|
| Status |
Public on Mar 04, 2010 |
| Title |
Dm_sn_kv_40 |
| Sample type |
RNA |
| |
|
| Source name |
Whole body
|
| Organism |
Drosophila montana |
| Characteristics |
isofemale line: O3F66 from Oulanka, Finland
|
| Treatment protocol |
Flies were maintained in vials in the laboratory under diapause-preventing conditions (continuous light, 19°C) since summer 2003.
|
| Growth protocol |
Female flies for the present study were collected under a light CO2 anaesthesia within one day after their emergence. Samples of young flies (three replicates, each including 13 flies) were transferred straight from the culturing conditions into liquid nitrogen for RNA extractions. The rest of the females were placed in vials (13-15 flies per vial) containing 7 ml of yeast-sucrose-agar medium with 1 mg of yeast sprinkled on top of the medium. The samples of reproducing females were obtained by maintaining the vials in a climate chamber in continuous light (24:0) for two weeks. Samples of diapausing females were maintained by keeping the vials in a climate chamber in a L:D cycle of 16:8 for the same time period.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the whole body of 13 individuals per sample, three replicates per sample. Qiagen RNA extraction kit with RNase-Free DNase treatment was used according to the manufacturer's protocols (Qiagen). The RNA was purified using Qiagen RNAeasy purification kit and the concentration and the purity of each sample was measured with NanoDrop ND-1000 spectrofotometer (NanoDrop Technologies, Wilmington, DE, USA).
|
| Label |
Cy3
|
| Label protocol |
350 ng total RNA of each sample was amplified and Cy3-labeled with Agilent’s Low RNA Input Linear Amplification Kit PLUS (One Color) and processed together with Agilent’s One-Colour RNA Spike-in Kit.
|
| |
|
| Hybridization protocol |
600 ng of each cRNA sample was hybridized to Agilent’s 8x15K D. montana custom arrays with Gasket slide at 65˚C overnight (17 h) using Agilent’s Gene Expression Hybridization Kit and washed with Agilent’s Gene Expression Wash Pack and Stabilization and Drying solution
|
| Scan protocol |
Arrays were scanned with Agilent Technologies Scanner (model G2505B) and numerical results were extracted with Feature Extraction version 9.5.3.1 using 018413_D_F_20071204 grid, GE1-v5_95_Feb07 protocol and GE1_QCM_Feb07 QC metric set.
|
| Description |
In addition to the 101 candidate genes we placed on our chip, we used seven genes, Ef1α48D, eIF-4a Gapdh1, Gapdh2, RpL11, RpL19 and RpL27A, which have been used as control genes in Drosophila whole genome microarrays (GeneChip Drosophila genome 2.0 Array, Affymetrix, 2006). These genes were added on the chip to select the best control genes for microarray validation with qRT-PCR analysis.
|
| Data processing |
The microarray data were normalized within the groups and across all samples using the VSN method. The statistical analyses were carried out using Bioconductor related Limma package [10] and the differentially expressed genes in each comparison were selected requiring a strict selection criteria of fold-change > 2.0 and false discovery rate (fdr) < 0.05.
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| |
|
| Submission date |
Oct 08, 2009 |
| Last update date |
Mar 04, 2010 |
| Contact name |
Maaria Kankare |
| E-mail(s) |
maaria.kankare@jyu.fi
|
| Organization name |
University of Jyvaskyla
|
| Street address |
Survontie 9
|
| City |
Jyvaskyla |
| ZIP/Postal code |
40014 |
| Country |
Finland |
| |
|
| Platform ID |
GPL9293 |
| Series (1) |
| GSE18475 |
Gene expression in Drosophila montana |
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