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| Status |
Public on Sep 30, 2011 |
| Title |
MOYO-R; 3 hr, replication 3 |
| Sample type |
RNA |
| |
|
| Source name |
MOYO-R; 3 hr
|
| Organism |
Aedes aegypti |
| Characteristics |
strain: MOYO-R
|
| Treatment protocol |
The starved females were provided with an artificial infectious blood meal, freshly prepared using defibrinated sheep blood (Colorado Serum Co., CS1122) mixed with (equal volume) a dengue viral suspension. DENV2 strain JAM1409 was cultured using C6/36 mosquito cells until 80-90% confluence in MEM-EBSS media (HyClone SH3002401) supplemented with 25 mM Hepes buffer, 1 mM sodium pyruvate, 0.025 mg/ml Gentamycin, 1x (0.01 mM) non-essential amino acids and 10% fetal bovine serum (heat inactivated). A 0.1 MOI (multiplicity of infection) was used for infecting the mosquito cells. The multiplicity of infection refers to the average number of viral particles that infect a single cell, which for our purpose, is equal to plaque-forming units (pfu) per cell. The flasks were incubated at 28°C for 7 days after which the viral supernatant was obtained by centrifugation at 1500 rpm for 5 min at 4ºC. All DENV infection work was performed in a BSL3 facility.
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| Growth protocol |
Fully engorged females were isolated and maintained at 26 °C with 84% humidity and in a 16-h light/8-h dark cycle. At 3 hr and 18 hr post blood feeding, DENV2 infected and control females were sampled for each strain. Three independent feeding experiments were done to obtain three biological replicates of samples for both the strains and the post-infection time points.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At the appropriate time point, RNA was extracted from 20 females per sample (the blood meal was removed from each female using a micro syringe needle prior to RNA extractions) using a Qiagen RNAeasy Kit as per manufacturer’s instructions. The RNA quality was assessed by a Bioanalyzer and quantified by a Nanodrop spectrophotometer.
|
| Label |
cy3
|
| Label protocol |
The cDNA labeling was performed by NimbelGene. The manufacturer protocol for labeling is as follows. First, the RNA samples are tested for quality control so that they are free of contamination and intact (not degraded). The RNA is then used to prepare first and second strand cDNA. The cDNA is cleaned up and checked for quality control. Then cy3 dye was used to label the cDNA as described in http://www.nimblegen.com/products/lit/exp_userguide_v3p2.pdf.
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| |
|
| Hybridization protocol |
The array hybridization and washing was performed by NimbleGen as per protocol described at http://www.nimblegen.com/products/lit/exp_userguide_v3p2.pdf
|
| Scan protocol |
Scanning was also performed by NimbleGen as described in http://www.nimblegen.com/products/lit/exp_userguide_v3p2.pdf
|
| Description |
Three independent feeding experiments were done to obtain three biological replicates of samples for both the strains and the post-infection time points. These consisted of four DENV2 infected samples (Moyo-S-3hr, Moyo-R-3hr, Moyo-S-18hr and Moyo-R-18hr, post-infection, respectively) and a control sample that consisted of RNA isolated at the same time points following uninfected blood meals and pooled across strains and time of sampling. Thus, a total of 15 independent samples were used for array hybridizations.
The Aedes aegypti strain MOYO-R was used. These mosquitoes show 19.56% susceptibility to dengue serotype-2. Early interactions between host and virus is important. So, we profiled the gene expression changes in dengue-2 infected MOYO-R females at 3 hr post infection. The control was prepared in parallel to the test. The control mosquitoes were mock transfected by normal blood meal without dengue.
|
| Data processing |
The raw data from array expressions were normalized using quintile normalization method and the Robust Multichip Average (RMA) algorithm by NimbleGen. The Statistical Analysis of Microarray (SAM) program was employed, using the Microsoft Excel addin v1.21, to determine differential expression between test and control samples. The control and infected samples were compared as two-class unpaired response types. The significant gene list was further subjected to filter for the genes that showed either positive (up-regulation) or negative (down-regulation) in both the Moyo-S and Moyo-R strains for both the post-infection time points. This step was performed because each of the test samples were compared to a common uninfected control (prepared by pooling equal amount of the individual control for these samples). This approach was adopted to ensure the identification of genes showing differential expression in response to DENV but not due to genetic differences between the two strains or developmental associated changes between the two observation time points.
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| |
|
| Submission date |
Jun 11, 2009 |
| Last update date |
Sep 30, 2011 |
| Contact name |
David W Severson |
| E-mail(s) |
severson.1@nd.edu
|
| Organization name |
University of Notre Dame
|
| Department |
Dept. of Biological Sciences
|
| Street address |
Galvin Life Science
|
| City |
NOTRE DAME |
| State/province |
IN |
| ZIP/Postal code |
46556 |
| Country |
USA |
| |
|
| Platform ID |
GPL8705 |
| Series (1) |
| GSE16563 |
MOYO-S and MOYO-R responsive genes to dengue-2 infection at 3hr and 18hr post-infection times |
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