|
| Status |
Public on Jan 13, 2016 |
| Title |
plasma_MDS_12 |
| Sample type |
RNA |
| |
|
| Source name |
peripheral blood plasma RNA, MDS patient no. 12
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
age: 64y
diagnosis: RAEB-2
iPSs risk category: int-2
sample type: blood plasma
|
| Treatment protocol |
The patients has not been subjected to the disease therapy or hematopoietic stem cell transplantation before the blood collection.
|
| Growth protocol |
Czech patients with primary MDS and healthy donors with no known history of previous malignancy, chemotherapy or radiation therapy were tested.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Plasma was separated from peripheral blood (centrifugation at 460 g, 4°C for 10 minutes) and stored at -80°C. After thawing on ice, plasma samples were centrifuged (12000 g, 4°C for 10 minutes) to dispose of cell debris. Samples (500 μl) were further homogenized with equal volume of Trizol LS reagent (Invitrogen, Basel, Switzerland), incubated at room temperature (5 minutes) and centrifuged (12000 g, 4°C for 10 minutes). Aqueous phase containing RNA was transferred to a fresh tube. To maximize RNA recovery, we reextracted the organic phase again. RNA was precipitated by 100% isopropanol (500 μl) with 100 μg of glycogen, incubated for 10 minutes at room temperature and subsequently centrifuged (12000 g, 4°C for 10 minutes). The pellet was washed with 75% ethanol, spinned at 7500 g, 4°C for 5 minutes and air dried. RNA was dissolved in RNase-free water and stored at -80°C.
|
| Label |
Cy3
|
| Label protocol |
Labeling was performed using Agilent miRNA Labeling and Hybridization kit according to the manufacturer's instructions. Total RNA (350 ng) was dephosphorylated with calf intestine alkaline phosphatase (CIP), followed by RNA denaturation with DMSO and 10 min incubation at 100°C. Dephosphorylated RNA was ligated with pCp-Cy3 mononucleotide and purified with MicroBioSpin 6 columns (Bio-rad, USA).
|
| |
|
| Hybridization protocol |
After purification, labeled samples were resuspended with Gene Expression blocking Reagent and Hi-RPM Hybridization buffer, followed by incubation at 100°C for 5 min. Finally, denatured labeled probes were pipetted onto assembled Agilent miRNA Microarray and hybridized for 20 hours at 5°C with 20 RPM rotating in Agilent Hybridization oven.
|
| Scan protocol |
The hybridized images were scanned using Agilent DNA microarray scanner and quantified with Agilent Feature Extraction Software.
|
| Data processing |
Quality control and background subtraction of raw microarray data were carried out by Agilent Feature Extraction Software. Only those miRNAs whose signal was detected in more than 4 samples at least in one analyzed group were included for further analysis. Quantile normalization was performed in R statistical environment (www.r-project.org).
|
| |
|
| Submission date |
Jan 12, 2016 |
| Last update date |
Jan 13, 2016 |
| Contact name |
Michaela Dostalova Merkerova |
| E-mail(s) |
michaela.merkerova@uhkt.cz
|
| Organization name |
Institute of Hematology and Blood Transfusion
|
| Department |
Department of Genomics
|
| Street address |
U nemocnice 1
|
| City |
Prague 2 |
| ZIP/Postal code |
12820 |
| Country |
Czech Republic |
| |
|
| Platform ID |
GPL18402 |
| Series (1) |
| GSE76775 |
Low plasma levels of miR-451a, miR-223-3p, and miR-27a-3p predict adverse prognosis in myelodysplastic syndromes |
|
