|
| Status |
Public on Jun 01, 2010 |
| Title |
T00219254: cardiomyocytes, 48 hour conditioned |
| Sample type |
RNA |
| |
|
| Source name |
cardiomyocytes, 48 hour conditioned
|
| Organism |
Rattus norvegicus |
| Characteristics |
Rat neonatal cardiomyocytes harvested from day 1 ventricles (n=24). Cells purified via serial Percoll gradients and plated at 2.5x10^5 cells per 100 mm sq dish.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Extracted using Invitrogen Trizol LS Reagent and Invitrogen's RNA isolation protocol supplied with the reagent.
|
| Label |
biotin
|
| Label protocol |
GE Healthcare Amersham CodeLink iExpress Assay Reagent Kit protocol
|
| |
|
| Hybridization protocol |
GE Healthcare Amersham CodeLink Gene Expression System: Single-Assay Bioarray Hybridization and Detection protocol
Detection dye: streptavidin - Alexa Fluor 647
|
| Scan protocol |
Scanned using a GenePix 4000B at 600 PMT and CodeLink software (version 5.0).
|
| Description |
Cardiac media was diluted 50% with conditioned hesc media while FBS and HS percentages were held constant at 5% and 10% respectively.
RNA extracted after 48 hours.
|
| Data processing |
The threshold was calculated by removing the top 10% and bottom 10% of negative controls and averaging the remaining intensities. The threshold was subtracted from each of the discovery probes. An average was computed for the following positive controls: LEUB, HISB, FIXB, GND, ENTF, and ARAB. The averages of each of these positive controls was grouped together with its average on all the other arrays in this series. By taking the median of each group, a global median was created which represents the median value for a positive control across all of the arrays. Correction factors for this array were calculated by dividing the average of each positive control by its corresponding global median. It was observed that the arrays behaved differently at high intensity levels compared to low intensity levels. As the positive controls increased in intensity, the correction factors also steadily increased or steadily decreased. Therefore, a correction factor derived from a low intensity positive control was not a good correction for high intensity probes. To accommodate for the effect of intensity, the correction factor for each positive control was graphed versus its intensity and a best-fit regression line was created. The equation of the best-fit line was used to calculate a correction factor for each individual discovery probe based on where it fit into the slope of the correction factors. Negative values were replaced by a placeholder of ".0001".
In summary, the data was normalized by comparing each positive control to its median value across arrays and correcting based on the slope of the positive controls versus intensity. Note that the data was never median normalized or log transformed.
|
| |
|
| Submission date |
Nov 19, 2008 |
| Last update date |
Jun 16, 2009 |
| Contact name |
John Michael Krill-Burger |
| E-mail(s) |
burgerm@upmc.edu
|
| Phone |
412-656-6727
|
| Organization name |
University of Pittsburgh Medical Center
|
| Street address |
Rm. WG21.3 Shadyside Hospital
|
| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15232 |
| Country |
USA |
| |
|
| Platform ID |
GPL2890 |
| Series (1) |
| GSE13708 |
Stem Cells Secrete Factors That Induce Proliferation In Differentiated Cardiomyocytes |
|
