Unknown,Transcriptomics,Genomics,Proteomics

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MicroRNA expression analysis in Uveal melanoma from formalin fixed paraffin embedded sections

ABSTRACT: miRNA expression analysis was done on Uveal melanoma, metastatic and non - metastatic case in formalin fixed paraffin embedded sections, identified from case registry and confirmed by Chromosome insitu hybridization harboring monosomy 3 aberration in metastaic case and no aberration in non metastatic case. 19 miRNAs were found to be expressed only in class I tumors (choroidal melanoma) and not in class II (liver metastasis) and 11 miRNAs were found to be expressed only in class II and not in class I. The tumors were found to harbor oncomirs in both choroidal melanoma and metastasizing melanoma targeting tumor suppressor genes and metastatic suppressor genes. None of the differentially expressed miRNAs in either case were found to be located on the chromosomes which have been proved to carry chromosomal abnormalities. Rather it was found that genes targeted by the miRNAs were found to be present in chromosomal regions that are often found to be deleted 8p22, 13q and 17p. Organism used: Homo sapiens. * Slides: Human miRNA V2 8x15k Arrays AMADID: 19118. * Starting material: 18 slides with formalin fixed Paraffin embedded sections-20 μm. * RNA Samples used: 225-94-DUP, 225-94-E, 727-07-DUP, 727-07 -E. * Labeling kit: Agilent miRNA labeling reagent and Hybridization Kit. * Labeling Method: Ligation of one cyanine3-pCp molecule to the 3' end of RNA molecule with greater than 90% efficiency that generates fluorescent miRNA. * Total RNA and cRNA Purification Kit: Biorad MicroBio Spin 6 columns. * Hybridization Kit: Agilent miRNA Hybridization kit. Hybridization protocol See Agilent Technologies website for miRNA microarray hybridization and wash protocol: http://www.chem.agilent.com/scripts/LiteraturePDF.asp?iWHID=50500 Scan protocol Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Description Microarrays were scanned on an Agilent scanner (G2505C) at 100% PMT with 10% for lower intensity (XDR Scanning).Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturer’s protocol Data processing Feature extracted data was analyzed using GeneSpring GX v 10.0.2 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended Percentile shift normalization (50th percentile) . Further quality control of normalized data was done using correlation based condition tree to eliminate bad experiments. High expressed miRNA's were clustered using gene tree to identify significant gene expression patterns.

ORGANISM(S): Homo sapiens

SUBMITTER: Sugandan Sivamani

PROVIDER: E-GEOD-17037 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

Project description: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).

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MicroRNA expression analysis in Uveal melanoma from formalin fixed paraffin embedded sections

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