|
| Status |
Public on Nov 11, 2019 |
| Title |
Resveratrol 10 min replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Treated cells: Experimental
|
| Organism |
Lactiplantibacillus plantarum |
| Characteristics |
strain: WCFS1
compound: Resveratrol 3mM
|
| Treatment protocol |
Cell cultures were centrifuged after 10 minutes of exposure to resveratrol and RNA was extracted and purified.
|
| Growth protocol |
Paired independent L. plantarum batch cultures (50 mL each) were grown in MRS lacking resveratrol to an OD600 ≈ 0.8-0.9, then one pair of the culture were exposed to resveratrol to bring their final concentration to 3 mM. Cell cultures were centrifuged after 10 minutes of exposure to resveratrol and RNA was extracted and purified.
|
| 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.
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| |
|
| Channel 2 |
| Source name |
Untreated cells: Control
|
| Organism |
Lactiplantibacillus plantarum |
| Characteristics |
strain: WCFS1
compound: without resveratrol
|
| Treatment protocol |
Cell cultures were centrifuged after 10 minutes of exposure to resveratrol and RNA was extracted and purified.
|
| Growth protocol |
Paired independent L. plantarum batch cultures (50 mL each) were grown in MRS lacking resveratrol to an OD600 ≈ 0.8-0.9, then one pair of the culture were exposed to resveratrol to bring their final concentration to 3 mM. Cell cultures were centrifuged after 10 minutes of exposure to resveratrol and RNA was extracted and purified.
|
| 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.
|
| Scan protocol |
Images were captured with a Agilent Technologies Scanner G2505C / GenePix 4000B (Axon) and spots quantified using GenPix software (Axon).
|
| Description |
Biological replicate 2 of 3
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).
|
| Data processing |
Background correction and normalization of expression data were performed using the methods normexp and loess in LIMMA. Linear model methods were used for determining differentially expressed genes. Each probe was tested for changes in expression over replicates by using an empirical Bayes moderated t-statistic. To control the false discovery rate p-values were corrected by using the method of Benjamani and Hochberg. 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. FC was calculated as the average of the fold change between significantly regulated probes.
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| |
|
| Submission date |
Nov 12, 2016 |
| Last update date |
Nov 11, 2019 |
| Contact name |
Felix Lopez de Felipe |
| E-mail(s) |
fxlopez@ictan.csic.es
|
| Organization name |
ICTAN-CSIC
|
| Street address |
Jose Antonio Novais
|
| City |
Madrid |
| ZIP/Postal code |
28040 |
| Country |
Spain |
| |
|
| Platform ID |
GPL22663 |
| Series (1) |
| GSE89785 |
Transcriptional profile Lactobacillus plantarum WCFS1 : Resveratrol treated cells |
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