|
| Status |
Public on Jan 01, 2017 |
| Title |
zic:HRAS_3d_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
zic:HRAS_3d_1
|
| Organism |
Danio rerio |
| Characteristics |
genotype/variation: zic:HRAS
developmental stage: larvae at 3 dpf
|
| Treatment protocol |
15 3dpf, 15 7dpf and 5 14dpf larvae were pooled to obtain each sample. Replicates for each experiment were obtained by pooling larvae obtained from different parents. Controls for 3-7-14 dpf larvae kita:HRAS and zic:HRAS were AB wild type embryos. Heat-shock induction of hsp70:RAS was performed at 37°C for 30min at 3dpf and RNA was isolated after 6 hours. Controls for hsp70:HRAS were AB wild type larvae heat-shocked at 37°C for 30min at 3dpf. Adult samples are individual fins with tumor from kita:HRAS fish. Control for adult samples are individual fins from AB wild type fish.
|
| 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 28,5°C in egg water (60µg/ml Instant Ocean sea salts).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Larvae or adult fins for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at -80°C. Total RNA was isolated using the miRNeasy Mini kit (qiagen) to preserve small RNA pieces.
|
| Label |
Hy3
|
| Label protocol |
RNA labeling was carried out with miRCURY™ LNA microRNA, Hy3™/Hy5™ Power Labeling kit (Exiqon) using 1 microgram of total RNA according to the manufacturer’s instructions.
|
| |
|
| Channel 2 |
| Source name |
control_for_zic:HRAS_3d_1
|
| Organism |
Danio rerio |
| Characteristics |
genotype/variation: control
developmental stage: larvae at 3 dpf
|
| Treatment protocol |
15 3dpf, 15 7dpf and 5 14dpf larvae were pooled to obtain each sample. Replicates for each experiment were obtained by pooling larvae obtained from different parents. Controls for 3-7-14 dpf larvae kita:HRAS and zic:HRAS were AB wild type embryos. Heat-shock induction of hsp70:RAS was performed at 37°C for 30min at 3dpf and RNA was isolated after 6 hours. Controls for hsp70:HRAS were AB wild type larvae heat-shocked at 37°C for 30min at 3dpf. Adult samples are individual fins with tumor from kita:HRAS fish. Control for adult samples are individual fins from AB wild type fish.
|
| 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 28,5°C in egg water (60µg/ml Instant Ocean sea salts).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Larvae or adult fins for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at -80°C. Total RNA was isolated using the miRNeasy Mini kit (qiagen) to preserve small RNA pieces.
|
| Label |
Hy5
|
| Label protocol |
RNA labeling was carried out with miRCURY™ LNA microRNA, Hy3™/Hy5™ Power Labeling kit (Exiqon) using 1 microgram of total RNA according to the manufacturer’s instructions.
|
| |
|
| |
| Hybridization protocol |
The dual colour hybridization of the microarray chips was performed according to Agilent protocol GE2-v5_95_Feb07 and GE2_105_Jan09 (www.Agilent.com) for two-color microarray-based gene expression analysis except that hybridization and washing was performed at 37 ◦C.
|
| Scan protocol |
The arrays were scanned with DNA Microarray Scanner G2565CA from Agilent Technologies. The arrays were scanned twice with 10% PMT and 100% PMT laser power.
|
| Description |
Biological replicate 1 of 3: zic:HRAS versus control, 3 dpf
252043010013_5u20b_S02_GE2_107_Sep09_1_4
|
| Data processing |
Microarray data was processed from raw data image files with Feature Extraction Software 9.5.3.1 (Agilent Technologies). The XDR function was used to extend the dynamic range. Processed data were subsequently imported into Rosetta Resolver 7.1 (Rosetta Biosoftware, Seattle, Washington) and subjected to default ratio error modelling. The Agilent Feature Extraction files contain the final log ratio data. The algorithms that were used to normalize the feature signal and calculate the log ratios are described in the Reference Guide for Agilent Feature Extraction Software v9.5. The standard settings as described for the GE2-v5_95 protocol in the Reference Guide were used. In order to calculate the normalized signal, the Rank Consistency Probes method was used and the Linear&LOWESSDyeNormFactor (= DyeNormalSignal/(BGSubSignal x LinearDyeNormFactor) was calculated as described on page 231 of the Reference Guide. The dye normalized signal was calculated as follows (page 232): DyeNormSignal = BGSubSignal x DyeNormFactor. The log10 ratio was subsequently calculated as follows (page 232): LogRatio = Log(rProcessedSignal/gProcessedSignal), where rProcessedSignal and gProcessedSignal are signals post dye normalization and post surrogate processing in the red and green channels, respectively. The surrogate values are calculated and used as the lowest limit of detection to replace the dye normalized signal when either the mean feature signal is less than the background signal or not significant when compared to the background signal, or when the mean signal is less than its background standard deviation (page 226).
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| |
|
| Submission date |
Apr 03, 2012 |
| Last update date |
Jan 01, 2017 |
| Contact name |
Annemarie H. Meijer |
| E-mail(s) |
a.h.meijer@biology.leidenuniv.nl
|
| Organization name |
Institute of Biology, Leiden University
|
| Department |
Animal Sciences and Health
|
| Street address |
Einsteinweg 55
|
| City |
Leiden |
| ZIP/Postal code |
2333 CC |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL15403 |
| Series (1) |
| GSE37015 |
Aberrant splicing of cdkn1a/p21 through micro-RNA mediated downregulation of JMJD6 |
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