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| Status |
Public on Apr 01, 2015 |
| Title |
581 MCL patient/AM6000 |
| Sample type |
RNA |
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| Source name |
MCL patient
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| Organism |
Homo sapiens |
| Characteristics |
disease state: mantle cell lymphoma
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| Treatment protocol |
Tumor samples were taken prior to treatment of patients. All patients subsequently underwent treatment with the 'Nordic Regimen': R-maxiCHOP, alternating Rituximab and cytarabine. Responders were treated with BEAM/BEAC followed by autologous stem cell support.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from all samples was isolated from four 10μm tumor tissue sections using the RecoverAll total nucleic acid isolation kit (Ambion, Carlsbad, CA, USA) according to the manufacturer’s guidelines. The samples were incubated in Xylene at 50°C to remove paraffin, followed by ethanol wash. Proteins were degraded by digestion buffer and protease at 50/80°C. Subsequently, isolation buffer and ethanol were added, samples were bound to a spin-column, and DNA was degraded by DNAse treatment. The filter was washed and total RNA was eluted in a 60 μL elution solution. Total RNA quantity (OD 260 nm) and quality (260/280 ratio) were measured by an ND-1000 spectrophotometer (Thermo Scientific, Delaware, USA).
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| Label |
Hy3
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| Label protocol |
For miRNA microarray profiling, 500 ng of total RNA from samples or reference (AM6000; common RNA pool for identifying a maximum number of expressed miRNAs in patient samples, Ambion, Carlsbad, CA, USA) was labeled with Hy3 and Hy5 fluorescent dyes, respectively, using the miRCURY LNA array power labeling kit (Exiqon A/S, Vedbæk, Denmark). Samples with RNA concentrations lower than 160 ng/µl were vacuum-centrifuged (speedvac) to obtain higher concentrations. All samples were labeled the same day with the same master mix to minimize technical variation
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| Hybridization protocol |
The hybridization was performed overnight at 56°C according to the manufacturer’s specifications using an HS4800 hybridization station (Tecan, Männendorf, Switzerland). Since it was not possible to hybridize all arrays at the same time point, samples were randomly split into six batches to minimize day-to-day variation in the hybridization process, and analyzed over six consecutive days. After hybridization, the microarray slides were scanned in an ozone free environment in order to prevent potential bleaching of the fluorescent Hy5 dye.
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| Scan protocol |
Scanning was done using a G2565BA Microarray Scanner system (Agilent Technologies, Santa Clara, CA, USA) at 10-µm resolution
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| Data processing |
Resulting TIFF images were analyzed using the GenePix 6.1 software on standard settings. Due to a confounding batch effect in the background (Hy5) intensities, we ended up only using the Hy3 signal and as such we treat the array data as single channel.
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| Submission date |
Feb 25, 2015 |
| Last update date |
Apr 01, 2015 |
| Contact name |
Ulrik Ralfkiaer |
| E-mail(s) |
ralfkiaerulrik@gmail.com
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| Phone |
+4522765232
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| Organization name |
Copenhagen University Hospital
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| Department |
Hematology
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| Lab |
Epi-genome
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| Street address |
Blegdamsvej 9
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| City |
Copenhagen |
| State/province |
N/A |
| ZIP/Postal code |
2100 |
| Country |
Denmark |
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| Platform ID |
GPL19128 |
| Series (1) |
| GSE66299 |
miR-18b overexpression identifies mantle cell lymphoma patients with poor outcome and improves the MIPI-B prognosticator |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM1619063_581.gpr.gz |
1.2 Mb |
(ftp)(http) |
GPR |
| Processed data are available on Series record |
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