ABSTRACT: 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.
