|
| Status |
Public on Feb 14, 2015 |
| Title |
Ovol1 KO, biological rep1 (no calcium) |
| Sample type |
RNA |
| |
|
| Source name |
isolated keratinocytes from mouse skin
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: Skin
cell type: keratinocyte
gender: female
age: E18.5
genotype/variation: Ovol1 KO
|
| Treatment protocol |
Control (Ovol2flox/+; Ovol1+/- or Ovol2flox/+;K14-Cre, 2 mice/array) and Ovol1 knockout (Ovol1-/-, 2 mice/array) and Ovol1/2 double conditional knockout (Ovol2flox/-;K14-Cre; Ovol1-/-, 2 mice/array)
|
| Growth protocol |
Littermates were raised in the same cages.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA followed by column purification (Qiagen) was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
The Ambion WT expression kit (Life Technologies, Carlsbad, CA) was used to prepare RNA samples for whole transcriptome microarray analysis. Briefly, random hexamers that are tagged with a T7 promoter are used in first strand synthesis of cDNA. Then using the T7 promoter, second strand synthesis is performed and the double stranded cDNA is subsequently used as a template in an in vitro transcription reaction to generate many copies of antisense cRNA. 10ug of antisense cRNA is input into a second cycle cDNA reaction using reverse transcriptase and random hexamers to produce single stranded DNA in the sense orientation. The single-stranded DNA is fragmented to an average length of 70 bases and then labeled using a recombinant terminal deoxynucleotidyl transferase (TdT) and an Affymetrix proprietary DNA labeling reagent that is covalently linked to biotin.
|
| |
|
| Hybridization protocol |
3.3ug of the labeled, fragmented single-stranded cDNA is hybridized at 45oC with rotation for 17 hours (Affymetrix GeneChip Hybridization Oven 640) to probe sets present on an Affymetrix GeneChip 1.0ST array. The GeneChip arrays were washed and then stained with streptavidin-phycoerythrin on an Affymetrix Fluidics Station 450 (Fluidics protocol FS450_007).
|
| Scan protocol |
Arrays were scanned using GeneChip Scanner 3000 7G and Command Console Software v. 3.2.3 to produce .CEL intensity files.
|
| Description |
Gene expression data from isolated keratinocytes from skin
Ovol2flox/+; Ovol1+/- or Ovol1-/- or Ovol2flox/-;K14-Cre; Ovol1-/-
|
| Data processing |
The probe cell intensity files (*.CEL) were analyzed in Affymetrix Expression Console software v1.1.1 using the PLIER algorithm to generate probe level summarization files (*.CHP). The settings used were algorithm-PLIER v2.0; quantification scale-Linear; quantification type-signal and detection p-value; background-PM-GCBG; normalization method-sketch-quantile.
|
| |
|
| Submission date |
Feb 17, 2014 |
| Last update date |
Feb 14, 2015 |
| Contact name |
Xing Dai |
| E-mail(s) |
xdai@uci.edu
|
| Organization name |
UC Irvine
|
| Street address |
Med Sci I D288
|
| City |
Irvine |
| State/province |
Ca |
| ZIP/Postal code |
92692 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (2) |
| GSE55072 |
Expression data from isolated keratinocytes from mouse skin with or without Ovol1 and Ovol2 expression |
| GSE55075 |
Ovol as gatekeepers of epithelial adhesion and differentiation |
|
