genotype/variation: wild type growth condition: Listeria monocytogenes 10403S grown at 37°C to Log phase (OD600 = 0.4) then exposed to heat (50°C for 10 min)
Extracted molecule
total RNA
Extraction protocol
RNA was isolated from frozen bacterial pellets with the Qiagen RNeasy Midi kit according to the manufacturer’s protocol for enzymatic and mechanical disruption, except that cells treated with lysozyme followed by sonication on ice for three 30 s bursts at 18–21 W (30 s pauses between bursts). RQ1 DNase treatment of total RNA in the presence of RNasin Plus RNA inhibitor (Promega) was performed according to the manufacturer’s protocol, followed by subsequent phenol:chloroform and chloroform extractions. RNA was ethanol-precipitated overnight, centrifuged and washed with 70% ethanol before resuspension of the final RNA pellet in DEPC-treated water. Total nucleic acid concentration and purity were estimated using absorbance readings (260/280 nm, 260/230 nm) on a NanoDrop ND-1000 spectrophotometer. RNA integrity was checked by agarose gel electrophoresis.
Label
Alexa Fluor 647,Alexa Fluor 555
Label protocol
cDNA was synthesized from purified total RNA and labeled with Alexa Fluor dyes using the SuperScript Plus Indirect cDNA Labeling System according to the manufacturer's protocol with following modifications. 10 μg total RNA was combined with 1 μl of random primers included in the kit, incubated at 70°C for 5 min. Following a 5 min on ice, 6 μl 5X First-Strand buffer, 1.5 μl 0.1M dithiothreitol (DTT), 1.5 μl dNTP mix containing aminoallyl-dATP and aminohexyl-dTTP, 1 μl RNaseOUT and 2 μl SuperScript III Reverse Transcriptase were added to the reaction tube. The reverse transcription reaction was carried out at 42ºC overnight. Remaining RNA was hydrolyzed by addition of 15 μl 1N NaOH (70ºC for 10 min) and then neutralized with 15 μl 1N HCl. Each cDNA sample was purified with the QIAquick PCR purification kit (Qiagen) using RNase-free water for the final elution step. cDNA samples were dried in a SpeedVac until reduced to ~3 μl. Each cDNA sample was combined with 2X Coupling Buffer and then added to a vial of DMSO-suspended Alexa Fluor® Reactive Dye and the dye coupling reaction was carried out at room temperature overnight. Labeled cDNA samples were purified with the QIAquick PCR purification kit with an additional 35% Guanidine-HCl wash step and a final elution step in RNase-free water. Purified, labeled cDNA was quantified with UV spectrophotometer readings at wavelengths appropriate to determine the frequency of incorporation (FOI), which is defined as the number of labeled nucleotides incorporated per 1,000 nucleotides of cDNA. cDNA samples with FOIs between 20-50 were used for microarray hybridization.
genotype/variation: C3-113 (sigC null mutant) growth condition: Listeria monocytogenes C3-113 grown at 37°C to Log phase (OD600 = 0.4) then exposed to heat (50°C for 10 min)
Extracted molecule
total RNA
Extraction protocol
RNA was isolated from frozen bacterial pellets with the Qiagen RNeasy Midi kit according to the manufacturer’s protocol for enzymatic and mechanical disruption, except that cells treated with lysozyme followed by sonication on ice for three 30 s bursts at 18–21 W (30 s pauses between bursts). RQ1 DNase treatment of total RNA in the presence of RNasin Plus RNA inhibitor (Promega) was performed according to the manufacturer’s protocol, followed by subsequent phenol:chloroform and chloroform extractions. RNA was ethanol-precipitated overnight, centrifuged and washed with 70% ethanol before resuspension of the final RNA pellet in DEPC-treated water. Total nucleic acid concentration and purity were estimated using absorbance readings (260/280 nm, 260/230 nm) on a NanoDrop ND-1000 spectrophotometer. RNA integrity was checked by agarose gel electrophoresis.
Label
Alexa Fluor 647,Alexa Fluor 555
Label protocol
cDNA was synthesized from purified total RNA and labeled with Alexa Fluor dyes using the SuperScript Plus Indirect cDNA Labeling System according to the manufacturer's protocol with following modifications. 10 μg total RNA was combined with 1 μl of random primers included in the kit, incubated at 70°C for 5 min. Following a 5 min on ice, 6 μl 5X First-Strand buffer, 1.5 μl 0.1M dithiothreitol (DTT), 1.5 μl dNTP mix containing aminoallyl-dATP and aminohexyl-dTTP, 1 μl RNaseOUT and 2 μl SuperScript III Reverse Transcriptase were added to the reaction tube. The reverse transcription reaction was carried out at 42ºC overnight. Remaining RNA was hydrolyzed by addition of 15 μl 1N NaOH (70ºC for 10 min) and then neutralized with 15 μl 1N HCl. Each cDNA sample was purified with the QIAquick PCR purification kit (Qiagen) using RNase-free water for the final elution step. cDNA samples were dried in a SpeedVac until reduced to ~3 μl. Each cDNA sample was combined with 2X Coupling Buffer and then added to a vial of DMSO-suspended Alexa Fluor® Reactive Dye and the dye coupling reaction was carried out at room temperature overnight. Labeled cDNA samples were purified with the QIAquick PCR purification kit with an additional 35% Guanidine-HCl wash step and a final elution step in RNase-free water. Purified, labeled cDNA was quantified with UV spectrophotometer readings at wavelengths appropriate to determine the frequency of incorporation (FOI), which is defined as the number of labeled nucleotides incorporated per 1,000 nucleotides of cDNA. cDNA samples with FOIs between 20-50 were used for microarray hybridization.
Hybridization protocol
Microarray slide blocking was performed prior to hybridization. Slides were incubated for 10 min in a solution containing methyl pyrrilidone, succinic anhydride and sodium borate, and then washed in fresh methyl pyrrilidone for 10 min. Successive 1-min washes in nanopure water (once), 0.1% SDS (3 times), nanopure water (5 times), and 95% EtOH (once). The slides were dried by centrifugation and immersed for 1 h in blocking solution (0.1% BSA, 5X SSC, 0.1% SDS) at 42°C. After blocking, the slides were washed for 1 min each in nanopure water (5 times) and 95% EtOH (2 times) and then dried by centrifugation. For the strains to be compared, labeled cDNAs were combined and dried, then resuspended in 50μl of hybridization buffer (5X SSC, 0.1% SDS, 0.1mM dithiothreitol (DTT), 0.5X formamide, 0.06 μg/μl salmon sperm DNA in DEPC-treated water), denatured at 95°C for 5 min then applied to the slides using mSeries LifterSlips™ (Erie Scientific). Following overnight hybridization at 42°C, the slides were washed in 2X SSC+0.1% SDS (5 min at 42˚C), with subsequent 5-min washes at room temperature in 2X SSC+0.1% SDS, 2X SSC, and 0.2X SSC, followed by final rinse in nanopure water. Slides were dried by centrifugation.
Scan protocol
Images were scanned using a GenePix 4000B scanner (Molecular Devices Corp.). Raw TIFF images were automatically gridded and analyzed using GenePix Pro 6.0 software.
Description
Expression profiles were measured from three independent RNA extractions, resulting in six replicate spots per gene (array contains duplicate spots).
Data processing
Analyses of microarray data were performed in R with Bioconductor using LIMMA software (Smyth, 2005). Background correction was performed using the “normexp” method. For normalization within arrays, the data were weighted for the genomic DNA controls and normalized using print-tip loess method (Smyth & Speed, 2003). The data were then scale normalized between arrays. The correlation between duplicate spots was calculated (Smyth et al., 2005) and a linear model was fitted to the normalized log ratios and empirical Bayes statistics were applied for differential expression analysis.
