miRNA Microarray

For the miRNA microarray experiments, formalin-fixed paraffin-embedded blocks of SPN specimens were obtained from the archives of the medical center's Institute of Pathology. The samples were reviewed, and ten micrometer-thick sections were cut from the tumor block and transferred onto glass slides. A single glass slide containing a block sample was stained with hematoxylin and eosin, and tumor margins were marked by the pathologist (E.B.). Microdissection was performed manually to extract tumoral tissue with a sharp scalpel. Total RNA, including miRNA, was extracted using an microRNeasy formalin-fixed paraffin-embedded kit (Qiagen, Hilden, Germany). Total RNA concentrations were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA) (19). The miRNA microarray experiments were utilized for microRNA hybridization using GeneChip microRNA 4.1 Array strip (Affymetrix) (a total of 11 arrays). Samples (500 ng) were labeled with the Genisphere FlashTag Biotin Labeling Assay, which utilizes the 3DNA technology. The 3DNA dendrimers are ligated to samples to allow multiple biotins (~15) to bind to each poly-A-tailed RNA molecule. After FlashTag ligation, the samples were hybridized overnight on the Affymetrix GeneChip microRNA array. The arrays were washed and immediately scanned with a GenePix 4000B array scanner (20).