Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Microarray analysis of microRNA expression in cutaneous squamous cell carcinoma

ABSTRACT: MicroRNAs (miRNAs) are a novel class of short RNAs which have shown to be dysregulated in a variety of cancers including squamous cell carcinoma (SCC) of the head&neck. Microarray based miRNA expression profiles of cutaneous SCC (cSCC) however have not been investigated so far. Seven patients with cutaneous SCC were enrolled in the study. Tumor biopsies (n=7) were taken from the center of the tumor. Adjacent healthy skin (n=7) was biopsied as a control (intraindividual control). miRNA expression profiles of all specimen were detected by mircroarray miRNA expression profiling based on miRBAse 16 and compared. In 7 patients biopsy specimens of cutaneous SCC and control specimens from an adjacent healthy skin site near the tumor border (intraindividual control) were obtained during surgical removal of the tumor. Specimes were immediately stored in RNAlater (Qiagen, HIlden, Germany) at -80 M-BM-0C. Total RNA including miRNAs were isolated with miRNeasy Mini Kit (Quiagen, Hilden, Germany) according to the manufacturerM-bM-^@M-^Ys protocol. For RNA quality control purposes we determined RNA concentration, purity and RNA integrity number (RIN) with Agilent 2100 Bioanalyzer, RNA 6000 NanoLabCHip Kits (both Agilent Technologies, Santa Clara, USA) and the NanoDrop ND-1000 spectral photometer (Peqlab, Erlangen, Germany). All of the following steps described were carried out according to the manufacturerM-BM-4s protocol. In order to enable assessment of labeling and hybridization efficiencies total RNA samples were spiked with MicroRNA Spike-In Kit (Agilent Technologies, Santa Clara, USA). After treatment with calf intestine phosphatase (CIP), a labeling reaction was started with 100 ng total-RNA per sample. For labeling dephosphorylated RNA, T4 RNA ligase, incorporating Cyanine 3-Cytidine biphosphate (miRNA Complete Labeling and Hyb Kit, Agilent Technologies, Santa Clara, USA) was used. The Cyanine-3-labeled miRNA samples were then prepared for One-Color based hybridization (Complete miRNA Labeling and Hyb Kit, Agilent Technologies, Santa Clara, USA). Hybridization was performed at 55M-BM-0C for 20 hrs with Human miRNA Microarrays Release 16.0, 8x60K format (Agilent Technologies, Santa Clara, USA). Microarray slides were washed (Gene Expression Wash Buffers, Agilent Technologies, Santa Clara) and dried with acetonitrile (Sigma-Aldich, St.Louis, USA). Fluorescent signal intensities were detected with Agilent DNA Microarray Scanner (Agilent Technologies, Santa Clara, USA) and Scan Control A.8.4.1 Software (Agilent Technologies, Santa Clara, USA). Extraction of data was done by using the Feature Extraction 10.7.3.1 Software (Agilent Technologies, Santa Clara, USA).

ORGANISM(S): Homo sapiens

SUBMITTER: Michael Sand

PROVIDER: E-GEOD-34536 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Microarray analysis of microRNA expression in cutaneous squamous cell carcinoma.

Sand Michael M Skrygan Marina M Georgas Dimitrios D Sand Daniel D Hahn Stephan A SA Gambichler Thilo T Altmeyer Peter P Bechara Falk G FG

Journal of dermatological science 20120913 3

<h4>Background</h4>MicroRNAs (miRNAs) are a novel class of short RNAs that are capable epigenetically regulating gene expression in eukaryotes. MicroRNAs have been shown to be dysregulated in a variety of cancers. The data on miRNA expression in cutaneous squamous cell carcinoma (cSCC) are very limited, and microarray-based miRNA expression profiles of cSCC have not yet been determined.<h4>Objective</h4>To describe differentially expressed miRNAs in cSCC.<h4>Methods</h4>Seven patients with cSCC ...[more]

PMID: 23026055

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Project description:Perturbations in the expression profiles of microRNAs (miRNAs) have been reported for a variety of different cancers. Basal cell carcinoma (BCC) of the skin has not been systematically evaluated regarding differentially expressed miRNAs. Patients with BCC (n=7) were included in the study. Punch biopsies were harvested from the center of the tumors (lesional, n=7) and from sites of adjacent non-lesional skin (intraindividual control, n=7). Microarray miRNA expression profiles were detected based on an Agilent platform and miRBase 16. For validation purposes the expression of a subset of dysregulated miRNAs were measured by means of TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR). Seven patients with basal cell carcinoma (BCC) were enrolled in the present study. While BCCs were excised with cold steel under local anaesthesia, a 4-mm punch biopsy was taken from the center of the tumor and from adjacent non-lesional epithelial skin (control), immediately stored in RNAlater (Qiagen, HIlden, Germany) and stored at -80 M-BM-0C. miRNAs were isolated with miRNeasy Mini Kit (Quiagen, Hilden, Germany) according to the manufacturerM-bM-^@M-^Ys protocol. RNA concentration, purity and RNA integrity number (RIN) were determined with NanoDrop ND-1000 spectral photometer (Peqlab, Erlangen, Germany), Agilent 2100 Bioanalyzer and the RNA 6000 NanoLabCHip Kits (both Agilent Technologies, Santa Clara, USA). total RNA samples were spiked with the MicroRNA Spike-In Kit (Agilent Technologies, Santa Clara, USA) for assessment of labeling and hybridization efficiencies. After the spiked total RNA was treated with alkaline calf intestine phosphatase (CIP), a labeling reaction was started with 100 ng total-RNA per sample. T4 RNA ligase, incorporating Cyanine 3-Cytidine biphosphate (miRNA Complete Labeling and Hyb Kit, Agilent Technologies, Santa Clara, USA) was used to label the dephosphorylated RNA. Cyanine-3-labeled miRNA samples were subsequently prepared for One-Color based hybridization (Complete miRNA Labeling and Hyb Kit, Agilent Technologies, Santa Clara, USA). Hybridization of the labeled miRNA samples with Human miRNA Microarrays Release 16.0 (Agilent Technologies, Santa Clara, USA) was done at 55M-BM-0C for 20 hrs. After washing microarray slides with increasing stringency (Gene Expression Wash Buffers, Agilent Technologies, Santa Clara) they were dried with acetonitrile (Sigma-Aldich, St.Louis, USA). Agilent DNA Microarray Scanner (Agilent Technologies, Santa Clara, USA) was used detect fluorescent signal intensities. They were detected with the Scan Control A.8.4.1 Software (Agilent Technologies, Santa Clara, USA) and extracted from images by using the Feature Extraction 10.7.3.1 Software (Agilent Technologies, Santa Clara, USA). All the steps described were carried out according to the manufacturerM-BM-4s instructions.

Project description: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. 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 M-BM-0C until RNA isolation. Total RNA including miRNAs was isolated using the miRNeasy Mini Kit (Quiagen, Hilden, Germany) according to the manufacturerM-bM-^@M-^Ys 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 M-bM-^IM-% 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 manufacturerM-BM-4s 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 manufacturerM-BM-4s instructions. Labeled miRNA samples were hybridized at 55M-BM-0C 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 manufacturerM-BM-4s instructions.

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Dataset Information

Microarray analysis of microRNA expression in cutaneous squamous cell carcinoma

Publications

Microarray analysis of microRNA expression in cutaneous squamous cell carcinoma.

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