|
| Status |
Public on Jul 19, 2012 |
| Title |
scrambled siRNA biological replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
primary human normal neonatal keratinocytes (NHEK)
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: primary human normal neonatal keratinocytes
treatment: scrambled siRNA
|
| Treatment protocol |
Cells were transfected with either scrambled control or GRHL3 siRNA (Ambion) by reverse transcription using RNAi Max (Life Technologies). 24 hours post-transfection medium was changed to DermaLife containing 1.8mM Calcium
|
| Growth protocol |
NHEK cells were grown in DermaLife medium (LifeLine Cell Technologies) at 37C, 5%CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Isolated total RNA samples were processed as recommended by Affymetrix, Inc. (Affymetrix GeneChip Whole Transcript Sense Target Labeling Assay Manual, Affymetrix, Inc., Santa Clara, CA). In brief, total RNA was initially isolated using TRIzol Reagent (Gibco BRL Life Technologies, Rockville, MD), and then put through an RNeasy spin column (Qiagen, Chatsworth, CA) for further clean up. Eluted total RNAs were quantified by NanoDrop (ThermoScientific, Wilmington, DE) and the concentrations of sample aliquots were adjusted to 100ng/ul. Total RNA samples were assessed for quality prior to performing target preparation/processing steps by loading approximately 25-250ng of each sample onto a RNA 6000 Nano LabChip and evaluated on an Agilent Biolanalyzer 2100 (Agilent Technologies, Palo Alto, CA).
|
| 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 |
2ug 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.
|
| 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 |
Apr 04, 2012 |
| Last update date |
Jul 19, 2012 |
| Contact name |
Amelia Soto Hopkin |
| E-mail(s) |
[email protected]
|
| Phone |
9498249372
|
| Organization name |
University of California Irvine
|
| Department |
Biological Chemistry & Medicine
|
| Lab |
Bogi Andersen
|
| Street address |
250 Sprague Hall
|
| City |
Irvine |
| State/province |
CA |
| ZIP/Postal code |
92617 |
| Country |
USA |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE37049 |
Analysis of gene expression in differentiated human primary keratinocytes depleted for Grainyhead like 3 (GRHL3) |
|
