|
| Status |
Public on Mar 01, 2018 |
| Title |
DMSO vs. Pyo, 8 h Timepoint, Replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Pooled A. coluzzii Ngousso third instar larvae
|
| Organism |
Anopheles coluzzii |
| Characteristics |
gender: mixed
strain: Ngousso
tissue: whole-body
developmental stage: third instar larvae
exposure time: 8 h
exposure compound: DMSO
exposure compound concentration: 50 µM Pyo equivalent volume
replicate: 2
|
| Treatment protocol |
Fifteen third instar larvae (ca. 6 days old) were incubated with 1-HP, Pyo (50 µM) or an equivalent volume of DMSO in single wells of 6-well tissue culture plates for 4 h or 8 h.
|
| Growth protocol |
Anopheles coluzzii Ngousso strain was maintained at 28 ± 2°C, 85 ± 5% humidity and 12 h day/night cycle. Larvae were reared in 0.1% NaCl/ddH2O and fed with generic cat food pellets.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with TRIzol (Life Technologies) according the supplier’s protocol using Glycogen RNA carrier. Quality control and quantification of total RNA was assessed using an Agilent 2100 Bioanalyzer with a RNA Nano 6000 microfluidics kit (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
RNA was labeled with a two-color Low Input Quick Amp Labeling Kit according the supplier’s recommendations (Agilent Technologies). In brief, mRNA was reverse transcribed and amplified using an oligo-dT-T7 primer, and labeled with Cyanine 3-CTP or Cyanine 5-CTP. After precipitation, purification, and quantification, 1.25 μg of each labeled cRNA was fragmented and hybridized to A. gambiae whole genome 4x44K multipack microarrays (Design ID 020449) according to the manufacturer’s protocol (Agilent Technologies).
|
| |
|
| Channel 2 |
| Source name |
Pooled A. coluzzii Ngousso third instar larvae
|
| Organism |
Anopheles coluzzii |
| Characteristics |
gender: mixed
strain: Ngousso
tissue: whole-body
developmental stage: third instar larvae
exposure time: 8 h
exposure compound: Pyo
exposure compound concentration: 50 µM
replicate: 2
|
| Treatment protocol |
Fifteen third instar larvae (ca. 6 days old) were incubated with 1-HP, Pyo (50 µM) or an equivalent volume of DMSO in single wells of 6-well tissue culture plates for 4 h or 8 h.
|
| Growth protocol |
Anopheles coluzzii Ngousso strain was maintained at 28 ± 2°C, 85 ± 5% humidity and 12 h day/night cycle. Larvae were reared in 0.1% NaCl/ddH2O and fed with generic cat food pellets.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with TRIzol (Life Technologies) according the supplier’s protocol using Glycogen RNA carrier. Quality control and quantification of total RNA was assessed using an Agilent 2100 Bioanalyzer with a RNA Nano 6000 microfluidics kit (Agilent Technologies).
|
| Label |
Cy5
|
| Label protocol |
RNA was labeled with a two-color Low Input Quick Amp Labeling Kit according the supplier’s recommendations (Agilent Technologies). In brief, mRNA was reverse transcribed and amplified using an oligo-dT-T7 primer, and labeled with Cyanine 3-CTP or Cyanine 5-CTP. After precipitation, purification, and quantification, 1.25 μg of each labeled cRNA was fragmented and hybridized to A. gambiae whole genome 4x44K multipack microarrays (Design ID 020449) according to the manufacturer’s protocol (Agilent Technologies).
|
| |
|
| |
| Hybridization protocol |
Microarray experiments were performed as dual-color hybridizations with color-swap dye-reversal.
|
| Scan protocol |
Scanning of microarrays was performed at 5 μm resolution using a G2565CA high-resolution laser microarray scanner (Agilent Technologies) with XDR extended range.
|
| Description |
Pyo vs. DMSO, 8 h Timepoint, Replicate 2. A. coluzzii Ngousso third instar larvae incubated with 50 µM Pyo compared to DMSO control. Color-swap dye-reversal.
|
| Data processing |
Microarray image data were analyzed and extracted with the Image Analysis/Feature Extraction software G2567AA v. A.11.5.1.1 (Agilent Technologies) using default settings.
|
| |
|
| Submission date |
Nov 14, 2016 |
| Last update date |
Mar 01, 2018 |
| Contact name |
Philip Adrian Huegli |
| E-mail(s) |
huegli@mpiib-berlin.mpg.de
|
| Organization name |
Max Planck Institute for Infection Biology
|
| Department |
Vector Biology
|
| Street address |
Charitéplatz 1
|
| City |
Berlin |
| State/province |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13155 |
| Series (1) |
| GSE89829 |
Anopheles coluzzii larval response to two purified Pseudomonas aeruginosa phenazine pigments 1-HP and Pyo. |
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