|
| Status |
Public on Oct 06, 2011 |
| Title |
PIC_G_P2 |
| Sample type |
RNA |
| |
|
| Source name |
Brain_PIC_withT_replicate2
|
| Organism |
Danio rerio |
| Characteristics |
age: Adults
tissue: Whole Brain
treatment: PIC_withT
|
| Treatment protocol |
For microarray screening of fever in zebrafish, RNA extracted of zebrafish brains injected or not with 1mg*Kg-1 of Poly (I:C): zebrafish in thermal gradient (control, n = 9), zebrafish PBS injected in thermal gradient (PBS, n = 9), zebrafish Poly I:C injected without thermal gradient (PIC_sG, n = 9), and zebrafish Poly I:C injected with thermal gradient (PIC_sG, n = 9). Individuals RNAs were grouped into pools from 3 individual for each treatment (3 pools by treatment).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from brains using 500uL of TriReagent (Molecular Research Center) following the manufacturer’s instructions. RNA concentration was quantified using Nanodrop ND-1000 and RNA integrity and quality was assessed using a Bioanalyzer 2100 with the RNA 6000 Nano LabChip kit (Agilent Technologies). The RNA integrity number (RIN) was calculated for each sample using the Agilent 2100 Expert software, only RNAs with a RIN number > 7 were processed (to reduce experimental biases). Total RNA (2μg) was used to synthesize cDNA with SuperScript III Transcriptase (Invitrogen) and oligo-dT primer (Promega).
|
| Label |
Cy3
|
| Label protocol |
A loop microarray design approach was taken for the study, all experimental RNA samples were labelled with one dye Cy3 and each stimulated sample was compared to the control sample (pool without stimulation) labelled with the same dye (Cy3). Denatured samples of RNA were reversed transcribed and indirectly labelled with Cy3. RNA labelling, hybridizations, and scanning were performed according to manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
Briefly, total RNA (500 ng) was amplified and Cy3-labeled with Agilent’s One-Color Microarray-Based Gene Expression Analysis (Quick Amp Labelling kit) along with Agilent’s One-Color RNA SpikeIn Kit. Each sample (1.65 μg of RNA) was hybridized to Zebrafish array (ID 026437, Agilent) at 65 °C for 17 hours using Agilent’s GE Hybridization Kit. Washes were conducted as recommended by the manufacturer using Agilent’s Gene Expression Wash Pack with stabilization and drying solution.
|
| Scan protocol |
Microarrays slides were scanned with Agilent Technologies Scanner model G2505B. Spot intensities and other quality control features were extracted with Agilent’s Feature Extraction software version 10.4.0.0. One-channel TIFF images were imported into the Gene GeneSpring software GX 11.0.
|
| Description |
Gene expression after 24 of ip injection with Poly (I:C) with thermal gradient
|
| Data processing |
Percentile shift normalization was made to adjust all spot intensities in an array. This normalization takes each column in an experiment independently, and computes the median expression values for this array, across all spots. It then subtracts this value from the expression value of each entity (Bolstad et al, 2003). After Percentile normalization, all data were filtered by comparison of the standard deviation expression among groups (filter by expression). The entities that had values lesser or greater than the standard deviation value were retained. This filter procedure allowed selected genes that have outlier samples, and filter out genes that have a low variation in expression values across all samples.
|
| |
|
| Submission date |
Oct 05, 2011 |
| Last update date |
Oct 06, 2011 |
| Contact name |
Sebastian Boltana |
| E-mail(s) |
sboltana@udec.cl
|
| Phone |
93 581 3473
|
| Organization name |
Universidad de Concepcion
|
| Department |
Oceanografia
|
| Lab |
Evolutionary Immunology
|
| Street address |
Edmundo Larenas s/n, Barrio Universitario. Biotechnology Center
|
| City |
Chile |
| State/province |
Bio BIO |
| ZIP/Postal code |
4030000 |
| Country |
Chile |
| |
|
| Platform ID |
GPL6457 |
| Series (1) |
| GSE32636 |
Behavioural fever and its underlying molecular mechanisms |
|
