|
| Status |
Public on Dec 31, 2011 |
| Title |
10dpiP1_10dpiC4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
A. gambiae P. breghei infected vs uninfected control 10 days post infection
|
| Organism |
Anopheles gambiae |
| Characteristics |
tissue: A. gambiae mosquitoes
treatment: infected with P. berghei
time: 10 days post infection
|
| Growth protocol |
Mosquitoes were infected with Plasmodium berghei NK65 redstar by feeding on infeced mice. Control mosquitoes were fed on clean mice. Fed mosquitoes were kept at 21 degree since blood meal, and unfed ones were discarded. 10 and 17 days post blood feeding, mosquitoes were harvested and total RNA were isolated.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the Rneasy Mini Kit (Qiagen) following the manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
50ng of isolated total RNA per sample (QCed for A260/280 using NanoDrop and integrity using Bio Analyzer) were reverse transcribed using T7 RNA polymerase and labeled with either Cy3 or Cy5 (dependig on channel - see sample descriptions for details) using the Agilent Low Input Quick Amp labeling procedure, following the manufacturer's instructions (Version 6.5, May 2010).
|
| |
|
| Channel 2 |
| Source name |
A. gambiae mosquitoes
|
| Organism |
Anopheles gambiae |
| Characteristics |
tissue: A. gambiae mosquitoes
treatment: uninfected controls
|
| Growth protocol |
Mosquitoes were infected with Plasmodium berghei NK65 redstar by feeding on infeced mice. Control mosquitoes were fed on clean mice. Fed mosquitoes were kept at 21 degree since blood meal, and unfed ones were discarded. 10 and 17 days post blood feeding, mosquitoes were harvested and total RNA were isolated.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the Rneasy Mini Kit (Qiagen) following the manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
50ng of isolated total RNA per sample (QCed for A260/280 using NanoDrop and integrity using Bio Analyzer) were reverse transcribed using T7 RNA polymerase and labeled with either Cy3 or Cy5 (dependig on channel - see sample descriptions for details) using the Agilent Low Input Quick Amp labeling procedure, following the manufacturer's instructions (Version 6.5, May 2010).
|
| |
|
| |
| Hybridization protocol |
Equal amounts of linearily amplified, Cy3/Cy5-labeled were fragmented by incubating the samples in fragmentation buffer for exactly 30 minutes in a water bath at 60C; fragmented cRNA were combined in 2x GEx hybridization buffer; hybridization solution was immediately loaded onto arrays and hybridized in a oven set to 65C rotating at 10rpm for 17h. Prior to scanning arrays were washed according go manufacturer's instructions (Agilent Low Input Quick Amp Labeling protocol Version 6.5 May 2010)
|
| Scan protocol |
Scanned with Agilent G2505C Microarray Scanner
The information about each probe on the array was extracted from the image data using Agilent Feature Extraction 10.9 (FE)
|
| Description |
Biological replicate 4 of 4; 10 mosquitoes per infected (visually confirmed by fluorescent microscopy) and uninfected sample used for extraction of total RNA
|
| Data processing |
Intensities were not background corrected. The median red (Cy5) and green (Cy3) intensities are used to calculate the ratio of expression for each element on the array, in terms of M (log2(Red/Green)) and A ((log2(Red) + log2(Green))/2)). The M values are then global loess normalized within each array using the LIMMA microarray processing package in “R” (Smyth, 2004).
|
| |
|
| Submission date |
Sep 18, 2011 |
| Last update date |
Dec 31, 2011 |
| Contact name |
Sebastian Kurscheid |
| E-mail(s) |
sebastian.kurscheid@anu.edu.au
|
| Organization name |
Australian National University
|
| Department |
JCSMR
|
| Lab |
Prof David Tremethick
|
| Street address |
57 Garran Road
|
| City |
Acton |
| State/province |
ACT |
| ZIP/Postal code |
2601 |
| Country |
Australia |
| |
|
| Platform ID |
GPL13157 |
| Series (1) |
| GSE32200 |
Effects of Plasmodium berghei infection on Anopheles gambiae during oocysts development |
|
