|
| Status |
Public on Sep 20, 2008 |
| Title |
Embryo_sptMO_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
75% epiboly embryo, spt MO-injected
|
| Organism |
Danio rerio |
| Characteristics |
Fish strain:ABC
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For each sample 20 embryos were dechorionated, vortexed in Trizol and phenol-chloroform extracted. They were treated with Dnase and cleaned up using a Qiagen RNeasy kit
|
| Label |
Biotin + Streptavidin-Alexa 647
|
| Label protocol |
The biotin-labeled cRNA target is prepared by a linear amplification method. Poly (A) RNA from 5 μg of total RNA is primed for reverse transcription by a DNA oligonucleotide containing a T7 RNA polymerase promoter 5′ to a d(T)24 sequence. After second-strand cDNA synthesis, the cDNA serves as the template in an in vitro transcription (IVT) reaction to produce the target cRNA. The IVT is performed in the presence of biotinylated nucleotides to label the target cRNA: Total RNA (5 μg) was incubated with T7 primer at 70 °C for 10 min before addition of Superscript reverse transcriptase reaction mix and further incubation at 37 °C for 1 h. The first-strand cDNA was added to polymerase mix and incubated in a TropiCooler incubator at 16 °C for 2 h.
Double-strand cDNA was precipitated in isopropanol at −80 °C for 20 min, centrifuged at 14k rpm for 20 min, and pellet resuspended in ddH2O after air-drying for 30 min. In vitro transcription was performed using the Ambion MEGAscript T7 kit. Biotinylated UTP, NTPs and enzyme reaction mix were added to dried cDNA and incubated at 37 °C overnight. Synthesized cRNA was purified using the Qiagen RNeasy kit. Concentration and quality of cRNA was measured by an Eppendorf Biophotometer. The target labeling by the KCC/TJU standard procedure results in a 50- to 200-fold linear amplification of the input poly(A) RNA.
|
| |
|
| Hybridization protocol |
The microarrays were hybridized in 6X SSPE with 50% formamide at 37 degree C for 20h, washed in 0.75X TNT at 46 degree C for 1 h, followed by blocking in TNB at RT for 30 min. and then processed by using direct detection method of the biotin-containing transcripts by Streptavidinin-Alexa 647 conjugated in TNB buffer (1:500) at RT for 30 min. Stained chips were washed in 1X TNT wash buffer at room temperature for 60 min with changing fresh buffer every 15 min.
|
| Scan protocol |
Processed chips were scanned by using a Perkin-Elmer ScanArray XL5000, software version 3.1, at a scan resolution of 10 microns. Images were quantified by Perkin-Elmer QuantArray Software 3.0. The fixed circle quantification method was used and output was given as total intensities.
|
| Description |
Embryo_sptMO_rep3
|
| Data processing |
The raw intensity for each probe was normalized to the median intensity for the probes on the array.
|
| |
|
| Submission date |
Sep 19, 2008 |
| Last update date |
Sep 25, 2008 |
| Contact name |
Aaron Garnett |
| E-mail(s) |
[email protected]
|
| Organization name |
UC Berkeley
|
| Street address |
555 LSA
|
| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94709 |
| Country |
USA |
| |
|
| Platform ID |
GPL7343 |
| Series (1) |
| GSE12857 |
Identification of direct T-box target genes in the developing zebrafish mesoderm |
|
