Vivaspin 500 3k

Total RNAs of tissue samples from two groups (Control, LTL (200 mg/kg) group) with three samples per group were isolated using Trizol according to the manufacturer’s protocol. Small RNA fragments were enriched by NanoSep 100K (Pall Corporation, New York, NY, USA) and de-salted by flowing through vivaspin 500 3k (Sartorius Stedim Biotech, Goettingen, Germany) from 2.5 μg total RNA. Fluorescent targets were prepared using a miRNA ULSTM Labeling Kit. Labeled Fluorescent targets were hybridized to pre-hybridized Mouse miRNA OneArray^® v5. After 16 h hybridization at 37 °C, non-specific binding targets were washed away, and the slides were dried by centrifugation and scanned by an Axon 4000B scanner (Molecular Devices, Sunnyvale, CA, USA). The Cy5 fluorescent intensities of each spot were analyzed by GenePix 4.1 software (Molecular Devices), which performed median normalization.

The small RNA fragments were enriched with NanoSep 100K (Pall Corporation, USA) and desalted by flowing through Vivaspin 500 3k (Sartorius Stedim Biotech) from 2.5 μg of total RNA. The fluorescent targets were prepared using miRNA ULSTM Labeling Kit (Kreatech Diagnostics, Netherlands). The labeled fluorescent targets were hybridized to prehybridized mouse miRNA OneArray v5 (Phalanx Biotech Group, Hsinchu, Taiwan). Nonspecific binding targets were washed away after 16 h hybridization at 37°C, Slides were dried via centrifugation and scanned using an Axon 4000B scanner(Molecular Devices, Sunnyvale, CA, USA). The Cy5 fluorescent intensities of each spot were analyzed using GenePix 4.1 software (Molecular Devices).
The signal intensity of each spot was processed by R program. The median value of the repeating spots was selected for analysis. We filtered out spots with flag that was <0 within all arrays. Spots that passed the criteria were normalized through invariant set normalization method. Normalized spot intensities were transformed to gene expression log2 ratios in the pairwise t-test between the control and treatment groups. The spots with |log2 ratio| ≥0.8 and P-value <0.05 were tested for further analysis. Target genes and functions prediction for selected differential miRNAs were analyzed by NTU miRSystem website (NTU, Taipei, Taiwan).

The cDNAs from PAXgene blood samples were sent to Phalanx Biotech Group for processing. These samples were purified via Nanosep 100k (Pall) and Vivaspin 500 3k (Sartorius Stedim Biotech). Purified cDNAs were examined by OD values (NanoDropTM 2000c), gel electrophoresis and capillary electrophoresis for quality control, and then labeled with Universal Linkage system (Kreatech) for hybridization on microRNA microarray (Phalanx) in NimbleGen Hybridization System. The images were scanned by the Molecular Devices AXON4000B scanner, and the figure signals were transformed to digital signals by GenePix™ 4.