|
| Status |
Public on Jul 04, 2012 |
| Title |
donor heart tissue [VC-F10] |
| Sample type |
RNA |
| |
|
| Source name |
human heart tissue from left ventricular free wall of organ donors with no previous history of heart disease
|
| Organism |
Homo sapiens |
| Characteristics |
rs1333049 genotype: GG
age: 52
Sex: F
batch: 9
|
| Treatment protocol |
Heart specimens were rapidly frozen in liquid nitrogen at the time of harvest and stored at -80°C until use.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA and genomic DNA was simultaneously extracted from frozen tissue after automated grinding (Retsch Mixer Mill MM301, Haan, Germany) in TRIzol® (Invitrogen, Carlsbad, CA) and chloroform. RNA was purified using RNeasy Midi columns (Qiagen, Valencia, CA) according to manufacturer’s instructions and tested for quality and quantity with an Experion (Bio-Rad Laboratories, Hercules, CA). Genomic DNA was extracted from the remaining organic and interphase layers of the TRIzol® extract (see protocol at http://genome-www.stanford.edu/DFSP/materials.shtml).
|
| Label |
biotin
|
| Label protocol |
Biotinylated cDNA was prepared according to the standard Affymetrix protocol from 100 ng of total RNA ( whole transcript(WT) sense target labeling assay Technical manual version 4 2007, Affymetrix)) using the non ribosomal RNA reduced option. (chapter 4)
|
| |
|
| Hybridization protocol |
Following fragmentation, 5.5 ug of single stranded cDNA was hybridized for 17 hr at 45C on GeneChip Human Gene 1.0 ST Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner G7 plus.
|
| Description |
Gene expression and genotype in normal heart
|
| Data processing |
The data were analysed using R version 2.8.1 with Bioconductor. Aroma Affymetrix was used for background correction, quantile normalization and fitting GCRMA probe level models. Batch effects were corrected with Combat. Limma was used to calculate differential expression and apply Benjamini and Hochberg corection for multiple testing.
|
| |
|
| Submission date |
Jul 03, 2012 |
| Last update date |
Jul 04, 2012 |
| Contact name |
Anna P Pilbrow |
| E-mail(s) |
anna.pilbrow@otago.ac.nz
|
| Organization name |
University of Otago Christchurch
|
| Department |
Medicine
|
| Lab |
Christchurch Cardioendocrine Research Group
|
| Street address |
2 Riccarton Avenue
|
| City |
Christchurch |
| ZIP/Postal code |
8041 |
| Country |
New Zealand |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE22253 |
Gene expression and genotype in normal heart |
|
