|
| Status |
Public on Jul 01, 2013 |
| Title |
noLac_vs_DL-Lac |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
TF101 no lactate added
|
| Organism |
Lactiplantibacillus plantarum |
| Characteristics |
strain: NCIMB8826
treatment: control
|
| Treatment protocol |
Cells were harvested by centrifugation (5,000xg, 10 minutes). Cell pellets were stored at -20°C until extraction.
|
| Growth protocol |
A mutant of L. plantarum NCIMB8826 deficient for NAD-dependent L-lactate activity (TF101; Ferain et al. 1994. 176:596), and thus producing no L-lactate, was grown in MRS medium at 28°C until mid-exponential phase (OD600nm 0.75). The culture was then divided into 3 sub-cultures. Optically pure sodium L-lactate (200 mM) was added to the first sub-culture (TF101 + L-lac 200 mM). An equimolar mixture of sodium D- and L-lactate (100 mM each) was added to the second sub-culture (TF101 + L/D-lac 200 mM). The third sub-culture was not treated (TF101). The three sub-cultures were further incubated at 28°C for 1h30 (a time known to be sufficient for induction of lactate racemase activity by L-lactate).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1.Resuspend the frozen cell pellet thoroughly in TE buffer 2. Aliquot the resuspended cell pellet (400 µl per tube) in screw-cap tubes containing 500 µl phenol/Chloroform -30 µl10% SDS -30 µl 3 M NaAc (pH 5.2) -500 mg glass-beads (75-150 µm) 3.Freeze the tube immediately in liquid nitrogen 4.Break cells in the Savant FastPrep FP120. Three times 40 seconds at speed 4.0 (cool cells between steps if necessary) 5.Centrifuge for 5 minutes at 4°C in an Eppendorf centrifuge at 10.000 rpm 6.Transfer 500 µl of the supernatant to a new tube. Add 400 µl chloroform (chloroform should be cooled at 4°C) 7.Centrifuge for 2 minutes in an Eppendorf centrifuge at full speed (4°C) 8.Continue with the High Pure RNA Isolation Kit from Roche (order # 1 828 665) 9.Incubate for 60 minutes with DnaseI, elute with 50 µl elution buffer 10.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 |
TF101 200 mM DL-lactate added
|
| Organism |
Lactiplantibacillus plantarum |
| Characteristics |
strain: NCIMB8826
treatment: L/D lac 200 mM
|
| Treatment protocol |
Cells were harvested by centrifugation (5,000xg, 10 minutes). Cell pellets were stored at -20°C until extraction.
|
| Growth protocol |
A mutant of L. plantarum NCIMB8826 deficient for NAD-dependent L-lactate activity (TF101; Ferain et al. 1994. 176:596), and thus producing no L-lactate, was grown in MRS medium at 28°C until mid-exponential phase (OD600nm 0.75). The culture was then divided into 3 sub-cultures. Optically pure sodium L-lactate (200 mM) was added to the first sub-culture (TF101 + L-lac 200 mM). An equimolar mixture of sodium D- and L-lactate (100 mM each) was added to the second sub-culture (TF101 + L/D-lac 200 mM). The third sub-culture was not treated (TF101). The three sub-cultures were further incubated at 28°C for 1h30 (a time known to be sufficient for induction of lactate racemase activity by L-lactate).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1.Resuspend the frozen cell pellet thoroughly in TE buffer 2. Aliquot the resuspended cell pellet (400 µl per tube) in screw-cap tubes containing 500 µl phenol/Chloroform -30 µl10% SDS -30 µl 3 M NaAc (pH 5.2) -500 mg glass-beads (75-150 µm) 3.Freeze the tube immediately in liquid nitrogen 4.Break cells in the Savant FastPrep FP120. Three times 40 seconds at speed 4.0 (cool cells between steps if necessary) 5.Centrifuge for 5 minutes at 4°C in an Eppendorf centrifuge at 10.000 rpm 6.Transfer 500 µl of the supernatant to a new tube. Add 400 µl chloroform (chloroform should be cooled at 4°C) 7.Centrifuge for 2 minutes in an Eppendorf centrifuge at full speed (4°C) 8.Continue with the High Pure RNA Isolation Kit from Roche (order # 1 828 665) 9.Incubate for 60 minutes with DnaseI, elute with 50 µl elution buffer 10.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)
|
| Data processing |
1.Used script to split 8-array imagene file of Cy3 intensity into eight separate Cy3 files. 2.Used script to split 8-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)
|
| |
|
| Submission date |
Jan 15, 2013 |
| Last update date |
Jul 01, 2013 |
| 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 |
GPL4318 |
| Series (1) |
| GSE43518 |
Identification of genes regulated by L-lactate and by DL-lactate in Lactobacillus plantarum |
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