|
| Status |
Public on Jan 27, 2014 |
| Title |
L. monocytogenes EGD, aerobic vs. anaerobic, biological rep. 6, technical rep. left |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
anaerobically grown cells
|
| Organism |
Listeria monocytogenes EGD |
| Characteristics |
od600 at harvest: 0.71
|
| Treatment protocol |
After reaching the final OD, cells were collected by centrifuging and the cell pellet was shock frozen immediately in liquid nitrogen. The cell pellet was stored at - 80 °C untill further processing.
|
| Growth protocol |
500 µl of an L. monocytogenes EGD overnight culure was used for inoculation of 50 ml BHI medium. The cultures were grown at 37 °C to an OD 600 of 0.70-0.75 either aerobically or anaerobically.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cell pellet was resuspendet in 1 ml TRIzol/TRI Reagent (Invitrogen/Sigma) and transferred into a 2 ml shredder-tube (Sarstedt) containing silic beads (diameter 0.1 mm). Solution was incubated at room temperature for 1 min, before cell walls were disrupted mechanically in a FastPrep cell disrupter. Silica beads were removed by centrifugation, RNA was extracted with chloroforme extraction, precipitated with isopropanol and the pellet was washed with 70 % EtOH. Residual DNA was removed by DNaseI digestion (Promega) according to manufacturer´s instructions. RNA was purified again follwoing the RNA clean-up protocol from the RNeasy Mini Kit (Quiagen), and an additional on column DNA digest was performed.
|
| Label |
Cy3
|
| Label protocol |
30 μg of bacterial RNA were mixed with the reverse transcription reaction mix (1 μl Superscript III Reverse Transcriptase (200 U/μl), 8 μl 5 x reaction buffer, 4 μl DTT (0.1 M) (Invitrogen), 3 µl random nonamer oligonucleotides (GE Healthcare), and 1 μl dNTP mix (20 mM dATP, 20 mM dGTP, 20 mM dTTP, and 16 mM dCTP (Fermentas)). Volume was adjusted to 38 μl with DEPC treated water, and 2 μl of a Cy3- or Cy5-labelled dCTP analogue were used for fluorescent labeling of the cDNA. Samples were incubated at 42 °C for 2 h. Then, 2 μl DNase-free RNase (> 30 U/mg, Roche Diagnostics) were added and the sample was incubated for 45 min at 37 °C and cDNA was purified using the QIAquick® PCR-Purification Kit (Qiagen) and eluted in 40 μl dH2O.
|
| |
|
| Channel 2 |
| Source name |
aerobically grown cells
|
| Organism |
Listeria monocytogenes EGD |
| Characteristics |
od600 at harvest: 0.72
|
| Treatment protocol |
After reaching the final OD, cells were collected by centrifuging and the cell pellet was shock frozen immediately in liquid nitrogen. The cell pellet was stored at - 80 °C untill further processing.
|
| Growth protocol |
500 µl of an L. monocytogenes EGD overnight culure was used for inoculation of 50 ml BHI medium. The cultures were grown at 37 °C to an OD 600 of 0.70-0.75 either aerobically or anaerobically.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cell pellet was resuspendet in 1 ml TRIzol/TRI Reagent (Invitrogen/Sigma) and transferred into a 2 ml shredder-tube (Sarstedt) containing silic beads (diameter 0.1 mm). Solution was incubated at room temperature for 1 min, before cell walls were disrupted mechanically in a FastPrep cell disrupter. Silica beads were removed by centrifugation, RNA was extracted with chloroforme extraction, precipitated with isopropanol and the pellet was washed with 70 % EtOH. Residual DNA was removed by DNaseI digestion (Promega) according to manufacturer´s instructions. RNA was purified again follwoing the RNA clean-up protocol from the RNeasy Mini Kit (Quiagen), and an additional on column DNA digest was performed.
|
| Label |
Cy5
|
| Label protocol |
30 μg of bacterial RNA were mixed with the reverse transcription reaction mix (1 μl Superscript III Reverse Transcriptase (200 U/μl), 8 μl 5 x reaction buffer, 4 μl DTT (0.1 M) (Invitrogen), 3 µl random nonamer oligonucleotides (GE Healthcare), and 1 μl dNTP mix (20 mM dATP, 20 mM dGTP, 20 mM dTTP, and 16 mM dCTP (Fermentas)). Volume was adjusted to 38 μl with DEPC treated water, and 2 μl of a Cy3- or Cy5-labelled dCTP analogue were used for fluorescent labeling of the cDNA. Samples were incubated at 42 °C for 2 h. Then, 2 μl DNase-free RNase (> 30 U/mg, Roche Diagnostics) were added and the sample was incubated for 45 min at 37 °C and cDNA was purified using the QIAquick® PCR-Purification Kit (Qiagen) and eluted in 40 μl dH2O.
|
| |
|
| |
| Hybridization protocol |
Differently labeled samples derived from one RNA set were combined. The volume was reduced to 30 μl in a SpeedVac Concentrater 5301 (Eppendorf). Then, 6 μl 20 x SSC and 4 μl 1 % SDS were added. Reaction mix was incubated 1 min at 94 °C and subsequently put on ice. Then, the reaction mix was applied to the microarray slide and the slide was covered with a cover slip. Hybridization was performed over night at 64 °C.
|
| Scan protocol |
Slides were scanned with the GenePix 4000B scanner (MDS Analytical Technologies) and data were analyzed via the GenePix 6.0 software (MDS Analytical Technologies).
|
| Description |
Sample 6a
|
| Data processing |
For normalization, the arithmetic mean of the ratios was set to one (GenePix Pro 6.0 software). In general, only spots with a fluorescence signal bigger than the local background plus one standard deviation were included in the final analysis. Spots with irregular morphology were in general excluded from the interpretation when the ratio of medians, the ratio of means and the regression ratio differed more than 30 %. To minimize the possibility of incorrect exclusion of spots due to automated interpretation, raw data of spots with a log2 ratio of medians ≥ 1 or ≤ -1 were further analyzed. If either the Cy5 or the Cy3 fluorescence intensity reached at least 50 % of the intensity averages of all spots of the respective array, the spot was included in the final analysis. As each experiment included technical duplicates, 12 data points were received for the calculation of the expression level of each gene. Genes were treated as up- or down-regulated, if (i) the log2 RTL was calculated based on at least 8 valid spots, each with a log2 ratio of medians (anaerobic/aerobic) ≤ -1.0 or ≥ 1.0 and (ii) the arithmetic mean of the log2 ratio of medians of valid spots was ≤ -1.0 or ≥ 1.0.
|
| |
|
| Submission date |
Nov 13, 2013 |
| Last update date |
Jan 27, 2014 |
| Contact name |
Stefanie Müller-Herbst |
| E-mail(s) |
[email protected]
|
| Organization name |
Technical University Munich
|
| Department |
ZIEL and Chair for Microbial Ecology
|
| Street address |
Weihenstephaner Berg 3
|
| City |
Freising |
| ZIP/Postal code |
85354 |
| Country |
Germany |
| |
|
| Platform ID |
GPL17774 |
| Series (1) |
| GSE52325 |
Transcriptional adaptation of L. monocytogenes EGD to oxygen availability |
|
