|
| Status |
Public on Apr 10, 2015 |
| Title |
ArrayE2.1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Larval head, 20 pool, D1D2MO
|
| Organism |
Danio rerio |
| Characteristics |
strain: Wild type WIK
age: 72 hours post fertilization
|
| Treatment protocol |
Zebrafish head from 20 larvae per replicate were pooled, snap frozen in dry ice and stored at -80C. Samples were homogenized in Qiazol using mortar and plastic pestle
|
| Growth protocol |
zebrafish embryos were microinjected with SCMO, D1D2MO and D3MO and kept in Danieu's solution at 28,5C until 72 hpf. 72 hpf-old zebrafish larvae were manually dissected in ice-cold RNAse free water to separate the head, abdomen and tail region.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Qiazol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
Total RNA was amplified and labeled using the Low Input Quick Amplification Labelling Kit (LIQA, Agilent, Diegem) according to the manufacturer’s instructions. In brief, 100 ng RNA was reverse transcribed into cDNA using oligo dT primers. Subsequently, cDNA was amplified and converted into cRNA and the resulting cRNA of each sample was labeled once with Cy3-CTP and once with Cy5-CTP. The RNeasy mini spin column kit (Qiagen, Venlo) was then used to purify each amplified and labeled cRNA sample. cRNA yield, quality and dye incorporation efficiency were verified using the nanodrop spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
Larval head, 20 pool, D3MO
|
| Organism |
Danio rerio |
| Characteristics |
strain: Wild type WIK
age: 72 hours post fertilization
|
| Treatment protocol |
Zebrafish head from 20 larvae per replicate were pooled, snap frozen in dry ice and stored at -80C. Samples were homogenized in Qiazol using mortar and plastic pestle
|
| Growth protocol |
zebrafish embryos were microinjected with SCMO, D1D2MO and D3MO and kept in Danieu's solution at 28,5C until 72 hpf. 72 hpf-old zebrafish larvae were manually dissected in ice-cold RNAse free water to separate the head, abdomen and tail region.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Qiazol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Total RNA was amplified and labeled using the Low Input Quick Amplification Labelling Kit (LIQA, Agilent, Diegem) according to the manufacturer’s instructions. In brief, 100 ng RNA was reverse transcribed into cDNA using oligo dT primers. Subsequently, cDNA was amplified and converted into cRNA and the resulting cRNA of each sample was labeled once with Cy3-CTP and once with Cy5-CTP. The RNeasy mini spin column kit (Qiagen, Venlo) was then used to purify each amplified and labeled cRNA sample. cRNA yield, quality and dye incorporation efficiency were verified using the nanodrop spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
825 ng Cy3 and 825 ng Cy5 labeled and purified cRNA were hybridized onto 4x44K full genome zebrafish (version 3, Agilent, Diegem) microarrays for 17 h at 65 °C in a rotating (10 rpm) hybridization oven (Agilent, Diegem). An A-optimal design was used as the hybridization design for each body region separately (Knapen et al., 2009) and 4 biological replicates of each condition were used, resulting in n=12 RNA samples per design. A total of 72 labelled cRNA samples was analyzed on 42 arrays.
|
| Scan protocol |
Microarray slides were scanned using a Genepix 4100A confocal scanner (Axon Instruments, Union City, CA, USA) at a resolution of 5 µm and excitation wavelengths of 635 nm and 532 nm in an ozone-free environment (NoZone scanner enclosure, SciGene, Sunnyvale, CA, USA). Images were analyzed for spot identification and for quantification of the fluorescent signal intensities using the Genepix Pro software 6.1 (Axon Instruments).
|
| Data processing |
Statistical analyses of raw microarray data were performed using the R package Limma as described by Vergauwen et al. (2010). Both knockdown conditions were contrasted against SCMO. Spots for which the criterion FG < BG + 2SD (FG: foreground, BG: background, SD: standard deviation of the local background of the entire array) was true for all arrays in the dataset, were excluded from analysis. Background correction was carried out using a normal-exponential convolution model . Within-array adjustment was done by Loess normalization, which performs an intensity-dependent normalization of the ratio of red and green. Linear models were fitted to intensity ratios, after which probes were ranked in order of evidence of differential transcription using an empirical Bayes method. The contrasts summarized across all replicates are available on the series record in the file: genes.all.contrasts_head.txt.
Contrasts were fitted to the linear models and cut off at FDR < 0.05 (false discovery rate, multiple testing correction using the Benjamini-Hochberg procedure) and |log2FC| > 0.585 (log2 fold change, corresponding to a fold induction of at least 1.5 or -1.5).
|
| |
|
| Submission date |
Sep 22, 2014 |
| Last update date |
Apr 10, 2015 |
| Contact name |
Lucia Vergauwen |
| E-mail(s) |
lucia.vergauwen@uantwerpen.be
|
| Phone |
003232652791
|
| Organization name |
University of Antwerp
|
| Department |
Veterinary Sciences
|
| Lab |
Zebrafishlab
|
| Street address |
Universiteitsplein 1
|
| City |
Wilrijk |
| ZIP/Postal code |
2160 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL14664 |
| Series (2) |
| GSE61622 |
Transcriptional profile of the head of zebrafish deiodinase knockdown |
| GSE61625 |
Transcriptional profile of deiodinase knockdown in zebrafish head, abdomen and tail |
|
