|
| Status |
Public on Jun 21, 2017 |
| Title |
Roemerine_60min_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
(-)-Roemerine treatment, 60 min, replicate 2
|
| Organism |
Escherichia coli |
| Characteristics |
strain: TB1
|
| Treatment protocol |
When OD600 of the culture reached 0.54±0.06, cells were treated with 100 µg/ml of (-)-Roemerine. Since the alkaloid was dissolved in DMSO, control cells were also treated with the same amount of DMSO.
|
| Growth protocol |
The pre-culture was the overnight propagated cells in 5 ml of LB medium at 37ºC and 180 rpm. The main bacterial culture was prepared by 1% inoculation from pre-culture when optical densities at 600 nm (i.e., OD600) reached 0.7. Growth was achieved in 50 ml of LB medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction was carried out based on the protocols given in the manual of QIAGEN - RNAprotect Bacteria Reagent Handbook, QIAGEN - RNeasy Mini Kit and QIAGEN - RNase-free DNase set. The concentration of each RNA sample was measured by Qubit® RNA BR Assay Kit (Invitrogen).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). cRNA was quantitated using NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1.
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| |
|
| Hybridization protocol |
10X Blocking Agent supplied with the Agilent Gene Expression Hybridization Kit and 2x GEx Hybridization buffer HI-RPM were used in preparation of hybridization assembly by following manufacturers instructions.Hybridization was carried out at 65oC for 17 hours. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent C Scanner using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 3 µg, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after 60 min. in Roemerine treated E.coli cells
|
| Data processing |
Raw Microarray data evaluation including preprocessing, normalization, grouping the differentially expressed genes and statistical analyses with Moderated T-test and Bonferroni FWER was carried out with GeneSpring 13.0 Software.
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| |
|
| Submission date |
Apr 29, 2016 |
| Last update date |
Jun 21, 2017 |
| Contact name |
Dilara Ayyıldız |
| E-mail(s) |
dilarayyildiz@gmail.com
|
| Phone |
+39 366 8933431
|
| Organization name |
University of Udine
|
| Street address |
p.le kolbe, 4
|
| City |
Udine |
| ZIP/Postal code |
33100 |
| Country |
Italy |
| |
|
| Platform ID |
GPL13359 |
| Series (1) |
| GSE80827 |
Transcriptional Changes in Escherichia coli Upon Treatment with (-)-Roemerine |
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