ND-1000 spectrophotometer.
RNA was isolated from samples using the RNeasy Mini Kit (Qiagen). The lysis and digestion protocol was followed with two 50 μl ddH20 elutions. Each sample was treated with 2 μl of DNase (Ambion, Austin, TX) at 37°C for 30 min. Samples were purified and concentrated using Microcon YM-30 columns (Millipore, Billerica, MA), and the quantity and purity were determined using an ND-1000 spectrophotometer (Nanodrop, Wilmington, DE). Samples were determined to be free of contaminating genomic DNA by absence of a band on a DNA electrophoresis gel after 30 rounds of PCR.
Label
Cy 5
Label protocol
Targets were generated from 10 µg of DNase treated total RNA as follows. Ten µg of total RNA was combined with 1 μl of 10 µg/μl random hexamers (Integrated DNA Technologies) and ddH20 was added to bring the total reaction volume to 15.5 μl. Samples were incubated at 70°C for 10 min followed by 10 min incubation on ice. After incubation, 14.5 μl of a reaction mix (6 μl of 5x First Strand Buffer, 1.2 μl of 25x aa-dUTP/dNTP mix (Fermentas, Glen Burnie, MD), 3 μl 0.1M DTT, 2 μl Superscript III 200U/ μl (Invitrogen Corp., Carlsbad, CA), 2.4 μl RNase-free water) was added and reactions were incubated at 42°C for at least 2 hrs. After incubation, RNA was immediately hydrolyzed by incubation with 3 μl 0.1 M ETDA and 3.5 μl 0.1 M NaOH at 65°C for 10 min. The pH was neutralized with the addition of 36.5 μl of 500 mM HEPES (pH 7.0). Reactions were purified using the Mo Bio UltraClean PCR Clean-Up Kit (MO BIO Laboratories, Inc., Carlsbad, CA), quantified using the ND-1000 spectrophotometer, dried-down, and resuspended in 10 μl RNase-free water and 1.5 μl sodium bicarbonate (pH 8.7). After 10 min, 6 μl of Cy 3 (catalog number PA25001) or Cy 5 (catalog number PA23001) (GE Healthcare, Piscataway, NJ) were added according the microarray experimental design, and samples were incubated at room temperature for 3 h. Dyes were prepared by resuspending a single tube of dye in 72 μl of DMSO and storing in 6 μl aliquots at -70°C until use. Dye coupled reactions were purified in the same manner as cDNA synthesis reactions. Frequency of dye incorporation and yield was determined using the ND-1000 spectrophotometer.
Hybridization protocol
From each paired sample, 1.5 μg of labeled cDNA was combined, dried-down, resuspended in 60 μl Pronto! cDNA/long oligonucleotide hybridization solution (Corning), incubated at 95°C for 5 min and centrifuged at 13,000 × g for 2 min at room temperature. This solution was then pipetted on a freshly pre-hybridized microarray slide, covered with a 22 x 60 mm HybriSlip (Grace Bio Labs, Bend, OR), placed in a Corning hybridization chamber, and incubated in a 42°C water bath for 12 to 16 h. Array pre-hybridization was conducted using sodium borohydride as previously described (Raghavachari, et al., 2003). Slides were washed according to Corning’s UltraGAPS protocol and dried by centrifugation.
Scan protocol
Scanning, image segmentation, and normalization were conducted as previously described (Madsen, et al., 2006).
Description
To better understand the impact of heat shock on E. coli O157:H7, global transcript levels of strain EDL933 cells shifted from 37°C to 50°C for 15 min were compared to cells left at 37°C using microarrays.
Data processing
Mixed model analysis was conducted as previously described (Madsen, et al., 2006) excluding slide region and slide-by-region interaction effects. The value column was not used for analysis or in publication. The normalized values for duplicate spots were averaged within each array to produce one normalized measure of expression for each of the probe sequences and each of the RNA samples. [mean log transformed median centered lowess normalized signal intensity]
