Quantitative Analysis of miR-29b Expression

RNA extraction, reverse transcription (RT), and quantitative real-time amplification (q-RT-PCR) were performed as previously described [32 (link)]. Briefly, total RNA was isolated from MM cells by using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. The integrity and purity of total RNA isolated were measured by using Nanodrop (Celbio Nanodrop Spectrophotometer nd-2000, Thermo Fisher Scientific, Waltham, MA, USA).
The single-tube TaqMan miRNA assay (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) was used to detect and quantify mature miR-29b (Assay ID: 000413, Applied Biosystems, Life Technologies) according to the manufacturer’s instructions, by the use of QuantStudio 12K Flex reader (Thermo Fisher Scientific, Waltham, MA, USA). miR-29b expression was normalized on RNU44 (Applied Biosystems, assay ID: 001094).