|
| Status |
Public on Jan 01, 2010 |
| Title |
reactor 1 (resp) vs reactor 2 (resp)_2514964100131a |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
L. plantarum Reactor 1 resp
|
| Organism |
Lactiplantibacillus plantarum |
| Characteristics |
strain: WCFS1
|
| Treatment protocol |
heme +K2
|
| Growth protocol |
non-pH controlled Batch cultivation MRS-broth
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Quenching Buffer 66.7 mM HEPES (pH 6.5) 60%Methanol Extraction Mixture (Prepared in a screw-cap tube) -500 µl phenol/Chloroform -30 µl10% SDS -30 µl 3 M NaAc (pH 5.2) -500 mg glass-beads (75-150 µm) -400 µl TE 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) 12.Continue with the High Pure RNA Isolation Kit from Roche (order # 1 828 665) 13.Incubate for 60 minutes with DnaseI, elute with 50 µl elution buffer 14.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 |
10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 3 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy3-label
|
| |
|
| Channel 2 |
| Source name |
L. plantarum Reactor 2 resp
|
| Organism |
Lactiplantibacillus plantarum |
| Characteristics |
strain: WCFS1
|
| Treatment protocol |
heme +K2
|
| Growth protocol |
non-pH controlled Batch cultivation MRS-broth
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Quenching Buffer 66.7 mM HEPES (pH 6.5) 60%Methanol Extraction Mixture (Prepared in a screw-cap tube) -500 µl phenol/Chloroform -30 µl10% SDS -30 µl 3 M NaAc (pH 5.2) -500 mg glass-beads (75-150 µm) -400 µl TE 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) 12.Continue with the High Pure RNA Isolation Kit from Roche (order # 1 828 665) 13.Incubate for 60 minutes with DnaseI, elute with 50 µl elution buffer 14.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 |
10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 3 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy3-label
|
| |
|
| |
| Hybridization protocol |
Agilent hybridization protocol for Two-color microarray-based Gene expression analysis. Version 5.5
|
| Scan protocol |
Scanned on an Agilent G2565AA scanner. Images were quantified using Agilent Feature Extraction Software (version A.7.5)
|
| Description |
reactor 1 (resp) vs reactor 2 (resp)
|
| Data processing |
1.Used script to split 2-array imagene file of Cy3 intensity into eight separate Cy3 files. 2.Used script to split 2-array imagene file of Cy5 intensity into eight 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). Values below 0 (higher background than foreground) were disregarded 5. LOWESS normalization was performed using the BASE plugin 6. Normalised data was further processed using an R package (limma) to get to sample to sample comparisons with p-values (false discovery rate)
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| |
|
| Submission date |
Oct 06, 2009 |
| Last update date |
Oct 07, 2009 |
| Contact name |
Michiel Wels |
| E-mail(s) |
michiel.wels@nizo.com
|
| Organization name |
NIZO food research
|
| Street address |
Kernhemseweg 2
|
| City |
Ede |
| ZIP/Postal code |
6718 ZB |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL9359 |
| Series (1) |
| GSE18432 |
Aerobic cultivation vs Respiratory conditions |
|
