|
| Status |
Public on Dec 20, 2008 |
| Title |
Polysome Control Rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
Polysome fraction from lysates treated with control siRNAs
|
| Organism |
Chlorocebus aethiops |
| Characteristics |
kidney derived from CV-1 transformed with an origin-defective mutant of SV-44
Size fractionated RNA
|
| Treatment protocol |
For RHA downregulation , COS cells were transfected with siRNAs (Dharmacon) targeted to the coding region (nucleotides 2701-2721 and 2784-2803) of human RHA (NCBI accession code NM_001357), or scrambled RHA siRNAs to a final concentration of 0.5 nM.
|
| Growth protocol |
Seven 100 mm plates seeded with 1 x 106 cells were transfected with siRNAs targeting RHA or scrambled control siRNA. Incubation for 4 hours at 37°C in medium without serum was followed by a 24 hour incubation in 10% FBS medium. Cells were split and 24 hours later transfected again with siRNAs as above and incubated at 37°C for 24 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
The Ovation™ Biotin RNA Amplification and Labeling System (NuGen Technologies, Inc., San Carlos, CA) was used to prepare amplified, biotin-labeled cDNA from total RNA for following manufacturer’s instructions. Briefly, first strand cDNA was synthesized from 25 ng of total RNA using a unique first strand DNA/RNA chimeric primer and reverse transcriptase. Following double strand cDNA generation, amplification of cDNA was achieved by utilizing an isothermal DNA amplification process that involves repeated SPIA™ DNA/RNA primer binding, DNA duplication, strand displacement and RNA cleavage. The amplified SPIA™ cDNA was purified and subjected to a two-step fragmentation and labeling process. The fragmented/biotinylated cDNA content was measured in a ND-1000 spectrophotometer and the quality was analyzed on an RNA 6000 Nano LabChip (Agilent) using Agilent Bioanalyzer 2100.
|
| |
|
| Hybridization protocol |
Following fragmentation, labeled cRNA was hybridized to the Rhesus Macaque Genome Array. Hybridization was allowed to continue for 16 hours at 45ºC followed by washing and staining of microarrays in a Fluidics Station 450 (Affymetrix Inc., USA).
|
| Scan protocol |
GeneChips were scanned in a GeneChip Scanner 3000 (Affymetrix Inc., USA)
|
| Description |
RNAs in the polysome fraction under normal levels of RHA
|
| Data processing |
CEL files were generated from DAT files using GeneChip® Operating Software (GCOS) software (Affymetrix Inc., USA). The probe set signals were generated using the RMA algorithm in ArrayAssist 3.4 (Stratagene) and were used to determine differential gene expression by pair-wise comparisons. The genes that were altered by two-fold either ways were sorted and used for further interpretation of the microarray data.
|
| |
|
| Submission date |
Dec 19, 2008 |
| Last update date |
Dec 19, 2008 |
| Contact name |
Kathleen Boris-Lawrie |
| E-mail(s) |
Kathleen.Boris-Lawrie@cvm.osu.edu
|
| Phone |
(614) 292-1392
|
| Fax |
(614) 292-6473
|
| URL |
http://www.vet.ohio-state.edu/KathleenBoris-Lawrie.htm
|
| Organization name |
The Ohio State University
|
| Department |
Department of Veterinary Biosciences
|
| Street address |
1925 Coffey Rd
|
| City |
Columbus |
| State/province |
OH |
| ZIP/Postal code |
43210 |
| Country |
USA |
| |
|
| Platform ID |
GPL3535 |
| Series (2) |
| GSE14055 |
Changes in polysome loading as a consequence of RHA downregulation |
| GSE14057 |
Identification of transcripts regulated by RNA helicase A (RHA) |
|
