|
| Status |
Public on Feb 07, 2014 |
| Title |
Control Morholino (POOL2) |
| Sample type |
RNA |
| |
|
| Source name |
whole embryo
|
| Organism |
Danio rerio |
| Characteristics |
strain: GL and Tubingen
sample type: 2 days old whole embryo lysate
developmental stage: one-cell stage embryo
treatment: control
|
| Treatment protocol |
Pools of one hundred one-cell stage embryos were microinjected with morpholino and control morpholino. Fish were grown for two days before RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted by Trizol (Invitrogen, Italy) method according to manufacturer's instructions. DNAse digestion was performed to reduced genomic DNA contamination. All samples were quantitated using a NanoDrop ND-1000 RNA samples were checked for integrity by capillary electrophoresis (RNA 6000, NanoLabChip Analyzer Agilent Techologies).
|
| Label |
Cy3-dCTP
|
| Label protocol |
1 μg of total RNA was labeled with “Agilent One-Color Microarray-Based Gene Expression protocol” according to the manufacturer's protocol. The synthesized cDNA was transcribed into aRNA and labeled with Cy3-dCTP. Labeled aRNA was purified with RNeasy Mini columns (Qiagen, Valecia, CA, USA). The quality of each aRNA sample was verified by total yield and specificity calculated with NanoDrop ND-1000 spectrophotometer measurements (Nanodrop, Wilmington, DE, USA).
|
| |
|
| Hybridization protocol |
1.65 μg of labeled aRNA was used in each reaction and hybridization was carried out at 65°C for 17 hours in a hybridization oven rotator (Agilent, Santa Clara, CA, USA). The arrays were washed using Agilent Gene expression washing buffers and Stabilization and Drying Solution, as suggested by the supplier.
|
| Scan protocol |
Slides were scanned on an Agilent microarray scanner (model G2565CA) and Agilent Feature Extraction software version 10.5.1.1 was used for image analysis. Gene expression data were performed on three biological replicates for each condition.
|
| Description |
Fourty-eight hours post-morpholino injection fish were sacrificed and total RNA extracted for gene expression analysis
|
| Data processing |
Inter-array normalization of expression levels (gProcessedSignal) was performed with quantile method to correct possible experimental distortions. Normalization function was applied to the expression data of all the experiments and the values for within-arrays replicate spots were then averaged (according the same ProbeName ID). Feature Extraction Software, which provided spot quality measures, was used to evaluate the quality and reliability of the hybridization. In particular, the flag “positive and significative” (set to 1 if the spot had an intensity value significantly different from the local background and to 0 when otherwise) was used to filter out unreliable probes: the flag equal to 0 was to be noted as “not available (NA)” the spot intensity and therfore not used in the average calculation. Probes with a high proportion of NA values were removed from the dataset in order to carry out a more solid and unbiased statistical analyses. Thirty-three percent of NA was used as the threshold in the filtering process. Differentially expressed genes identification (One-way ANOVA), hierarchical clustering analysis (Sperman Rank Correlation or Euclidean distance) using average linkage method were performed using the TMev suite. Gene Ontology analysis of differentially expressed genes was performed on web tool GeneCodis3. Cytoscape tool in association with the Agilent Literature Search plug-in (http://apps.cytoscape.org/apps/agilentliteraturesearch (accessed on 19 December)) was used to produce network of differentially expressed genes. Network was analyzed using Network Analyzer and CentiScaPe plug-in. Supervised pathway analysis was performed by GSEA implemented in the Graphite web tool.
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| |
|
| Submission date |
Feb 06, 2014 |
| Last update date |
Feb 07, 2014 |
| Contact name |
Enrico Moro |
| E-mail(s) |
moroe@bio.unipd.it
|
| Phone |
+390498276341
|
| Organization name |
University of Padova
|
| Department |
Molecular Medicine
|
| Street address |
Via U. Bassi 58/B
|
| City |
PADOVA |
| ZIP/Postal code |
35121 |
| Country |
Italy |
| |
|
| Platform ID |
GPL6563 |
| Series (1) |
| GSE54754 |
Differential gene expression in 2 days old zebrafish after glucocerebrosidase (GBA1) morpholino-mediated knockdown |
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