|
| Status |
Public on Apr 19, 2017 |
| Title |
Patient F5959 |
| Sample type |
RNA |
| |
|
| Source name |
Orbicularis Oris Muscle Mesenchymal Stem Cells Primary Cultures (OOMMSC)
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Orbicularis Oris Muscle
gender: Male
phenotype: Nonsyndromic cleft lip/palate (NSCL/P)
|
| Growth protocol |
We established OOMMSC primary cultures according to previously published protocol [Bueno et al. 2009] and expanded to 70-90% confluence. We confirmed the mesenchymal immunophenotype of a representative sample of cell cultures through flow cytometry analysis with fluorescent antibodies for markers CD29 (adhesion), CD31 (endothelial), CD45 (hematopoietic), CD73 or CD166, CD90, and CD105 (mesenchymal).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation was performed with NucleoSpin RNA II kit (Macherey-Nagel), following manufacturer’s recommendations. RNA quality and concentration were assessed using Nanodrop 1000 and agarose gel electrophoresis. Only RNA samples with absorbance ratio 260/280 > 1.8, preserved rRNA ratio (28S/18S) and no signs of degradation were used.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip®Human Gene 1.0 ST Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450 and hybridized for 16 hr in the Affymetrix Hybridization Oven 640.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G
|
| Description |
Gene expression data from mesenchymal stem cells derived from OOM.
|
| Data processing |
Data were analyzed with Expression Console (Affymetrix) and normalized by RMA method. We excluded from the expression data the Affymetrix controls and probe sets classified as ‘rescue->FLmRNA->unmapped’ (poorly aligning to transcriptome, Homo sapiens NetAffx annotation release 32, 2011-06-21), also removing probe sets with cross hybridization potential.
|
| |
|
| Submission date |
Aug 17, 2016 |
| Last update date |
Apr 19, 2017 |
| Contact name |
Cibele Masotti |
| Organization name |
University of Sao Paulo
|
| Department |
Genetics and Evolutionary Biology
|
| Lab |
Human Development Genetics Lab
|
| Street address |
Rua do Matao, 277.
|
| City |
Sao Paulo |
| State/province |
São Paulo |
| ZIP/Postal code |
05508-090 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL6244 |
| Series (2) |
| GSE85748 |
MRPL53, a New Candidate Gene for Orofacial Clefting, Identified Using an eQTL Approach [expression array] |
| GSE85817 |
MRPL53, a New Candidate Gene for Orofacial Clefting, Identified Using an eQTL Approach |
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