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Sample GSM286745 Query DataSets for GSM286745
Status Public on May 08, 2008
Title H2O2_Sig54vsSig54_B treatment
Sample type RNA
 
Channel 1
Source name Sig54_H2O2_B
Organism Lactiplantibacillus plantarum
Characteristics Sig54_H2O2_B
Extracted molecule total RNA
Extraction protocol Collecting cells Quenching Buffer 66.7 mM HEPES (pH 6.5) 60%Methanol Extraction Mixture (Prepared in a screw-cap tube) -500 µll phenol/Chloroform -30 µl10% SDS -30 µl3 M NaAc (pH 5.2) -500 mg glass-beads (75-150 µm) -400 µlTE buffer 1.For each sample prepare a tube with Extraction Mixture 2.Harvest cells and quench them immediately in Quenching Buffer. Add 4 volumes of Quenching Buffer ( 40°C) to 1 volume of cell culture 3.Mix and store the sample at -40°C 4.Centrifuge cells at -20 C at 9000 rpm for 10 minutes (Centrifuge RC5B) 5.Discard the supernatant; keep the cell pellet cooled and work quickly to prevent condensation of water. Transfer the pellet with a pre-cooled spatula to a screw-crap tube with Extraction Mixture 6.Mix the cell pellet thoroughly with Extraction Mixture by shaking by hand 7.Freeze the tube immediately in liquid nitrogen 8.Break cells in the Savant FastPrep FP120. Three times 40 seconds at speed 4.0 (cool cells between steps if necessary) 9.Centrifuge for 5 minutes at 4°C in an Eppendorf centrifuge at 10.000 rpm 10.Transfer 500 µl of the supernatant to a new tube. Add 400 µl chloroform (chloroform should be cooled at 4°C) 11.Centrifuge for 2 minutes in an Eppendorf centrifuge at full speed (4°C) 5.Continue with the "High Pure RNA Isolation Kit" from Roche (order # 1 828 665) 6.Incubate for 60 minutes with DnaseI, elute with 50 µl elution buffer 7.Store the RNA in at least 2 aliquots, one of 20 µl (for concentration and quality analysis and labelling) and one back-up of 30 µl at -80°C.
Label Cy3
Label protocol Annealing Mix x µl RNA preparation (=5µg) y µl nuclease-free water 1 µl Random Nonamers 11 µl TOTAL 1. mix Annealing Mix in a 200 µl PCR tube gently by pipetting up and down 2. incubate for 5’ 70°C 3. cool at room temperature for 10’ (annealing) 4. spin down the mixture to the bottom of the tube 5. place on ice Reverse transcription Reverse Transcription Mix 4 µl 5x buffer 2 µl 0.1 M DTT 1 µl dNTP-mix 1 µl AA-dUTP 1 µl TMIII (keep only briefly outside 20°C) 20 µl TOTAL 1. Mix by very gently by pipetting up and down (vigorous pipetting will denature the enzyme!) 2. Incubate for 3 hrs at 42°C (in PCR-machine) 3. Cool on ice for immediate purification or place at 20°C for storage Degradation of mRNA 1. Add 2 µl 2.5 M NaOH 2. Mix (vortex) and spin down 3. Incubate for 15 min. at 37°C 4. Add 10 µl 2 M HEPES free acid 5. Mix (vortex) and spin down 6. Ready for purification or store at 20°C Labelling and purification of cDNA with cyanine dyes Followed Ge Healthcare kit "Amersham CyDye (Cy3/Cy5)Reactive Dye Protocols for Post Labelling Aminoallyl-cDNA and Aminoallyl-aRNA" code RPN5661 Labelling of amino allyl-modified cDNA with CyDye 1. Add the amino allyl modified cDNA (in 0.1 M sodium bicarbonate) directly into one aliquot of CyDye NHS Ester. Resuspend the ester completely by pipetting several times and transfer the solution to an amber 1.5 ml tube 2. Incubate at room temperature, in the dark for 60 to 90 minutes 3. Add 15 µl 4 M Hydroxylamine to each coupling reaction 4. Mix by stirring and incubate at room temperature, in the dark, for 15 minutes 5. Proceed directly to purification of CyDye-labelled cDNA Purification of CyDye labelled cDNA Followed Ge Healthcare kit "Amersham CyDye (Cy3/Cy5)Reactive Dye Protocols for Post Labelling Aminoallyl-cDNA
 
Channel 2
Source name Sig54_B
Organism Lactiplantibacillus plantarum
Characteristics Sig54_B
Extracted molecule total RNA
Extraction protocol Collecting cells Quenching Buffer 66.7 mM HEPES (pH 6.5) 60%Methanol Extraction Mixture (Prepared in a screw-cap tube) -500 µll phenol/Chloroform -30 µl10% SDS -30 µl3 M NaAc (pH 5.2) -500 mg glass-beads (75-150 µm) -400 µlTE buffer 1.For each sample prepare a tube with Extraction Mixture 2.Harvest cells and quench them immediately in Quenching Buffer. Add 4 volumes of Quenching Buffer ( 40°C) to 1 volume of cell culture 3.Mix and store the sample at -40°C 4.Centrifuge cells at -20 C at 9000 rpm for 10 minutes (Centrifuge RC5B) 5.Discard the supernatant; keep the cell pellet cooled and work quickly to prevent condensation of water. Transfer the pellet with a pre-cooled spatula to a screw-crap tube with Extraction Mixture 6.Mix the cell pellet thoroughly with Extraction Mixture by shaking by hand 7.Freeze the tube immediately in liquid nitrogen 8.Break cells in the Savant FastPrep FP120. Three times 40 seconds at speed 4.0 (cool cells between steps if necessary) 9.Centrifuge for 5 minutes at 4°C in an Eppendorf centrifuge at 10.000 rpm 10.Transfer 500 µl of the supernatant to a new tube. Add 400 µl chloroform (chloroform should be cooled at 4°C) 11.Centrifuge for 2 minutes in an Eppendorf centrifuge at full speed (4°C) 5.Continue with the "High Pure RNA Isolation Kit" from Roche (order # 1 828 665) 6.Incubate for 60 minutes with DnaseI, elute with 50 µl elution buffer 7.Store the RNA in at least 2 aliquots, one of 20 µl (for concentration and quality analysis and labelling) and one back-up of 30 µl at -80°C.
Label Cy5
Label protocol Annealing Mix x µl RNA preparation (=5µg) y µl nuclease-free water 1 µl Random Nonamers 11 µl TOTAL 1. mix Annealing Mix in a 200 µl PCR tube gently by pipetting up and down 2. incubate for 5’ 70°C 3. cool at room temperature for 10’ (annealing) 4. spin down the mixture to the bottom of the tube 5. place on ice Reverse transcription Reverse Transcription Mix 4 µl 5x buffer 2 µl 0.1 M DTT 1 µl dNTP-mix 1 µl AA-dUTP 1 µl TMIII (keep only briefly outside 20°C) 20 µl TOTAL 1. Mix by very gently by pipetting up and down (vigorous pipetting will denature the enzyme!) 2. Incubate for 3 hrs at 42°C (in PCR-machine) 3. Cool on ice for immediate purification or place at 20°C for storage Degradation of mRNA 1. Add 2 µl 2.5 M NaOH 2. Mix (vortex) and spin down 3. Incubate for 15 min. at 37°C 4. Add 10 µl 2 M HEPES free acid 5. Mix (vortex) and spin down 6. Ready for purification or store at 20°C Labelling and purification of cDNA with cyanine dyes Followed Ge Healthcare kit "Amersham CyDye (Cy3/Cy5)Reactive Dye Protocols for Post Labelling Aminoallyl-cDNA and Aminoallyl-aRNA" code RPN5661 Labelling of amino allyl-modified cDNA with CyDye 1. Add the amino allyl modified cDNA (in 0.1 M sodium bicarbonate) directly into one aliquot of CyDye NHS Ester. Resuspend the ester completely by pipetting several times and transfer the solution to an amber 1.5 ml tube 2. Incubate at room temperature, in the dark for 60 to 90 minutes 3. Add 15 µl 4 M Hydroxylamine to each coupling reaction 4. Mix by stirring and incubate at room temperature, in the dark, for 15 minutes 5. Proceed directly to purification of CyDye-labelled cDNA Purification of CyDye labelled cDNA Followed Ge Healthcare kit "Amersham CyDye (Cy3/Cy5)Reactive Dye Protocols for Post Labelling Aminoallyl-cDNA
 
 
Hybridization protocol Two individual differentially labeled cDNAs were incubated at 95° C for 3' cooled down to 68° C and mixed (final-volume 20 µl). To these mixed cDNAs 180 µl of pre-heated (68° C) Slidehyb#1 hybridization buffer (Ambion Austin USA) was added and the resulting solution was applied on a pre-heated slide (68° C). Slides were then hybridized at 44° C for 16 hours. Subsequently slides were washed at 42°C once in 1 x SSC/0.2% SDS and twice in 1 x SSC and dried by centrifugation (1 x SSC is 0.15 M NaCl and 0.15 M Sodium Citrate).
Scan protocol Using the Scan Array Express microarray scanner. 1.Turn on the scanner and the computer (in this order) 2.Login: scanner 3.Double click the 'ScanArray Express' icon 4.Switch on lasers 1 and 3 at least 15 minutes prior to scanning arrays 5.Click the 'File' button This is to prevent you from accidentally turning off the laser after the 15 minutes warming-up phase 6.Insert the slide with the array-side up and the label towards the outside. On Agilent slides the array side is the side with the label that has the word Agilent"" on it 7.Press 'Scan | Prescan' 8.Put resolution at 50 m select both labels used and set PMT values for both channels 9.Press 'Start' 10.Press 'Palette ' 'Green' as soon as projection of the image has started (first dye) select the red colour as soon as scanning of the second layer (second dye) has started 11.Adjust PMT values to balance signals obtained for both channels. If necessary do a few low-resolution scans to obtain a optimal balance 12.Click the 'Scan' button again 13.Select frame for high resolution scan adjust resolution (to 10 m) and scan the array for both dyes 14.Press 'File | Save' to save the files 'Save all' saves both dye layers ," Create a new folder for each array 'Save all' saves the signals from both layers in individual files 15.Switch off the lasers
Description H2O2_Sig54vsSig54_B treatment mid exp Sigma54 KO 1 30 min, 3.5 mM H2O2
Data processing 1.-Used script to split 10-array imagene file of cy3 intensity into 10 separate cy3 files. 2.-Used script to split 10-array imagene file of cy5 intensity into 10 separate cy5 files. 3.-For each indidivual array a merge file was produced consisting of merging the cy3 and cy5 intensity files created in step 1 and 2. The merged file is provided as supplementary file for each array. 4.-Background correction (Mean values)
 
Submission date May 06, 2008
Last update date May 07, 2008
Contact name Douwe Molenaar
E-mail(s) douwe.molenaar@falw.vu.nl
Organization name Vrije Universiteit Amsterdam
Department Systems Bioinformatics
Street address De Boelelaan 1085
City Amsterdam
ZIP/Postal code 1081 HV
Country Netherlands
 
Platform ID GPL6368
Series (1)
GSE11351 Peroxide stress in Sigma54 mutant and Wt

Data table header descriptions
ID_REF Spot identifier, references platform spot identifier
SIGNAL_CH1
SIGNAL_CH2
VALUE Log2 of ratio (Channel1 normalized signal/Channel2 normalized signal)
F_CH1_MEAN Channel1 mean of spot pixel intensities
B_CH1_MEAN Channel1 mean of spot background pixel intensities
F_CH2_MEAN Channel2 mean of spot pixel intensities
B_CH2_MEAN Channel2 mean of spot background pixel intensities

Data table
ID_REF SIGNAL_CH1 SIGNAL_CH2 VALUE F_CH1_MEAN B_CH1_MEAN F_CH2_MEAN B_CH2_MEAN
1 7208.05 14970.4 -1.05443255 8000.13 792.073 15788.6 818.176
3 12436.9 17576 -0.49898066 13425.8 988.916 18761.9 1185.84
4 -5.9234 36.4361 665.863 671.786 750.468 714.032
5 2509.44 3156.52 -0.33097057 3220.63 711.186 3932.29 775.771
6 2729.84 4530.27 -0.73078044 3578.82 848.979 5395.06 864.789
7 660.485 775.35 -0.23132109 1367.03 706.542 1592.25 816.9
8 3455.85 5359.58 -0.63307971 4197.9 742.053 6194.68 835.096
9 191.33 223.187 -0.22218708 847.161 655.831 955.83 732.643
11 4326.11 15977.8 -1.88492519 5093.29 767.177 16881.8 904.011
12 1636.02 5544.31 -1.76081667 2335.36 699.336 6348.54 804.224
13 625.199 1919.81 -1.61857600 1314.94 689.745 2652.27 732.459
14 -8.01978 -4.6532 644.355 652.375 666.895 671.548
15 584.707 1122.1 -0.94041056 1234.18 649.474 1784.26 662.164
16 4926.86 5222.93 -0.08419291 5623.45 696.597 5911.48 688.548
17 17828.4 22665.7 -0.34633173 18577.8 749.383 23365.1 699.452
19 5393.21 12386.7 -1.19957525 6197.58 804.37 13214.9 828.229
20 7301.26 13799.8 -0.91842762 8079.3 778.047 14612.1 812.301
22 74.3748 68.0908 0.12735519 753.057 678.682 786.959 718.869
23 2009.66 5471.93 -1.44509978 2740.37 730.708 6277.81 805.877
24 2941.36 7307.13 -1.31282304 3656.76 715.401 8067.86 760.729

Total number of rows: 6114

Table truncated, full table size 379 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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