|
| Status |
Public on Jan 03, 2008 |
| Title |
epithelial; pair 4 |
| Sample type |
RNA |
| |
|
| Source name |
Pair 4-Mus musculus E9.5 yolk sac epithelial cells
|
| Organism |
Mus musculus |
| Characteristics |
FVB/N 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, and 1 epithelial cell was collected per pulse on LCM HS Capsure caps. Epithelial cells were collected from the same microscope slides immediately following procurement of the erythroid cells, and were used as the non-erythroid control. 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 4 different pairs of erythroid and epithelial 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 or epithelial 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 or epithelial cRNA 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 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 |
Gene expression data from pair 4 E9.5 yolk sac epithelial cells
|
| Data processing |
Quality control parameters such as scaling factors used to normalize the chips, average background and noise were analyzed and similar, thus making all comparisons valid. 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 Microarray Suite version 5.0 (MAS5) algorithm. MAS5 expression summaries were obtained using the mas5 function in the affy package in R/Bioconductor. Significance score (S-score) method was used to compare 4 sets of paired GeneChips to determine significant changes in gene expression between erythroid and epithelial cells. A paired set consisted of an erythroid and epithelial GeneChip, for which the two different cell types were isolated from the same yolk sacs. It uses an error-based model to determine the variances for probe pair signals and follows a normal standard distribution. 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 were combined using Fisher’s procedure for combining p-values.
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| |
|
| Submission date |
Dec 21, 2007 |
| Last update date |
Mar 18, 2009 |
| Contact name |
Latasha C Redmond |
| E-mail(s) |
[email protected]
|
| Phone |
807-628-2183
|
| Fax |
804-827-0810
|
| Organization name |
Virginia Commonwealth University
|
| Department |
Human Genetics
|
| Lab |
Dr. Joyce A. Lloyd
|
| Street address |
401 College Street
|
| City |
Richmond |
| State/province |
VA |
| ZIP/Postal code |
23298 |
| Country |
USA |
| |
|
| Platform ID |
GPL8321 |
| Series (1) |
| GSE10002 |
Identification of Erythroid-Enriched Gene Expression in the Mouse Embryonic Yolk Sac using Microdissected Cells |
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