Genepix 4000b microarray scanner

Microarray miRNA expression profiling experiments were performed for each sample using one-color hybridizations on μParaflo™ microfluidic biochip miRNA microarray platform (LC Sciences, Houston, TX, USA) through LC Sciences’ miRNA microarray service. The array contained 2019 sequence-specific probes covering mature human miRNAs (hsa-) listed in Sanger miRBase Release v.19.0 and 44 Epstein-Barr virus miRNA-specific probes. Hybridized microarrays were scanned using an Axon GenePix 4000B Microarray Scanner and images digitized using Array-Pro Analyzer (MediaCybernetics, Rockville, MD, USA). Microarray data files were subjected background subtraction and to robust LOWESS (locally weighted regression) multi-array average background correction normalization. Data adjustment included signal significance analysis and data filtering that removed miRNAs with undetectable intensity values in more than half of control samples (Additional file 2). The log2-fold change values were calculated for each miRNA between any two participant groups. The threshold value used to define upregulation or downregulation of miRNA was a log2 ratio > ± 0.5 in expression in comparison to HC group and a value of P < 0.05 by one-way analysis of variance (ANOVA) with Bonferroni post tests (log2-transformed intensity values) among participant groups.

Total RNA was extracted from 20 mg of liver tissue from the MCD diet-fed mice (n=3 mice per group) and the control diet-fed mice (n=2 mice per group) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The μParaflo MicroRNA Microarray Assay was performed using a service provider (LC Sciences, Houston, TX, USA). The assay started with the 3′-extension with a poly (A) tail of 4 to 8 μg of total RNA using poly (A) polymerase. An oligonucleotide tag was then ligated to the poly (A) tail for later fluorescent dye staining. Hybridization was performed overnight on a μParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies, Houston, TX, USA). After RNA hybridization, the tag-conjugating Cy3 dye was circulated through the microfluidic chip for dye staining. Fluorescence images were collected using GenePix 4000B Microarray Scanner Molecular Device, Sunnyvale, CA, USA and digitized using Array-Pro image analysis software (Media Cybernetics, Rockville, MD, USA). The data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (locally weighted regression).