RNA samples were obtained from two biological replicates at time4h (pH= 5.8; ≈ 3.8 X 107 cfu/ml)
Extracted molecule
total RNA
Extraction protocol
One-hundred ml aliquots of L. acidophilus cells growing in SM were harvested by centrifugation for 10 min at 3,220 x g, and frozen immediately in a dry ice/ethanol bath (the total time of each sampling process was 5 min). RNA isolation was performed as described previously (Azcarate-Peril et al., 2005) with the following modifications: 5 ml Trizol® Reagent (Invitrogen, Carlsband, CA) was added to the cell pellets and they were homogenized in a Mini-Beadbeater-8 cell disruptor (Biospec Products, Bartlesville, OK) for seven 40-sec cycles (and chilled on ice for 40 sec between cycles), the phases were separated by centrifugation (3,220 x g, 20 min, 4º C). The aqueous phase was removed to a fresh tube and 2.1 ml of Trizol and 1 ml of chloroform were added. The mixture was vortexed for 15 s and centrifuged to separate the phases. The Trizol step was repeated twice and RNA was precipitated from the final aqueous phase by adding 1 volume of isopropanol, followed by incubation at room temperature for 10 min and centrifugation (11,600 x g 10 min, 4º C). Purity and quality of RNA samples were determined by electrophoresis on agarose gels. NanoDrop (ND-3300, NanoDrop Technologies, DE) was used as an UV/VIS spectrophotometer for quantification of the RNA samples.
Label
Cy3
Label protocol
Reverse transcription and labeling of identical amounts (20 µg) of DNAse treated (Invitrogen, Carlsband, CA) RNA was performed with the SuperScript™ Indirect cDNA Labeling System for DNA Microarrays (Invitrogen) according to the manufacturer’s directions. Coupling of the Cy3 and Cy5 dyes (Amersham Biosciences, Piscataway, NJ) to the AA-dUTP labeled cDNA and hybridization of samples to microarrays were performed according to the protocols outlined in the TIGR protocols website (www.tigr.org/tdb/microarray/protocolsTGR.shtml) and described previously (Azcarate-Peril et al., 2005).
RNA samples were obtained from two biological replicates at time 8h (pH= 5.4; ≈ 7.1 X 107 cfu/ml)
Extracted molecule
total RNA
Extraction protocol
One-hundred ml aliquots of L. acidophilus cells growing in SM were harvested by centrifugation for 10 min at 3,220 x g, and frozen immediately in a dry ice/ethanol bath (the total time of each sampling process was 5 min). RNA isolation was performed as described previously (Azcarate-Peril et al., 2005) with the following modifications: 5 ml Trizol® Reagent (Invitrogen, Carlsband, CA) was added to the cell pellets and they were homogenized in a Mini-Beadbeater-8 cell disruptor (Biospec Products, Bartlesville, OK) for seven 40-sec cycles (and chilled on ice for 40 sec between cycles), the phases were separated by centrifugation (3,220 x g, 20 min, 4º C). The aqueous phase was removed to a fresh tube and 2.1 ml of Trizol and 1 ml of chloroform were added. The mixture was vortexed for 15 s and centrifuged to separate the phases. The Trizol step was repeated twice and RNA was precipitated from the final aqueous phase by adding 1 volume of isopropanol, followed by incubation at room temperature for 10 min and centrifugation (11,600 x g 10 min, 4º C). Purity and quality of RNA samples were determined by electrophoresis on agarose gels. NanoDrop (ND-3300, NanoDrop Technologies, DE) was used as an UV/VIS spectrophotometer for quantification of the RNA samples.
Label
Cy5
Label protocol
Reverse transcription and labeling of identical amounts (20 µg) of DNAse treated (Invitrogen, Carlsband, CA) RNA was performed with the SuperScript™ Indirect cDNA Labeling System for DNA Microarrays (Invitrogen) according to the manufacturer’s directions. Coupling of the Cy3 and Cy5 dyes (Amersham Biosciences, Piscataway, NJ) to the AA-dUTP labeled cDNA and hybridization of samples to microarrays were performed according to the protocols outlined in the TIGR protocols website (www.tigr.org/tdb/microarray/protocolsTGR.shtml) and described previously (Azcarate-Peril et al., 2005).
Hybridization protocol
Coupling of the Cy3 and Cy5 dyes (Amersham Biosciences, Piscataway, NJ) to the AA-dUTP labeled cDNA and hybridization of samples to microarrays were performed according to the protocols outlined in the TIGR protocols website (www.tigr.org/tdb/microarray/protocolsTGR.shtml) and described previously (Azcarate-Peril et al., 2005).
Scan protocol
After acquisition of fluorescence intensities at 10-µm resolution using a ScanArray 4000 Microarray Scanner (Packard Biochip BioScience; Biochip Technologies LLC, Billerica, MA),
Description
signal intensities (stored as TIFF files) were quantified, and the local background was subtracted using the QuantArray 3.0 software package (Packard BioScience).
Data processing
data was log2 transformed and imported into JMP Genomics (SAS, Cary, NC). Loess normalization was applied to pre-processed data. Following normalization, gene-specific effects were modeled in terms of the residuals. A Mixed Model Analysis was applied to the pre-normalized data (Cui & Churchill, 2003). False discovery rate (FDR) was selected to adjust for the large number of multiple hypothesis tests across all genes. The default value of alpha (0.05) was used. The model-based approach, step-down quadratic regression method was applied to 791 significant genes for pattern recognition (Liu et al., 2005).
