cell type: MM.1S cells activation agent: subjected to drug treatment treatment: RGB-286638 (50 nM) time: 8 hours
Treatment protocol
miRNA expression profiling was performed on MM.1S cells cultured 8 hours in control media or 50nM RGB-286638 with or without BMSCs.
Growth protocol
MM.1S MM cells were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS; Sigma Chemical, St Louis, MO), 2 mM L-glutamine, 100 U/mL penicillin, and 100 g/mL streptomycin (GIBCO, Grand Island, NY). Generation of bone marrow stromal cells (BMSCs) was performed from BM specimens from MM patients obtained after appropriate IRB approved informed consent. Mononuclear cells (MNCs) separated by Ficoll-Hipaque density sedimentation were used to establish long-term BMSCs cultures. When an adherent cell monolayer had developed, cells were harvested in Hanks buffered saline solution containing 0.25% trypsin and 0.02% EDTA, washed, and collected by centrifugation.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from MM cell pellets, using mirVana miRNA Isolation Kit (Ambion, Inc) according to manufacturer’s protocol.
Label
FAM
Label protocol
miRNA expression profiling was performed at Shannon McCormack Advanced Molecular Diagnostics Laboratory Research Services. TaqMan® Low-Density Array (TLDA) using human miRNA version 2.0A and version 3.0B cards (Applied Biosystems, Foster City, CA) were applied to examine the global change of miRNA expression levels in MM.1S cells when co-cultured with BMSCs with or without RGB-286638 treatment. A total of 756 mature miRNA updated in the Sanger miRBase v.15.0 were quantified according to the manufacturer's instructions. Briefly, total RNA was converted into cDNA using a single megaplex reverse transcriptase reaction. The megaplex reaction contained a specific stem-loop primer for each of the mature target microRNA's. The cDNA was subjected to a pre-amplification PCR. The diluted pre-amplification products served as substrates for TaqMan® quantitative PCR reactions.
Hybridization protocol
n/a
Scan protocol
n/a
Description
Test
Data processing
Relative quantification of miRNA expression was calculated with the 2−ΔΔCt Ct method using the ddCt program (Shannon McCormack Advanced Molecular Diagnostics Laboratory Research Services). The data was presented as log10 of the relative quantity of each miRNA. miRNAs with Ct values higher than 35 were excluded from the analysis. Normalization was carried out with the mean of RNU44 and RNU48.
