Unknown,Transcriptomics,Genomics,Proteomics

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MicroRNAs expression profiling during breast cancer progression

ABSTRACT: MicroRNAs (miRNAs) are known to be deregulated in human breast cancer (BC). The purpose of the current study was to investigate the expression of miRNAs in different stages of BC to assess their biological value in BC progression. MiRNA expression was assessed in a series of BC patients (n=7) with distinct stages of tumour progression (Normal, in-situ (DCIS), primary invasive BC and nodal metastases) to evaluate miRNA differential expression. We used an Agilent miRNA microarray based platform which uses miRBase 16 to screen for 1205 Homo sapiens (hsa) and 144 human viral miRNA candidates. To validate the microarray data, the expression of two deregulated miRNAs was measured by TaqMan quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). This study was conducted Formalin Fixed and Paraffin Embedded (FFPE) specimens from 7 female patients. Twenty M-NM-2 but <3. This is expected for FFPE breast samples and is acceptable by Agilent Technologies, Santa Clara, USA, for FFPE samples to undergo miRNA microarray analysis. MiRNAs extracted from laser microdissected tissue components were profiled using Agilent microRNA microarray profiling system (Agilent Technologies, Santa Clara, USA). Total RNA samples were spiked using the MicroRNA Spike-In Kit (Agilent Technologies, Santa Clara, USA) to evaluate efficiencies of labelling and hybridisation. Total RNA was treated with Calf Intestinal Alkaline Phosphatase (CIP), and then 100 ng of total RNA was used per sample to initiate a labelling reaction. Ligation master mix for T4 RNA ligase, which includes Cyanine 3-Cytidine biphosphate (Cyanine 3-pCp) (Complete miRNA Labelling and Hyb Kit, Agilent Technologies, Santa Clara, USA) was used to label the dephosphorylated RNA. Cyanine-3-labelled miRNA samples were hybridised to human miRNA microarrays (Release 16.0, 8x60K) (Agilent Technologies, Santa Clara, USA) at 55M-BM-0C for 20 hrs. Microarray slides were washed with increasing stringency (Gene Expression Wash Buffers, Agilent Technologies, Santa Clara), and subsequently dried with acetonitrile (Sigma-Aldrich, St. Louis, USA). Fluorescent signal intensities were detected on an Agilent Microarray Scanner (Agilent Technologies, Santa Clara, USA) using the Scan Control A.8.4.1 Software (Agilent Technologies, Santa Clara, USA) and extracted from the images with the Feature Extraction 10.7.3.1 Software (Agilent Technologies, Santa Clara, USA).

ORGANISM(S): Homo sapiens

SUBMITTER: Sarkawt Khoshnaw

PROVIDER: E-GEOD-38867 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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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.

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MicroRNAs expression profiling during breast cancer progression

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