Multiple myeloma (MM) is a plasma cell malignancy where the hypoxic bone marrow microenvironment influences clinical outcomes. Our preliminary results show that in vitro culturing of MM cells under hypoxia downregulates CD138 expression (flow cytometry and qPCR). Notably, the CD138low fraction constitutes putative MM cancer stem cells and hypoxia-inducible factors (HIFs) regulate the expression of stem cell transcription factors. This study is designed to identify deregulated micro-RNA (miR) expression to explore novel therapeutic avenues to target the clonogenic MM fractions. Using four cell lines, TaqMan Array Human miRNA Card A was run, normalized against RNU48 and relative miRNA levels were determined using the ΔΔCt method. Among various candidate miRs, miR-96 (34-fold downregulation, P < 0.05) was selected. qPCR validation using individual TaqMan miRNA assay showed that 72 h culture in hypoxia downregulated miR-96 expression in MM.1S, OPM2, H929, and U266 cells by 2.1, 1.8, 1.9, and 1.4 fold, respectively. The hsa-miR96 precursor was PCR amplified from the pSilencer 4.1-CMV neo vector (Ambion, TX, gift from Amendt lab, UI) using primers (5'-TATCT GTTAACTACCGAAGGGCCATAAACAG-3' and 5'-TCTATCTCGAGACCCAGCAGTAAGCCAGATG-3') to introduce XhoI and HpaI restriction enzyme sites. This 340 bp PCR fragment was cloned into pJET1.2/blunt cloning vector (Thermo Scientific, NY), verified by DNA sequencing, and termed hsa-miR96-pJET1.2 plasmid. Next, hsa-miR96-pJET1.2 and pLL3.7 lentivirus vector (Addgene, MA) were digested with HpaI and XhoI and ligated. Once hsa-miR96-pLL3.7 construct is verified, we will generate lentivirus particles to overexpress hsa-miR96 in MM cell lines. Future studies will evaluate if hsa-miR96 overexpression decreases stemness and chemo-resistance in MM.