Expression beadchip

RNA was extracted with TRIZOL (Life Technologies) according to the manufacturer's instructions. RNA quantity and quality were assessed with NanoDrop ND 1000 spectrophotometer (NanoDrop Tech) and LabChip GX (Perkin Elmer) respectively before loading the samples on a HumanHT-12 v4.0 (mir-1 overexpression, non-targeting control) or HumanHT-12 v3.0 (all residual samples) Expression BeadChip (Illumina) in 4–6 biological replicates. Raw data is available at the GEO repository under accession number GSE55822.
All data were imported and normalized using the Base server (http://base.thep.lu.se). Empirical cumulative distribution function (ECDF) plots as introduced by Grimson et al. [12] (link) were created with the array results. Downloaded 3′ UTR sequences from the SylArray [22] (link) analysis were searched for words containing either an internal seed (IS) or canonical seed (CS). A single factor was generated for each array probe calling if it had 1 or more IS, 1 or more CS or 1 or more of both seed types. The log fold changes are plotted as in Grimson et al. to illustrate and compare the strength of the different seed types.
The RNAduplex 2.1.1 function [23] (link) from the Vienna RNA Package 2.0 was used to calculate the energy of duplex structures formed between the microRNA mimic and the section of mRNA interaction. Values are given in (kcal/mol).

Purified RNA samples from aSAT (Pre: n = 12; Post: n = 12) and gSAT (Pre: n = 12; Post: n = 12) were analyzed before and after exercise training using Illumina Expression BeadChip (Epicentre Biotechnologies, Madison, WI, USA). One array per tissue sample, for each SAT depot and for each time point (i.e. 4 arrays per participant × 12 participants) was used for the gene expression analysis (total of 24 arrays for gSAT samples and 24 arrays for aSAT samples). Each total RNA aliquot of 250 ng was ethanol precipitated with GlycoBlue (Invitrogen) as a carrier and dissolved at a concentration of 100–150 ng/µl before probe synthesis using the TargetAmp-Nano Labeling Kit for Illumina Expression BeadChip. 750 ng of cRNA were hybridized to Human HT-12 v4 Expression BeadChips (Illumina, San Diego, CA, USA) and scanned on the Illumina iScan instrument according to the manufacturer’s specifications. Raw expression intensities of 47,323 probes were extracted using Illumina GenomeStudio (Version 1.9.0) and the normalization, background correction and analysis were executed using R statistic software (version 3.6.1).

IC₅₀ concentrations of NPs (Zn₃BO₆: 66.50 mg/L and TiB₂: 104.34 mg/L) were applied to the cell cultures and total RNA isolation procedure was performed at the end of a 72 h duration. RNA isolations were performed by using a PureLink™ RNA Mini Kit (Invitrogene^®, Waltham, MA, USA) and isolated RNA quality was investigated by using a bio-analyzer (Agilent Technologies, Santa Clara, CA, USA) and UV-visible spectrophotometer (NanoDrop^®, ThermoFisher^®, Waltham, MA, USA). A TargetAmp-Nano Labeling kit was used to amplify isolated RNAs and an Illumina Expression BeadChip (EPICENTRE, Madison, WI, USA) was utilized to obtain biotinylated cRNAs. By using a T7 oligo (dT) primer 500 ng of total RNA was reverse-transcribed to cDNA, second-strand cDNA was synthesized, in vitro transcribed, and labeled with biotin-NTP and cRNA was quantified using the ND-1000 Spectrophotometer. Human HT-12 v4.0 Expression Beadchip (Illumina Inc., SanDiego, CA, USA) hybridization was used to label cRNAs at 58 °C for 17 h. Amersham fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) bead array was used to detect array signals. Array signals were read by using the Illumina bead array reader.

Total RNA of cells was isolated by Nucleospin RNA Kit according to manufacturer’s instructions (Machery Nagel, Düren, Germany) and used for whole-genome gene expression array analysis (Illumina Expression Bead Chip, Illumina Inc., San Diego, California) or validation of the array by qRT-PCR. cDNA was synthesized with the RevertAid First strand cDNA Synthesis Kit (Thermo Scientific, Schwerte, Germany) and oligo dT₁₈-primers. The expression of the different genes was quantified by using the SensiMix™ SYBR® & Fluorescein Kit (Bioline, Luckenwalde, Germany) and the C1000 Touch™ Thermal Cycler (Bio-Rad, Munich, Germany). The specific primer sequences used in this work can be found in the sumpplemtary part (metabion international AG, Planegg, Germany). The expression was quantified by the ΔΔct-method [35 (link)].

As Parkinson Disease (PD) is a disorder of the central nervous system, we selected eQTL data in brain for a case study from Gibbs et al.21 (link) and Myers et al.22 (link). In Gibbs et al.’s study, four frozen tissue samples of the cerebellum (CRBLM), frontal cortex (FCTX), caudal pons (PONS) and temporal cortex (TCTX) were obtained from 150 neurologically normal Caucasian subjects resulting in 600 tissue samples. SNP genotyping was performed using Infinium HumanHap 550 beadchips (Illumina) for 561,466 SNPs. Profiling of 22,184 mRNA transcripts was performed using HumanRef-8 (link) Expression BeadChips (Illumina). For each of the four brain regions, a regression analysis was performed using Plink23 (link). After eQTL analysis in each brain regions, we integrated the results. In Myers et al.’s study, whole-genome genotyping for 366,140 SNPs and expression analysis of 14,078 genes were carried out on a series of 193 neurologically normal human brain samples using the Affymetrix GeneChip Human Mapping 500 K Array Set and Illumina HumanRefseq-8 Expression BeadChip platforms. A one-degree-of-freedom allelic test of association analysis was performed using Plink23 (link). We integrated the results from these 2 studies. Finally, we got 51,131 significant correlations between 22,740 SNPs and 7,161 genes with the threshold of FDR < 0.05.