|
| Status |
Public on Nov 12, 2013 |
| Title |
Control Whole embryo, stage 9, Replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Embryos injected with control morpholino
|
| Organism |
Xenopus laevis |
| Characteristics |
Stage: Late blastula (9)
tissue: Whole embryo
|
| Treatment protocol |
Embryos were injected using 60ng of morpholino in each blastomere using either Control morpholino or PVD2 morpholino mixture.
|
| Growth protocol |
Whole embryos were injected at two cells stage and cultured in 1/10 normal amphibian medium until stage 9 and 10. Animal caps were dissected from injected embryos at stage 8, cultured in 3/4 normal amphibian medium and collected at stage 9 and 10.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from 3 whole embryos or 25 animal caps using Qiagen Rneasy Kit according to manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
2.5µg of total RNA were processed using Agilent’s Quick Amp Labelling Kit. Labelled cRNA were purified using Rneasy Mini kit (Qiagen)
|
| |
|
| Channel 2 |
| Source name |
pooled whole embryo
|
| Organism |
Xenopus laevis |
| Characteristics |
stages: 2, 4, 6, 8, 9, 10, 11, 12, 15, 18, and 20 pooled
tissue: Whole embryo
|
| Treatment protocol |
Embryos were injected using 60ng of morpholino in each blastomere using either Control morpholino or PVD2 morpholino mixture.
|
| Growth protocol |
Whole embryos were injected at two cells stage and cultured in 1/10 normal amphibian medium until stage 9 and 10. Animal caps were dissected from injected embryos at stage 8, cultured in 3/4 normal amphibian medium and collected at stage 9 and 10.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from 3 whole embryos or 25 animal caps using Qiagen Rneasy Kit according to manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
2.5µg of total RNA were processed using Agilent’s Quick Amp Labelling Kit. Labelled cRNA were purified using Rneasy Mini kit (Qiagen)
|
| |
|
| |
| Hybridization protocol |
Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. Hybridization and washed were performed according to manufacturer's instructions.
|
| Scan protocol |
Slides were scanned with an Agilent DNA Microarray Scanner (model G2505-64120) at 100% PMT for Cy3 channel and 10% PMT for Cy5 channel with a scan resolution of 5µm
|
| Description |
embryos injected at two-cells stage with control morpholino oligos
|
| Data processing |
Data are extracted with Agilent Feature Extraction Software.The data were further processed with NIA ANOVA tool utilities.See http://lgsun.grc.nia.nih.gov/ANOVA for details.The data is normalized with the following method:(1) Take values from the columns gDyeNormSignal,rDyeNormSignal in the raw files and log-transform them using: log10(max(x,1)). These values will be referenced below as Xgi and Xri where i is array number. (2) Take average of Xri's for the oligo among 48 arrays in the series: AverXr = average(Xri). (3) For each array estimate Yi = Xgi-Xri+AverXr (4) Round Yi to 4 decimal digits. This is used as the normalized VALUE for each spot.
|
| |
|
| Submission date |
May 31, 2013 |
| Last update date |
Nov 12, 2013 |
| Contact name |
Joshua Brickman |
| E-mail(s) |
Joshua.Brickman@sund.ku.dk
|
| Organization name |
University of Copenhagen
|
| Department |
The Danish Stem Cell Centre (DanStem)
|
| Street address |
Blegdamsvej 3B
|
| City |
Copenhagen |
| ZIP/Postal code |
DK-2200 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL11258 |
| Series (1) |
| GSE47578 |
Xenopus laevis embryos and animal caps: Control vs PouV morpholino |
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