|
| Status |
Public on Jun 02, 2014 |
| Title |
Scramble treated embryos biological replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
Day 5 murine blastocyst embryos
|
| Organism |
Mus musculus |
| Characteristics |
sample: whole blastocyst
strain: B6C3F1
treatment: 400nM of Scramble-Alexa 488 for 72 hours
|
| Growth protocol |
After embryos are four times washed in fresh CCM they were extracted immediately.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To ensure that total RNA was recovered we performed RNA extraction using the Arcturus PicoPure RNA Isolation Kit (Life Technologies S.A. Madrid, Spain). RNA extracted was quantified using a NanoDrop (Thermo Fisher Scientific Inc, MA, USA) spectrophotometer and the RNA quality was evaluated using the total eukariote Pico RNA LabChip in the BioAnalyzer 2100, (Agilent Technologies Inc, DE, USA)
|
| Label |
Cy3-CTP
|
| Label protocol |
50ng of total RNA were mixed with spike and mixed with T7 promoter primer for 10' at 65º then cooled at 4ºC. Next to cDNA synthesis, First strand buffer, DTT, dNTP and RNAse block mix were added and incubated for 2 hours at 40ºC followed by 15' at 70ºC. Next for cRNA synthesis, transcription buffer, DTT, NTP, T7 RNA polymerase and Cy3-CTP were added and incubated for 2 hours at 40ºC and cooled for 5'. Then cRNA was purified according to Qiagen RNEasy kit protocol specifications. Yield and [cRNA] stimated. Then 1.65 ug de cRNA was used to be fragmented in presence of blocking agent and fragmentation buffer for 30º at 60ºC and cooled. Then sample was mixed with hybridization buffer.
|
| |
|
| Hybridization protocol |
1.65 ug of cRNA was used to be fragmented in presence of blocking agent and fragmentation buffer for 30º at 60ºC and cooled. Then sample was mixed with hybridization buffer.
|
| Scan protocol |
Microarrays slides were washed and scanned in Genepix Personal 4100A Scanner and scanned in Genepix Pro Software with a spot resolution of 5um in the green channel
|
| Description |
each sample was previous tested for total RNA quality using total pico RNA Labchip in an agilent bioanalyzer and with RIN above 9.0
|
| Data processing |
Median intensity value of each spot data was log2 transformed and normalized using R software and libraries from bioconductor database. Next, replicated probes were merged by mean using GEPAS.
|
| |
|
| Submission date |
Feb 27, 2013 |
| Last update date |
Jun 02, 2014 |
| Contact name |
Juan M Moreno-Moya |
| E-mail(s) |
jmmormoy@gmail.com
|
| Organization name |
FIVI
|
| Street address |
Catedrático Agustín Escardino
|
| City |
Valencia |
| ZIP/Postal code |
46980 |
| Country |
Spain |
| |
|
| Platform ID |
GPL10333 |
| Series (1) |
| GSE44730 |
Maternal microRNAs secreted by the endometrium act as transcriptomic regulators of the pre-implantation embryo (mouse) |
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