|
| Status |
Public on Jun 11, 2009 |
| Title |
Normal Puerto Rican control 3 |
| Sample type |
RNA |
| |
|
| Source name |
skin fibroblasts, passages #2 and 4
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
genotype: Wild type for TWIST2
ethnicity: Puerto Rican
|
| Biomaterial provider |
Coriell Cell Repositories http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=GM00037
|
| Growth protocol |
Skin biopsies were taken from patients and normal controls from the forearm region, digested with trypsin-EDTA (0.025%) at 37oC for 1 hour in a Petri Dish, trypsin was removed, and 1% collagenase type I in RPMI 1640 was added. The biopsy was cut in small pieces with a sterile scalpel and incubated 3 hrs at 37oC. Then, the tissue was centrifuged at 1200 rpm for 10 minutes at room temperature, the supernatant was discarded and replaced with growth media. We grew skin fibroblasts in DMEM supplemented with 10% Fetal Bovine Serum and L-glutamine, penicillin and streptomycin, in a tissue culture incubator at 5% CO2 and 37oC. We grew the cells until flasks were 50-80% confluent until RNA was prepared.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed directly on the culture flask by adding the appropriate amount of RLT solution from the RNeasy kit (Qiagen), as recommended by the manufacturer. RNA was quantified in a Nanodrop system or a Pharmacia spectrophotometer. RNA quality was assessed using the Agilent Bioanalyzer using the RNA Nanoassay.
|
| Label |
biotin
|
| Label protocol |
Double-stranded cDNA synthesis, in vitro transcription/target labeling, hybridization/control mixture preparation, chip-hybridization, and post-processing procedures were performed using standard protocols from Affymetrix by the Mount Sinai School of Medicine Microarray Shared Research Facility.
|
| |
|
| Hybridization protocol |
Gene expression profiles of normal and SS patient fibroblasts were examined by microarray analysis using Affymetrix U133plus2.0 chips. The hybridization steps were performed by the Mount Sinai School of Medicine Microarray Shared Research Facility
|
| Scan protocol |
GeneChips scans were performed by the Mount Sinai School of Medicine Microarray Shared Research Facility on an Affymetrix GeneChip system.
|
| Description |
1:1 Pool of two control cell lines, one is GM00037, the other is from a normal donor
|
| Data processing |
A summarized dataset of the Affymetrix chips was obtained by GC Robust Multi-Chip Average (gcRMA) analysis for the background correction and normalization (Wu et al, 2004). We combined the Affymetrix data to assess consistent differences between the mutant and wild type fibroblasts gene expression and used the following three criteria to find candidate genes: a) the moderated t-statistic is not less than 332 (Gordon 2004); b) the average fold change is no less than two fold increase (or decrease); c) The maximum of the log 2 based signal intensities across the arrays was greater than 3.5, which was approximately twenty percentile of all log 2 based signal intensities. This step was done to filter out genes with extremely low expression levels.
|
| |
|
| Submission date |
Jun 09, 2009 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Carmen Lydia Cadilla |
| E-mail(s) |
carmen.cadilla@upr.edu
|
| Phone |
7877544366
|
| Organization name |
University of Puerto Rico School of Medicine
|
| Department |
Biochemistry
|
| Lab |
A640
|
| Street address |
University of Puerto Rico Medical Sciences Campus, Area of Medical Center
|
| City |
San Juan |
| State/province |
PR |
| ZIP/Postal code |
00936 |
| Country |
Puerto Rico |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE16524 |
Expression data from skin fibroblasts derived from Setleis Syndrome patients and normal controls |
|
| Relations |
| Reanalyzed by |
GSE119087 |
