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| Status |
Public on Nov 02, 2012 |
| Title |
Comparative microarray analysis of microRNA expression profiles in primary cutaneous malignant melanoma, cutaneous malignant melanoma metastases and benign melanocytic naevi |
| Organism |
Homo sapiens |
| Experiment type |
Non-coding RNA profiling by array
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| Summary |
Perturbations in microRNA (miRNA) expression profiles has been reported for cutaneous malignant melanoma (CMM). With regards to a rapidly growing number of newly discovered miRNA sequences, the availability of up-to-date miRNA expression profiles for primary cutaneous malignant melanoma (PCMM), cutaneous malignant melanoma metastases (CMMM) and benign melanocytic naevi (BMN) is limited. Patients with PCMM (n=9), CMMM (n=4) and BMN (n=8) were included in the study. Specimens were obtained during surgery from the center of the tumors (lesional). An exploratory microarray analysis was performed by miRNA expression profiling based on miRBase V.16. Additionally, the expression levels of selected miRNA candidates were confirmed by TaqMan real-time quantitative polymerase chain reaction (RT-PCR). New miRNA candidates previously not described to be associated with CMM were found to be potentially dysregulated in CMM.
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| Overall design |
Nine patients with PCMM (PCMM1-PCMM9), four patients with CMMM (CMMM1-CMMM4) and eight patients with BMN (BMN1-BMN8) were enrolled in the present study. All cutaneous specimens were harvested in the operating room, immediately stored in RNAlater (Quiagen, Hilden, Germany) and kept at - 80 °C until RNA isolation. Total RNA including miRNAs was isolated using the miRNeasy Mini Kit (Quiagen, Hilden, Germany) according to the manufacturer’s protocol. RNA concentration and purity was determined by NanoDrop ND-1000 spectral photometer (Peqlab, Erlangen, Germany). RNA integrity control (RIN) was determined by means of capillary electrophoresis with the 2100 Bioanalyzer and the RNA 6000 NanoLabCHip Kits (both Agilent Technologies, Santa Clara, USA). To be further considered for Microarray analysis a RIN ≥ 7.0 indicating medium to good RNA quality was defined as inclusion criteria. For assessment of labeling and hybridization efficiencies total RNA samples were spiked with in-vitro synthesized oligonucleotides by using the MicroRNA Spike-In Kit (Agilent Technologies, Santa Clara, USA). A total of 100 ng total RNA per sample were introduced into a labeling reaction. The spiked total RNA was treated with alkaline calf intestine phosphatase (CIP). The dephosphorylated RNA was labeled with the miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, USA) according to the manufacturer´s instructions using T4 RNA ligase, incorporating Cyanine 3-Cytidine biphosphate (pCp). The Cyanine-3-labeled miRNA samples were prepared for One-Color based hybridization with Complete miRNA Labeling and Hyb Kit (Agilent Technologies, Santa Clara, USA) according to the manufacturer´s instructions. Labeled miRNA samples were hybridized at 55°C for 20 hrs on separate Human miRNA Microarrays Release 16.0 (8x60K format, (Agilent Technologies, Santa Clara, USA). Afterwards, microarrays were washed with increasing stringency using Gene Expression Wash Buffers (Agilent Technologies, Santa Clara, USA) followed by drying with acetonitrile (Sigma-Aldich, St.Louis, USA). Fluorescent signal intensities were detected with Scan Control A.8.4.1 Software (Agilent Technologies, Santa Clara, USA) on the Agilent DNA Microarray Scanner and extracted from the images using Feature Extraction 10.7.3.1 Software (Agilent Technologies, Santa Clara, USA). All the steps described were carried out according to the manufacturer´s instructions.
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| Contributor(s) |
Sand M, Skrygan M, Sand D, Georgas D, Gambichler T, Hahn SA, Altmeyer P, Bechara FG |
| Citation(s) |
23111773 |
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| Submission date |
Dec 14, 2011 |
| Last update date |
Mar 08, 2013 |
| Contact name |
Michael Sand |
| Organization name |
Ruhr-University Bochum
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| Department |
Depatment of Dermatology, Venereology and Allergology
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| Street address |
Gudrunstr. 56
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| City |
Bochum |
| State/province |
NRW |
| ZIP/Postal code |
44791 |
| Country |
Germany |
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| Platforms (1) |
| GPL15019 |
Agilent-031181 Unrestricted_Human_miRNA_V16.0_Microarray 030840 (miRBase release 14.0 miRNA ID version) |
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| Samples (21)
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| Relations |
| BioProject |
PRJNA151195 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE34460_RAW.tar |
162.5 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data included within Sample table |
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