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 Overall design: RNA was extracted from cells in the presence of 1a,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 5'-phosphate and 3'-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 1a,25(OH)2D3 (100 nM, 8h)