|
| Status |
Public on Aug 29, 2008 |
| Title |
BG-1 pre-mir-424 2 |
| Sample type |
RNA |
| |
|
| Source name |
Ovary
|
| Organism |
Homo sapiens |
| Characteristics |
poorly differentiated papillary carcinoma
Gender: female
|
| Treatment protocol |
Transfected with precursor miRNA hsa-miR-424 (Ambion) using siPORT NeoFX (Ambion)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy Mini Kit (Qiagen)
|
| Label |
Cy3
|
| Label protocol |
Streptavidin-Cy3 bound to cRNA amplified with biotin-16-UTP using the Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791).
A 0.5ug aliquot of total RNA from each sample was labeled using the Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791). RNA was first converted into single-stranded cDNA using reverse transcription and an oligo-dT primer containing the T7 RNA polymerase promoter then the single-stranded cDNA was copied to produce double-stranded cDNA molecules. The double-stranded cDNA was used in an overnight in vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporating biotin-16-UTP.
|
| |
|
| Hybridization protocol |
A total of 0.85ug of biotin-labeled cRNA was hybridized for 16 hours to Illumina's Sentrix MouseRef-8 Expression BeadChips (Illumina, San Diego, CA). Each BeadChip has 24,000 well-annotated RefSeq transcripts per sample with approximately 30-fold redundancy. The arrays were washed at both 55 degrees C and room temperature and then blocked. The biotinylated cRNA was detected with streptavidin-Cy3, washed a final time and the arrays were dried by spinning at 500 RPM for 4 minutes.
|
| Scan protocol |
Slides were scanned using Illumina's BeadStation 500X Genetic Analysis Systems scanner. The image data was extracted using BeadStudio software, version 3. (Illumina)
|
| Description |
Microarray expression data generated from Illumina Human_Ref-8_V2 Sentrix BeadChips hybridized and scanned using a single channel of Cy3 fluorescent label.
|
| Data processing |
Raw microarray data was analyzed using DIANE 6.0, a spreadsheet-based microarray analysis program. Microarray intensities were subjected to Z normalization. i.e. [(intensity of sample #1 from array A - avarage of all intensities from array A)/ standard diviation of all of intensities on array A]. Differently expressed cRNA species were identified between the different sample groups by the ratio of these normalized intensities.
|
| |
|
| Submission date |
Aug 29, 2008 |
| Last update date |
Jun 22, 2020 |
| Contact name |
Supriyo De |
| Organization name |
NIA-IRP, NIH
|
| Department |
Laboratory of Genetics and Genomics
|
| Lab |
Computational Biology & Genomics Core
|
| Street address |
251 Bayview Blvd
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21224 |
| Country |
USA |
| |
|
| Platform ID |
GPL6104 |
| Series (1) |
| GSE12615 |
Ovarian Cancer Cell Lines pre-microRNA Transfection |
|
