|
| Status |
Public on Jun 09, 2017 |
| Title |
Singapore blood + bacteria fed replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Aedes aegypti midgut tissue
|
| Organism |
Aedes aegypti |
| Characteristics |
strain: Singapore
treatment: Blood fed with blood + bacteria
|
| Treatment protocol |
Rockefeller and Singapore Aedes aegypti mosquitoes were reared in parallel and provided 3% sucrose + antibiotics [penicillin (100U/ml), streptomycin (100ug/ml), and gentamycin (75ug/ml)] upon eclosion. Five to seven days post eclosion, females from each strain were either (1) maintained on 3% sucrose, (2) given a sterile blood meal or (3) given a blood meal containing a bacterial cocktail. Approximately 12 hours after blood feeding, 15 midguts from all six strain/treatment combinations were dissected in sterile 1X PBS and transferred immediately to TRIzol reagent.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol reagent according to the manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
Labeling was performed using the Two-Color Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the Two-Color Microarray-Based Gene Expression Analysis Protocol (Agilent Technologies). 200ng of RNA from each sample was used for the labeling reaction and labeled cRNA was purified using RNeasy columns (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
Reference pool
|
| Organism |
Aedes aegypti |
| Characteristics |
sample type: Pool of all midgut samples from experiment (200ng/sample)
|
| Treatment protocol |
Rockefeller and Singapore Aedes aegypti mosquitoes were reared in parallel and provided 3% sucrose + antibiotics [penicillin (100U/ml), streptomycin (100ug/ml), and gentamycin (75ug/ml)] upon eclosion. Five to seven days post eclosion, females from each strain were either (1) maintained on 3% sucrose, (2) given a sterile blood meal or (3) given a blood meal containing a bacterial cocktail. Approximately 12 hours after blood feeding, 15 midguts from all six strain/treatment combinations were dissected in sterile 1X PBS and transferred immediately to TRIzol reagent.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol reagent according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Labeling was performed using the Two-Color Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the Two-Color Microarray-Based Gene Expression Analysis Protocol (Agilent Technologies). 200ng of RNA from each sample was used for the labeling reaction and labeled cRNA was purified using RNeasy columns (Qiagen).
|
| |
|
| |
| Hybridization protocol |
All samples were co-hybridized to the same reference sample, which was formed by pooling 200ng of RNA from 54 midgut samples collected for this experiment. The 18 samples included above were among the 54 samples, which included additional strains not analyzed here. The additonal samples were included in the reference to allow for comparisons across future experiments including additional samples from the total group of 54. All six samples from the same biological replicate were hybridized to six arrays on the same slide, and a different slide was used for each replicate, resulting in a complete block design. Hybridization was performed according to Agilent’s Two-Color Microarray-Based Gene Expression Analysis Protocol.
|
| Scan protocol |
Feature extraction was performed using an Agilent Scanner and Agilent Feature Extraction Software
|
| Data processing |
Data were analyzed using limma in R. Spots with at least one flag were excluded from analysis (flagged cells are empty in spreadsheet). Background correction was performed using the normexp method. Signals were normalized first within array using loess within-array normalization and then between arrays using quantile normalization.
|
| |
|
| Submission date |
Jun 08, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
George Dimopoulos |
| E-mail(s) |
gdimopo1@jhu.edu
|
| Organization name |
Johns Hopkins School of Public Health
|
| Department |
Molecular Microbiology and Immunology
|
| Lab |
Dimopoulos Group
|
| Street address |
615 N Wolfe St.
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL20714 |
| Series (1) |
| GSE99799 |
Aedes aegypti response to bloodfeeding and bacterial ingestion in midgut tissues of females from two strains |
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