Computational protocol: Let-7, Mir-98 and Mir-181 as Biomarkers for Cancer and Schizophrenia

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Protocol publication

[…] Total RNA purification that contained miRNA was carried out using the RNeasy Protect Animal Blood kit (Qiagen). All experiments that included sample RNA quality control, miRNA profiling and analysis were conducted by Exiqon Services (Vedbaek, Denmark). Briefly, RNA quality control and measurement were carried out using an Agilent 2100 Bioanalyzer and a nanodrop instrument. 300ng of total RNA from both sample and reference were then labeled with Hy3 and Hy5 fluorescent label, respectively, using the miRCURY LNA microRNA Hi-Power Labeling Kit, Hy3/Hy5 (Exiqon, Denmark), following the procedure described by the manufacturer. The Hy3-labeled samples and a Hy5-labeled reference RNA sample were mixed pair-wise and hybridized to the miRCURY LNA microRNA Array 7th (Exiqon, Denmark), which contains capture probes targeting all microRNAs for human, mouse or rat registered in the miRBASE 18.0. The hybridization was performed according to the miRCURY LNA microRNA Array Instruction manual using a Tecan HS4800 hybridization station (Tecan, Austria). Following hybridization the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes. The miRCURY LNA microRNA Array slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using the ImaGene 9 (miRCURY LNA microRNA Array Analysis Software, Exiqon, Denmark). The quantified signals were background corrected (Normexp with offset value 10) [] and normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm. Spike-in controls were added in various concentrations in both the Hy3 and the Hy5 labeling reactions to evaluate the labeling reaction, hybridization, and the performance of the array experiment. […]

Pipeline specifications

Software tools ImaGene, miRCURY LNA microRNA Array Analysis Software
Databases miRBase
Application miRNA array analysis
Organisms Homo sapiens
Diseases Neoplasms