|
| Status |
Public on Mar 02, 2011 |
| Title |
E9.5 FVB/N erythroid cells-7 |
| Sample type |
RNA |
| |
|
| Source name |
E9.5 FVB/N erythroid cells-7
|
| Organism |
Mus musculus |
| Characteristics |
genotype: wild type FVB/N
age: embryonic day 9.5
|
| Treatment protocol |
E9.5 yolk sacs tissues were rinsed once in 1:1 20% sucrose PBS:Optimal Cutting Temperature freezing media (OCT, Tissue-Tek), then OCT alone and frozen. Eight micron E9.5 sections were stained with the LCM frozen section staining kit (Molecular Devices, Mountain View, CA, USA) and used for laser capture dissection experiments. Using the PixCell II Laser Capture Microdissection System (Arcturus Bioscience), approximately 1 E9.5 yolk sac erythroid cell was collected for every 2 to 3 laser pulses. Approximately 12 to 50 microscope slides, using 2 to 4 yolk sacs from 2 different litters were used per biological replicate.
|
| Growth protocol |
Timed-pregnant FVB/N mice were anesthetized and sacrificed. E9.5 yolk sacs were dissected from the embryo and cryoprotected in PBS with 20% sucrose.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from wild type erythroid cells using reagents from the (ToTALLY RNA isolation kit; Ambion Inc., Austin, TX, USA) and quality assessment by capillary electrophoresis were performed as previously described (Redmond et al., Blood Cells Molecules and Diseases 2006, 37:27).
|
| Label |
biotin
|
| Label protocol |
Ten to 39 ng of erythroid RNA was used to generate double stranded cDNA with the T7-oligo (dT) primer according to the Affymetrix GeneChip Two cycle target labeling protocol (Affymetrix Inc., Santa Clara, CA, USA). Labeled cRNA yield and purity was determined as described (Redmond et al., 2006).
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| |
|
| Hybridization protocol |
Fifteen μg of labeled erythroid RNA was fragmented and 10 μg was hybridized to Affymetrix GeneChip Mouse Genome 430A 2.0 arrays for 18 hours (Affymetrix Inc.). The 8 microarrays were washed and stained with streptavidin–phycoerythrin as described (Redmond et al., Blood Cells Molecules and Diseases 2006, 37:27).
|
| Scan protocol |
Image analysis was performed on the E9.5 KLF2-/- erythroid yolk sac data set using the GeneChip Operating System version 1.4 (GCOS1.1, Affymetrix Inc.). The image of the scanned array was stored as a DAT file using the Affymetrix software.
|
| Description |
Comparison between mouse embryonic day 9.5 (E9.5) wild-type and KLF2-/- yolk sac microdissected erythroid precursors
|
| Data processing |
Quality control parameters such as scaling factors used to normalize the chips, average background and noise were also analyzed. The raw intensities for each probe set were stored in electronic formats by the GCOS software. Scaling factors used to normalize the chips to a target value (TGT), and in this study, the TGT was set at 100. Probe set expression summaries were calculated with the Probe set expression summaries were calculated with the Robust Multi-average (RMA) algorithm (Irizarry et al., 2003). RMA expression summaries were obtained for wildtype (GSE10002) and KLF2-/- erythroid GeneChips using the rma function in the affy package in R/Bioconductor. The Multi-Chip Significance score (S-score) method was used to compare the 4 different wildtype and KLF2-/- GeneChips to determine significant changes in gene expression between these types of erythroid cells (Kennedy et al., 2006). S-score values were obtained using the SScore function in the sscore package in Bioconductor. Probe sets with absolute values of the S-score greater or equal to 2.00 were considered to correspond to significant differences between the two types of samples. P-values were calculated from S-score values and then used to obtain the q-values. The q-value, a quantifiable measure of the false discovery rate (FDR), was used to identify differentially expressed genes between the KLF2-/- and WT microarrays. The FDR for a test is defined as the number of false positives among the total number of significant results. For this study, a false discovery rate of 5% was used.
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| |
|
| Submission date |
Mar 01, 2011 |
| Last update date |
Mar 02, 2011 |
| Contact name |
Latasha Redmond |
| E-mail(s) |
[email protected]
|
| Phone |
513-636-1002
|
| Organization name |
Virginia Commonwealth University
|
| Department |
Department of Human and Molecular Genetics
|
| Lab |
Dr. Joyce A. Lloyd
|
| Street address |
401 College Street
|
| City |
Richmond |
| State/province |
Virginia |
| ZIP/Postal code |
23298 |
| Country |
USA |
| |
|
| Platform ID |
GPL8321 |
| Series (1) |
| GSE27602 |
KLF2-Regulated Gene Expression in Mouse Embryonic Yolk Sac Erythroid Cells |
|
| Relations |
| Reanalysis of |
GSM252738 |
