|
| Status |
Public on Jul 30, 2012 |
| Title |
GlycophorinA positive cells_severe_Eb thalassemia |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral blood
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: Eb thalassemia
disease severity: severe
tissue: Peripheral blood
cell type: GlycophorinA positive cells
gender: Female
age: 25Yrs
|
| Treatment protocol |
PANCOLL (PAN Biotech GmbH) was used to isolate reticulocyte, light density erythrocyte and erythropoietic progenitor cells which were further purified by immmunomagnetic labelling using Glycophorin A (CD235+) MicroBeads (human, MACS Miltenyi Biotec) (Lot number 5090616269)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using using mirVana miRNA Isolation kit (Ambion, AM1560) following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
miRNA labeling was performed as described in Agilent miRNA microarray protocol version 2.0 using Agilent miRNA labeling kit. 100 ng of total RNA was dephosphorylated with calf intestine alkaline phosphatase for 30 min at 37°C. Denaturation was performed by adding DMSO and incubating at 100°C for 7 min and immediately transferred to ice water bath. Ligation was performed with pCp-Cy3 at 16 °C for 2 h. The labeled samples were dried completely in a vaccum concentrator and resuspended in 18 ul of nuclease free water.
|
| |
|
| Hybridization protocol |
The hybridization mixture [10X GE blocking agent (4.5 ul), 2X Hi-RPM hybridization buffer (22.5ul)] along with labeled miRNA sample was heated for 5 min at 100 °C and immediately cooled to 0 °C. Each 45 mL sample was hybridized onto a microarray at 55 °C for 20 h. Slides were washed 5 min in GE wash buffer 1 at RT and again for 5 min in GE wash buffer 2 at 37 °C, followed by an acetonitrile wash for 1 min at RT to dry the slides completely.
|
| Scan protocol |
Scanned on an Agilent G2505C scanner
|
| Description |
MicroRNA expression in GlycophorinA positive cells of severe Eb thalassemia patient
|
| Data processing |
Data extraction done using Agilent Feature extraction software v 10.5.1.1 and obtained background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Sep 29, 2010 |
| Last update date |
Jul 30, 2012 |
| Contact name |
Uma B Dasgupta |
| E-mail(s) |
dasgupta.uma@gmail.com
|
| Phone |
9007952774
|
| Organization name |
Calcutta University
|
| Department |
Biophysics, Molecular Biology & Bioinformatics
|
| Street address |
92, APC Road
|
| City |
Kolkata |
| ZIP/Postal code |
700009 |
| Country |
India |
| |
|
| Platform ID |
GPL7731 |
| Series (1) |
| GSE24428 |
MicroRNA expression profile in GlycophorinA positive cells of severe and intermedia Eb thalassemia |
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