|
| Status |
Public on Jun 02, 2014 |
| Title |
Ectopic Pregnancy (patient 7) |
| Sample type |
RNA |
| |
|
| Source name |
Trophoblast tissue
|
| Organism |
Homo sapiens |
| Characteristics |
type: tissue
age: 21
week of gestation: 5
status: ectopic pregnancy
|
| Growth protocol |
Endometrial fluid aspiration samples were immediately fast frozen at -80ºC until day of RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To ensure that miRNA fraction will be recovered we performed RNA extraction using the miRNeasy Kit (Qiagen, Valencia, CA, USA). RNA extracted was quantified using a NanoDrop (Thermo Fisher Scientific Inc, MA, USA) spectrophotometer and the proportion of miRNAs was evaluated using the Small RNA LabChip BioAnalyzer 2100, (Agilent Technologies Inc, DE, USA)
|
| Label |
Cy3-pCp
|
| Label protocol |
100ng of total RNA were mixed with spike and treated with phosphatase for 30' at 37º then cooled at 4ºC. Next DMSO was added and heated at 100ºC for 7.5' then cooled at 4ºC. Next mixed with Cy3-pCp and T4 RNA Ligase for 2 hours at 16ºC and cooled at 4ºC. Samples were dried in a vacuum for 3h at 45ºC. Then H2O added.
|
| |
|
| Hybridization protocol |
After H2O resuspended, samples were mixed with another Spike, mixed with blocking agent and Hi-RPM hybridization buffer and heated at 100ºC for 5' and cooled at 4ºC. Then samples were loaded onto gaskets slides and microarrays slides were put on them. Finally Samples were hybridized for 20h at 55ºC.
|
| 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 miRNAs using Small RNA Labchip in an agilent bioanalyzer
|
| Data processing |
Median intensity value of each spot data was normalized and log2 transformed 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 |
GPL14767 |
| Series (1) |
| GSE44731 |
Differential miRNA profiles of pregnancy tissue in ectopic pregnancies |
|
