6 protocols
array scanning and feature extraction protocol
The hybridization and washing of microarray slides was performed according to the manufacturers protocol. Upon washing, slides were dried by nitrogen gas at 23°C for 5 min. Scanning was performed immediately following hybridization at a resolution of 5 µm using the Tecan Powerscanner device
nucleic acid hybridization to array protocol
Labeled samples were hybridized to miRCURY LNA miRNA Array 7th generation microarray (Exiqon Inc., Woburn Massachusetts, USA) using the Tecan HS400 Pro device (Tecan Group Ltd., Männedorf, Switzerland). Experiments were performed in dye swap pairs with three biological replicates from transgenic MSA mice and age- and sex-matched control mice, for both SN and striatum
nucleic acid labeling protocol
For miRNA-labeling was perfomed by the Mercury LNA microRNA Hi-Power Labeling Kit from Exiqon. The standard protocol from Exiqon was employed with minor modifications. 1 µg of total RNA per sample was dephosphorylated, then split into two aliquots, which were subsequently fluorescently labeled with either Cy3 or Cy5 dyes to be used in dye swap experiments.
sample collection protocol
C57BL/6 wild type and multiple system atrophy transgenic mice (Tg(Plp1-SNCA)1Haa strain backcrossed in C57BL/6 in over 20 generations) at an age of 2-3 months were used in this study. The transgenic mice have a transgenic insertion of the human wild-type alpha synuclein driven by the proteolipid protein promoter. Transgenic protein is detected in oligodendrocytes but in no other brain cell type. The mice were sacrificed by cervical dislocation, brains were quickly extracted and the substantia nigra and striatum were dissected on ice. Three pools of substantia nigra and striatum, respectively, from 5 mice each were prepared and frozen in liquid nitrogen.
nucleic acid extraction protocol
Total RNA was isolated using Tri-Reagent (Sigma-Aldrich, St. Louis, Missouri, USA) according to the manufacturer's instructions and dissolved in DEPC-water. Quantity and quality of RNA preparations was determined by 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) measurement.
normalization data transformation protocol
Normalization was based on the net intensity values ((raw intensity) – (local background)) as calculated by the ArrayPro 6.3 software (Tecan Group Ltd., Männedorf, Switzerland). Subsequently, normalization was inspected by quality metrics [45]. A non-specific filtering step was conducted by employing the “rowSDS” and “shorth” functions of the genefilter package (version 1.48) for filtering by variance.