strain: WCFS1 compound: Olive oil 50% compound: without olive oil
Treatment protocol
Washed cells were suspended in 0.5 mL of 100 mM phosphate buffer pH 7.0 and then exposed to 0.5 mL of olive oil (final olive oil concentration 50 %) by vortex-shaking during 10 minutes. After exposure cells were centrifuged and RNA extracted and purified.
Growth protocol
Paired independent L. plantarum batch cultures (50 mL each) were grown in MRS to an OD600 ≈ 0.8-0.9, then one pair of the culture was centrifuged at 9,000 x g for 5 min at 20 ºC and cells washed twice in 100 mM phosphate buffer pH 7.0.
Extracted molecule
total RNA
Extraction protocol
For RNA extraction the celular pellet was mixed with 2 mL of quenching buffer (60% methanol, 66.7 mM HEPES, pH 6.5, -40 ºC) . Following quenching, the cells were pelleted by centrifugation at 9,000 x g for 10 min at -10 ºC, transferred to a screw cap tube containing an extraction mixture (500 uL 1:4 chloroform-acid phenol, 30uL of 10% SDS, 30uL Na-acetate 3M pH 5.2, 400uL Tris-EDTA buffer [10 mM Tris(hydroxymethyl)amino methane, 1 mM EDTA] pH 7.4, 15 mg of polyvinylpoly-pyrrolidone, and 500 mg of 75-150um glass beads). The cells were broken under frozen conditions in the Mini-BeadBeather (BIOSPEC Products, INC.) using three treatments of 4600 rpm for 40 seconds cycles (and chilled on dry ice for 60 seconds between cycles). The suspension was then centrifuged at 4 ºC at 10,000 x g for 2 min. Two extractions with 500 uL of chloroform were performed, and the supernatant containing the RNA was separated by centrifugation. The sample was immediately frozen in liquid nitrogen, and stored at -80 ºC. NanoDrop ND-1000 instrument was used for quantification of RNA. Continued with the QUIAGEN RNA isolation kit. Two treatments with DNase I (Ambion) were applied and the absence of genomic DNA was confirmed by PCR.
Label
Cy3
Label protocol
Fluorescently labelled cDNA for microarray hybridizations was obtained by using the SuperScript Indirect cDNA Labeling System (Invitrogen): 20 ug of total RNA was transformed to cDNA with SuperscriptTM III Reverse Transcriptase using anchored oligo(dT)20 primers, and including aminoallyl-modified nucleotides. After cDNA purification, the Cy3 and HyPer5 fluorescent dyes (Amersham Biosciences) were coupled to the aminoallyl-modified first-strand cDNA. Purification of Cy3- and HyPer5-labeled cDNA probes was carried out with the CyScribe GFX Purification Kit.
Washed cells were suspended in 0.5 mL of 100 mM phosphate buffer pH 7.0 and then exposed to 0.5 mL of olive oil (final olive oil concentration 50 %) by vortex-shaking during 10 minutes. After exposure cells were centrifuged and RNA extracted and purified.
Growth protocol
Paired independent L. plantarum batch cultures (50 mL each) were grown in MRS to an OD600 ≈ 0.8-0.9, then one pair of the culture was centrifuged at 9,000 x g for 5 min at 20 ºC and cells washed twice in 100 mM phosphate buffer pH 7.0.
Extracted molecule
total RNA
Extraction protocol
For RNA extraction the celular pellet was mixed with 2 mL of quenching buffer (60% methanol, 66.7 mM HEPES, pH 6.5, -40 ºC) . Following quenching, the cells were pelleted by centrifugation at 9,000 x g for 10 min at -10 ºC, transferred to a screw cap tube containing an extraction mixture (500 uL 1:4 chloroform-acid phenol, 30uL of 10% SDS, 30uL Na-acetate 3M pH 5.2, 400uL Tris-EDTA buffer [10 mM Tris(hydroxymethyl)amino methane, 1 mM EDTA] pH 7.4, 15 mg of polyvinylpoly-pyrrolidone, and 500 mg of 75-150um glass beads). The cells were broken under frozen conditions in the Mini-BeadBeather (BIOSPEC Products, INC.) using three treatments of 4600 rpm for 40 seconds cycles (and chilled on dry ice for 60 seconds between cycles). The suspension was then centrifuged at 4 ºC at 10,000 x g for 2 min. Two extractions with 500 uL of chloroform were performed, and the supernatant containing the RNA was separated by centrifugation. The sample was immediately frozen in liquid nitrogen, and stored at -80 ºC. NanoDrop ND-1000 instrument was used for quantification of RNA. Continued with the QUIAGEN RNA isolation kit. Two treatments with DNase I (Ambion) were applied and the absence of genomic DNA was confirmed by PCR.
Label
HyPer5
Label protocol
Fluorescently labelled cDNA for microarray hybridizations was obtained by using the SuperScript Indirect cDNA Labeling System (Invitrogen): 20 ug of total RNA was transformed to cDNA with SuperscriptTM III Reverse Transcriptase using anchored oligo(dT)20 primers, and including aminoallyl-modified nucleotides. After cDNA purification, the Cy3 and HyPer5 fluorescent dyes (Amersham Biosciences) were coupled to the aminoallyl-modified first-strand cDNA. Purification of Cy3- and HyPer5-labeled cDNA probes was carried out with the CyScribe GFX Purification Kit.
Hybridization protocol
Preparation of probes and hybridization was performed as described on the Two-Color Microarray Based Gene Expression Analysis Manual (Quick Amp Labeling) with Tecan HS Pro Hybridization (Ver. 5.7/Agilent Technologies): for each hybridization 300 ng of Cy3 and HyPer5 probes were added to 5 uL of 10x Blocking Agent, 1.2 uL of 25x Fragmentation Buffer and nuclease-free water to bring volume to 25 uL reaction and incubated at 60 ºC for 30 min. Then, 25 uL the 2x GEx Hybridization Buffer HI-RPM were added and loaded onto the arrays. The arrays were hybridized for 17 hours at 65ºC, and washed in GE Wash Buffer 1 and then in GE Wash Buffer 2. Finally, these were drained by centrifugation and scanned. Slide L. plantarum WCFS1 8x15K microarray GE Agilent G2509F Oligo Microarrays No. 026636 was custom designed and contains 60-mer probes that were taken at the gene expression omnibus database (GEO accession number GLP5874). The oligo-microarray contained an average of three probes per transcript. Each slide contains 8 arrays, these are numbered as follows: 1_1 (1st column upper row), 1_2 (2nd column upper row), 1_3 (3rd column upper row), 1_4 (4th column upper row), 2_1 (1st column lower row),2_2 (2nd column lower row), 2_3 (3rd column lower row), 2_4 (4th column lower row).
Scan protocol
Images from Cy3 and HyPer5 channels were captured with a GenePix 4000B (Axon) and spots quantified using GenPix software (Axon).
Description
Biological replicate 3 of 3
Data processing
Background correction and normalization of expression data were performed using the methods normexp and loess in LIMMA, respectively. The ratio of each probe (M) was defined as log [Cy3 intensity/HyPer5 intensity] where HyPer5 is the wild type. Fold change (FC) is defined as 2M. Each probe was tested for changes in expression over replicates by using an empirical Bayes moderated t-statistic. The expected False Discovery Rate (FDR) was controlled to be less than 5%. Genes were considered differentially expressed when nominal p-values were <0.05 and had a fold change (FC) equal or higher than ±1.5
