|
| Status |
Public on Mar 22, 2017 |
| Title |
Untreated vs toxin treated midgut 12hr 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Midgut untreated control at 12 hr
|
| Organism |
Culex quinquefasciatus |
| Characteristics |
developmental stage: larvae
treatment: control
time: 12 hr
tissue: Midgut
|
| Treatment protocol |
The third-instar C. quinquefasciatus larvae were starved for 3 h before feeding with the mixture of BinA and BinB at a 1:1 molar ratio. Four biological replicates of 20 third-instar Culex larvae were treated with an LC90 dose (80 ng/ml) of Bin toxin for 6, 12, or 18 h
|
| Growth protocol |
Mosquitoes were reared at 27°C with 12-h light/dark cycle.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Pools of at least 20 midguts were collected from each treatment type. RNA was extracted from dissected tissues using an RNeasy kit (Qiagen) according to standard protocol.
|
| Label |
Cy3
|
| Label protocol |
Cy5- and Cy3-labeled probes were synthesized from 200 ng of total RNA of each sample using Low Input Quick Amp Labeling Kit from Agilent Technologies
|
| |
|
| Channel 2 |
| Source name |
Midgut 12 hr after toxin treatment
|
| Organism |
Culex quinquefasciatus |
| Characteristics |
developmental stage: larvae
treatment: toxin
time: 12 hr
tissue: Midgut
|
| Treatment protocol |
The third-instar C. quinquefasciatus larvae were starved for 3 h before feeding with the mixture of BinA and BinB at a 1:1 molar ratio. Four biological replicates of 20 third-instar Culex larvae were treated with an LC90 dose (80 ng/ml) of Bin toxin for 6, 12, or 18 h
|
| Growth protocol |
Mosquitoes were reared at 27°C with 12-h light/dark cycle.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Pools of at least 20 midguts were collected from each treatment type. RNA was extracted from dissected tissues using an RNeasy kit (Qiagen) according to standard protocol.
|
| Label |
Cy5
|
| Label protocol |
Cy5- and Cy3-labeled probes were synthesized from 200 ng of total RNA of each sample using Low Input Quick Amp Labeling Kit from Agilent Technologies
|
| |
|
| |
| Hybridization protocol |
Probes prepared from the RNA of Bin toxin-treated mosquito midgut were co-hybridized with probes prepared from the RNA obtained from untreated mosquito midgut. The hybridizations were conducted on an Agilent-based microarray platform using custom-designed whole-genome 4 x 44K C. quinquefasciatus microarrays
|
| Scan protocol |
The arrays were scanned with an Agilent High Resolution Scanner (Agilent Technologies Inc., Santa Clara, CA, USA).
|
| Description |
LC9012hr2
|
| Data processing |
Spot intensity was quantified using Agilent Feature Extraction Software. The linear models for microarray data (limma) package in R program was used to process the gene expression data from microarray. The raw signal intensities of each array were corrected by backgroundCorrect then normalized within array by loess method. Replicate spots in each array were averaged with avereps function. Linear model was generated with lmFit function then the statistical significance of differential expression was analyzed by empirical Bayes moderated t-statistics. Finally, the raw p value was adjusted with Benjamini-Hochberg (BH) method at a significance level of adjusted p<0.05. Self-self hybridization was used to determine the intensity-dependent cutoff value for the significance of differentially expressed genes on these types of microarrays as 0.75 in log2 scale, which corresponds to a 1.68 fold-change in regulation. Gene expression data were described in terms of function by homology to functionally annotated genes in Culex gene databases such as Gene Ontology
|
| |
|
| Submission date |
Mar 21, 2017 |
| Last update date |
Mar 22, 2017 |
| Contact name |
George Dimopoulos |
| E-mail(s) |
[email protected]
|
| Phone |
4432870128
|
| Organization name |
Johns Hopkins School of Public Health
|
| Department |
Molecular Microbiology and Immunology
|
| Street address |
615 N Wolfe St.
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL10712 |
| Series (1) |
| GSE96838 |
Midgut transcriptome of Culex quinquefasciatus larvae treated with LC90 doses of Bin toxin from Lysinibacillus sphaericus |
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