|
| Status |
Public on Sep 25, 2017 |
| Title |
Lin-CD34-CD38- CML#5 |
| Sample type |
RNA |
| |
|
| Source name |
Chronic Myeloid Leukemia, Peripheral Blood
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: Chronic Myeloid Leukemia (CML)
subject id: patient 5
tissue: peripheral blood
cell markers: Lin-CD34-CD38-
|
| Treatment protocol |
Leukemic cells were obtained from 5 chronic phase Ph+ CML patients at diagnosis. Normal samples were leukapheresis products from 4 healthy donors receiving recombinant human granulocyte colony-stimulating factor (G-CSF; Lenograstim; Sanofi-Aventis).
|
| Growth protocol |
Cells were lysated right after isolation, no growth in culture
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was harvested from 1×10^5 cells from each sample using the miRNeasy Micro RNA isolation kit (QIAGEN), according to the manufacturer’s instructions. RNA samples concentration and purity (assessed as 260/280 nm and 260/230 nm ratios) were evaluated by NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies; Wilmington, DE), while RNA integrity was assessed by using the Agilent 2100 Bioanalyzer (Agilent Technologies; Waldbrunn, Germany).
|
| Label |
SYBR Green
|
| Label protocol |
miEP was performed by using the miRCURY LNATM Universal RT microRNA PCR system, Ready-to-use Human miRNome Panels I and II (Exiqon, Copenhagen, Denmark).
|
| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
n/a
|
| Description |
miEP of Lineage-Negative CD34-negative CD38-negative cells CML patient #5
Sample name: CML 5 34-38-
|
| Data processing |
RT-qPCR data were analysed using the Exiqon GenEx software for the inter-plate calibration, the definition of a Ct=40 as detection cutoff, the labeling of not-amplified wells as missing data and the setting of a cutoff for miRNAs with a low call-rate (i.e. assays with a detection rate <33,3%, i.e. assays not detected in 12 out of 18 samples, were excluded from analysis). For RT-qPCR data normalization, the comparative cycle threshold (CT) method was used by setting miR-23a-3p, miR-24-3p and miR-324-3p as reference miRNAs, as the best normalizers identified among the candidates by the NormFinder algorithm. The RQ value was expressed as 2−ΔΔCT.
Statistical analyses on ΔCT values between CML Lin-CD34-CD38- and Normal donor Lin-CD34-CD38- (set as calibrator) and between CML Lin-CD34+CD38- and Normal donor Lin-CD34+CD38- (set as calibrator) were performed by a two-tail unpaired t-Student test using the GenEx software. Fold Change (FC) worksheet reports the relative quantity (RQ) = 2^-ΔΔCt. To make the RQ value symmetric for up- and down-regulated genes, FC was used in the Tables for ease of interpretation (for RQ > 1, FC = RQ; for RQ < 1 FC = −1/RQ).
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| |
|
| Submission date |
Dec 01, 2016 |
| Last update date |
Sep 25, 2017 |
| Contact name |
Rossella Manfredini |
| E-mail(s) |
manfredini.rossella@unimore.it
|
| Phone |
+390592058065
|
| Organization name |
Centre for Regenerative Medicine
|
| Department |
Life Sciences
|
| Street address |
Via Gottardi 100
|
| City |
Modena |
| ZIP/Postal code |
41100 |
| Country |
Italy |
| |
|
| Platform ID |
GPL19066 |
| Series (1) |
| GSE90773 |
Real-time quantitative PCR analysis of microRNAs in CML stem cells |
|
