|
| Status |
Public on Dec 01, 2013 |
| Title |
Embryo_Control.Morpholino_Low_rep5 Reference |
| Sample type |
RNA |
| |
|
| Source name |
Common Reference
|
| Organism |
Danio rerio |
| Characteristics |
developmental stage: 30% epiboly
treatment: n.a.
|
| Treatment protocol |
Translation of traf6 was blocked by either one of two non-overlapping morpholinos (traf6 morpholino-1 and morpholino-2) specifically targeting the 5’UTR region. To be able to control for aspecific morpholino effects, a 5bp mismatch morpholino of traf6 morpholino-1 and a standard control morpholino (Gene Tools) were used. Morpholino oligonucleotides were diluted with Danieu buffer to a concentration of 0.5, 1 and 2 mM and embryos were injected at the one cell stage with 1nl of one of the above mentioned morpholinos. In addition, 1x Danieu’s buffer containing Phenol red was injected to control for the injection effect per se. All injections were performed in random order and the experiment was performed in quintuplicate.
|
| Growth protocol |
Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (http://ZFIN.org). Embryos were grown at 28°C in egg water (60µg/ml Instant Ocean sea salts).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Embryos of each treatment group were sampled at the 30% epiboly stage and snap frozen in liquid nitrogen. Total RNA from each sample was extracted using TRIZOL followed by a clean up procedure using the RNeasy Mini Kit (Qiagen) and removal of a residual genomic DNA by an RNase-free DNase treatment (Qiagen). RNA concentration was determined using a Nanodrop ND-1000 (Thermo Fisher Scientific) and RNA quality was assessed on a Agilent 2100 BioAnalyzer (Agilent Technologies). Total RNA samples with an RNA integrity number (RIN) >7 were used for further analysis. All assays were performed according to the manufacturer’s protocols.
|
| Label |
Cy5
|
| Label protocol |
0,5 μg of total RNA was amplified with the Amino-Allyl MessageAmp II kit (Ambion) according to manufacturer’s instruction. After aRNA synthesis, samples were labeled with Cy3-NHS mono-reactive dye (GE Healthcare) for the test samples and Cy5-NHS (GE Healthcare) mono-reactive dye for the common reference (pooled aRNA).
|
| |
|
| Hybridization protocol |
825 ng for each channel were combined and made to 55 μl with 10x Blocking Agent and 25x Fragmentation Buffer. 55 μl of 2x Hi-RPM Hybridization Buffer was added (Gene Expression Hybridization Kit, Agilent Technologies). 100 μl of hybridization mixture was loaded onto each array and allowed to hybridize at 65°C (10 RPM) for 17h. The slides were washed with Gene Expression Wash Buffers I and II (Gene Expression Wash Buffer Kit, Agilent Technologies).
|
| Scan protocol |
Slides were scanned in an ozone-free room with the Agilent G2565BA scanner as described in the Agilent Two-Color Microarray-Based Gene Expression Analysis guide version 5.5 (G4140-90050, Agilent technologies).
|
| Description |
The CR (common reference) was composed by combining 1 ug of cRNA from each sample.
|
| Data processing |
Data was extracted with Feature Extraction version 9.5.1.1. (Protocol GE2-v5_95) for two-color Agilent microarrays.
|
| |
|
| Submission date |
Dec 11, 2012 |
| Last update date |
Dec 01, 2013 |
| Contact name |
Oliver Stockhammer |
| E-mail(s) |
o.w.stockhammer@amc.uva.nl
|
| Organization name |
AMC
|
| Department |
Medical Microbiology
|
| Street address |
Meibergdreef 15
|
| City |
Amsterdam |
| State/province |
NH |
| ZIP/Postal code |
1105 AZ |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL7735 |
| Series (1) |
| GSE42856 |
Transcriptome analysis of Traf6 function in early zebrafish embryogenesis |
|
