|
| Status |
Public on Feb 01, 2012 |
| Title |
HeLa DGCR8 siRNA #2, mRNA |
| Sample type |
RNA |
| |
|
| Source name |
HeLa cells, DGCR8 siRNA-2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
cell type: cervical adenocarcinoma
treatment: DGCR8 siRNA-2
|
| Treatment protocol |
HeLa cells were transfected with siRNAs using RNAiMAX (Invitrogen) according to the manufacturer's recommendations.
|
| Growth protocol |
HeLa cells were grown in DMEM containing 10% FCS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol reagent according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
2ug of total RNA was converted to first-strand cDNA using Superscript II reverse transcriptase primed by a poly(T) oligomer that incorporated the T7 promoter. Second-strand cDNA synthesis was followed by in vitro transcription for linear amplification of each transcript and incorporation of biotinylated CTP and UTP.
|
| |
|
| Hybridization protocol |
The cRNA products were fragmented to 200 nucleotides or less, heated at 99C for 5 min and hybridized for 16 h at 45C to Affymetrix Human Gene 1.0 ST microarrays. The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
| Scan protocol |
An Affymetrix GCS3000 laser scanner was used to collect fluorescence signal at 3um resolution after excitation at 570 nm. The average signal from two sequential scans was calculated for each microarray feature.
|
| Description |
Gene expression data from DGCR8 siRNA #2-transfected HeLa cells.
|
| Data processing |
Affymetrix Microarray Suite 5.0 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. 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 total chip 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.
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| |
|
| Submission date |
Dec 22, 2011 |
| Last update date |
Feb 01, 2012 |
| Contact name |
Scott H Olejniczak |
| E-mail(s) |
olejnics@mskcc.org
|
| Phone |
716-912-2723
|
| Organization name |
Memorial Sloan Kettering Cancer Center
|
| Street address |
417 E68th St
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10044 |
| Country |
USA |
| |
|
| Platform ID |
GPL6244 |
| Series (2) |
| GSE34679 |
Effects of Ars2 or DGCR8 siRNA on gene expression |
| GSE34681 |
Effects of Ars2 or DGCR8 siRNA on gene and microRNA expression |
|
