|
| Status |
Public on Jul 10, 2017 |
| Title |
F66 |
| Sample type |
RNA |
| |
|
| Source name |
Retinoblastoma
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: Retinoblastoma
Sex: male
type: unilateral
|
| Growth protocol |
12 Rb from enucleated patients with no chemotherapy history were maintained in BSF 12% complemented RPMI medium for a week and collected in lysis buffered reagent. All samples came from patients of Children Hospital of Mexico “Federico Gomez” and Pediatric Hospital “Silvestre Frenk Freund” of Mexican Social Security Institute in Mexico City.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracter from each sample using TRIzol reagent and was suspended in nuclease free water and stored at -70°C.
|
| Label |
Biotin
|
| Label protocol |
Total RNA was tailing and biotinylated using Affymetrix Flash-tag biotin microarray (Affymetrtix, USA) for miRNAs and spike-in controls probes were added. Poli-A tailing on end 3’ was carried out at 37°C in 15ul reaction volume contained 1x reaction buffer, MnCl2 25mM, 1ul of 1:50 ATP mix and 1ul of phosphatidic acid phosphatase (PAP) polymerase. The biotin was added to this poli-A tails at 25°C adding 4ul of ligation mix containing biotin and 2ul of T4 DNA ligase making a total of 21ul then 2.5ul of stop solution was added. An Enzyme Linked Oligosorbent (ELOSA) was performed to confirm previous biotin labeling process. 2ul from sample were used to detect spike control oligos by colorimetric reaction revealed with HRP and measured UV absorbance. Every sample which absorbance is 1.5nm more than negative control is considered as successfully labeled and tailed.
|
| |
|
| Hybridization protocol |
The sample with hybridization cocktail was incubated at 99°C and 45°C for 5 minutes respectively. Finally a total of 130ul of sample labeled and prepared were injected in a new GeneChip® miRNA 4.0 array and hybridized at 48°C for 16-18 hours.
|
| Scan protocol |
Every sample was washed in the GeneChip® Fluidics Station 450 following FS450-0002 protocol and array fluorescence was measured by the GeneChip ® Scanner 3000 7G.
|
| Data processing |
Raw data from .CEL files were analyzed using Affymetrix® Expression ConsoleTM software to normalize fluorescence signals and to obtain the quality control data. To adjust background signal a Robust Multichip Analysis (RMA) was carried out and once completed we observed that the values of spike_in-control probes comply the quality standards established by the manufacturer.
|
| |
|
| Submission date |
Jul 22, 2016 |
| Last update date |
Jul 10, 2017 |
| Contact name |
Blanca E Castro |
| E-mail(s) |
blanqui86@gmail.com
|
| Organization name |
CMNSXXI
|
| Street address |
Cuauhtemoc 330
|
| City |
Mexico |
| State/province |
Mexico City |
| ZIP/Postal code |
06720 |
| Country |
Mexico |
| |
|
| Platform ID |
GPL21572 |
| Series (1) |
| GSE84747 |
RETINOBLASTOMA MICRORNA LANDSCAPE |
|
