Characterisation of VDR signaling in prostate cancer health disparities (small RNA-Seq)

To investigate the effect of 1,25(OH)2D3 activation on small RNA expression in prostate cell lines derived from European Americans and African Americans RNA was extracted from cells in the presence of 1α,25(OH)2D3 (100nM, 8hr) or EtOH in biological triplicate samples and analyzed by RNA-Seq. Cell lines were treated as RNA-Seq and library preparation included ligation of 5’ and 3’ RNA adapters to the mature miRNAs 5ʹ‐phosphate and 3ʹ‐hydroxyl groups and 11-13 PCR cycles using a universal primer and a primer containing one of 48 index sequences, which allowed pooling of libraries and multiplex sequencing. Prior to pooling, each individual sample’s amplified cDNA construct was visualized on a DNA-HS Bioanalyzer DNA chip (Agilent Technologies) for mature miRNA and other small RNA products (140-150bp). Successful constructs were purified using a Pippen prep (Sage Inc.), using 125 – 160 bp product size settings with separation on a 3% agarose gel. The purified samples were validated for size, purity and concentration using a DNA-HS Bioanalyzer chip. Validated libraries were pooled at equal molar to a final concentration of 10nM in Tris‐HCI 10 mM, pH 8.5, before 50 cycle sequencing on a MiSeq (Illumina, Inc.). Comparative small RNA gene expression profiling analysis of RNA-seq data for prostate cell lines treated with 1α,25(OH)2D3 (100 nM, 8h)