|
| Status |
Public on Jul 01, 2013 |
| Title |
FCL_Definite Mito Disease_RRM2B |
| Sample type |
RNA |
| |
|
| Source name |
fibroblast cell line (FCL)
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: fibroblast cell line
respiratory chain complex deficiency in muscle: Complex IV; 93% mtDNA depletion in muscle
gender: F
age (years): 0.3
informatic analysis group: Mito Disease Group
|
| Treatment protocol |
FCLs were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 1 g/L glucose and supplemented with 20% FBS (Gibco), 1 mM sodium pyruvate (CellGro), 2 mM L-glutamine, and 50 ug/mL uridine (Calbiochem).
|
| Growth protocol |
Fibroblast cell lines (FCLs) were obtained from prior skin biopsies when available and/or established in the Clinical CytoGenomics Laboratory from skin biopsies performed in Mitochondrial-Genetics Diagnostic Clinic at The Children’s Hospital of Philadelphia (M.J.F.), following informed consent.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from frozen FCL pellets per standard protocol by RNeasy Mini kit (Qiagen), after cells were placed in kit lysis buffer and processed by a QIAshredder homogenizer to increase RNA yield. RNA was analyzed by NanoDrop 1000 spectrophotometer and Agilent 2100 Bioanalyzer in the Nucleic Acid and Protein Core Facility at The Children’s Hospital of Philadelphia, with RNA integrity number (RIN) > 8 acceptable (maximum RIN is 10) for microarray analyses.
|
| Label |
biotin
|
| Label protocol |
Biotinylated amplified RNA (aRNA) was prepared from 200 ng total RNA using the standard protocol of the Ambion MessageAmp Premier Kit.
|
| |
|
| Hybridization protocol |
Following fragmentation, 4.6 ug of aRNA were hybridized for 16 hr at 45C on Affymetrix Human Exon 1.0ST cartridge arrays. The arrays were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000, 7G.
|
| Data processing |
Probe-level data in CEL files was normalized by RMA method implemented in the affy package of Bioconductor to get gene-level expression measurements. Alternative CDF file of the HuEx ST1.0 platform was used as library.
probe group file: huex_chop_alt1.cdf
meta-probeset file: annotation.csv
|
| |
|
| Submission date |
Dec 18, 2012 |
| Last update date |
Jul 01, 2013 |
| Contact name |
Marni J Falk |
| E-mail(s) |
falkm@email.chop.edu
|
| Phone |
215-590-4564
|
| Organization name |
CHOP
|
| Department |
Pediatrics/ Human Genetics
|
| Lab |
ARC 1002c
|
| Street address |
3615 Civic Center Blvd
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL16240 |
| Series (1) |
| GSE42986 |
Transcriptome profiling in human primary mitochondrial respiratory chain disease |
|
