|
| Status |
Public on Jul 01, 2013 |
| Title |
Skeletal Muscle_FlashFrozen_Suspected Mito Disease_immune disease |
| Sample type |
RNA |
| |
|
| Source name |
Skeletal muscle
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Skeletal muscle
respiratory chain complex deficiency: No Respiratory Chain Complex Deficiency
gender: F
age (years): 4
informatic analysis group: Mito Disease Group
|
| Growth protocol |
Residual skeletal muscle biopsy specimens were obtained from Clinical Pathology at The Children’s Hospital of Philadelphia or University of California San Diego following completion of all clinical diagnostic assays with the express participant consent of all living participants and/or families, or from decedents following IRB approval following completion of all clinical diagnostic assays with the express participant consent of all living participants and/or families, or from decedents following IRB approval.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from liquid nitrogen flash-frozen or isopentane frozen skeletal muscle tissues by Norgen RNA/DNA/Protein purification kit (Norgen Biotek Corporation, Ontario, Canada), with slight modification. 10-20 mg of frozen muscle tissue was transferred into a plastic tube with tight fitting pestle and 300 ul of kit lysis buffer, homogenized, and mixed by vortexing after 600 ul of RNAse free water was added. Lysates were incubated with intermittent vortexing at 550C x15 min with 20 ul Proteinase K, centrifuged x10 min, supernatants were mixed with equal volumes of 70% alcohol, and RNA extraction was completed per published protocol (Norgen protocol booklet step 1.B.h). 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 and real-time qPCR 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 |
|
