|
| Status |
Public on Apr 01, 2012 |
| Title |
Heterozygote biol rep 1 |
| Sample type |
RNA |
| |
|
| Source name |
Th3_het_fetal_liver_erythroblasts
|
| Organism |
Mus musculus |
| Characteristics |
strain background: 129SV background and backcrossed onto C57BL/6 for 9 generations
developmental stage: E14.5
genotype/variation: Th3/+ heterozygote
tissue: fetal liver
cell type: FACS-purified CD71+Ter119+FSChigh erythroblasts
|
| Growth protocol |
E14.5 fetal livers from Th3/+ x Th3/+ crosses were disaggregated, and CD71+Ter119+FSChigh cells purified by FACS
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizo lLS RNA samples were prepared using Qiagen miRNAeasy kit according to manufacturer instructions
|
| Label |
biotin
|
| Label protocol |
35ng of RNA was amplify with the WT-Ovation Pico RNA amplification System from NuGen, ST -cDNA generation was performed with the WT-Ovation Exon Module starting 4ug of cDNA, 5 ug of ST -cDNA was fragmented and labeled with the Encore Biotin Module.
|
| |
|
| Hybridization protocol |
Affymetrix standard protocol
|
| Scan protocol |
A confocal scanner was used to collect fluorescence signal after excitation at 570 nm.
|
| Description |
Th3_het_1
Gene expression data from Th3/+ E14.5 fetal liver CD71+Ter119+FSChigh erythroblasts
|
| Data processing |
Affymetrix Command Console was used to quantitate expression levels for targeted genes; default values provided by Affymetrix were applied to all analysis parameters. Border pixels were removed, and the average intensity of pixels within the 75th percentile was computed for each probe. The average of the lowest 2% of probe intensities occurring in each of 16 microarray sectors was set as background and subtracted from all features in that sector. Probe pairs were scored positive or negative for detection of the targeted sequence by comparing signals from the perfect match and mismatch probe features using the MAS5.0 algorithm. The number of probe pairs meeting the default discrimination threshold (tau = 0.015) was used to assign a call of absent, present or marginal for each assayed gene, and a p-value was calculated to reflect confidence in the detection call. A weighted mean of probe fluorescence (corrected for nonspecific signal by subtracting the mismatch probe value) was calculated using the One-step Tukey's Biweight Estimate. This signal value, a relative measure of the expression level, was computed for each assayed gene. Global scaling was applied to allow comparison of gene signals across multiple microarrays: after exclusion of the highest and lowest 2%, the average feature signal was calculated and used to determine what scaling factor was required to adjust the chip average to an arbitrary target of 150. All signal values from one microarray were then multiplied by the appropriate scaling factor
|
| |
|
| Submission date |
Dec 02, 2011 |
| Last update date |
Apr 01, 2012 |
| Contact name |
Mitchell J Weiss |
| Organization name |
Children's Hospital of Philadelphia
|
| Department |
Hematology
|
| Lab |
316 Abramson
|
| Street address |
3615 Civic Center Blvd
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE34125 |
Expression data from murine beta thalassemia erythroblasts |
|
