Viia 7 dx multicolor detection system

Total RNA was extracted from cells using TRIzol^® reagent (Gibco, Life Technologies, Carlsbad, CA), following the manufacturer’s instructions. The RNA quantity and quality was assessed through NanoDrop^® (ND-1000 Spectrophotometer). To evaluate gene expression levels, 1000 ng of total RNA were reverse transcribed to cDNA using the “High Capacity cDNA Reverse Transcription Kit” (Applied Biosystems, Carlsbad, CA). The single-tube TaqMan assays (Applied Biosystems, Carlsbad, CA) were used to detect and quantify genes: EZH2 (Hs01016789_m1), MALAT1 (Hs00273907_s1) according to the manufacturer’s instructions, using Viia 7 Dx multicolor detection system (Applied Biosystems, Carlsbad, CA). The obtained Threshold Cycle (CT) values were normalized on GAPDH (Hs03929097_g1). The single-tube TaqMan miRNA assay (Applied Biosystems, Assay id 000413) was used to detect and quantify mature miR-29b expression, that was normalized on RNU44 (Applied Biosystems, Assay id 001094). Comparative real-time polymerase chain-reaction (RT-PCR) was carried out in triplicate, including no-template controls. Relative expression was calculated using the comparative cross threshold (Ct) method.

Total RNA extraction from MM cells and qRT-PCR were performed as previously described [26 (link)]. Briefly, total RNA was extracted from cells using TRIzol^® reagent (Gibco, Life Technologies, Carlsbad, CA, USA), following the manufacturer’s instructions. The RNA quantity and quality was assessed through NanoDrop^® (ND-1000 Spectrophotometer). To evaluate gene expression levels, 1000 ng of total RNA was reverse transcribed to cDNA using the “High Capacity cDNA Reverse Transcription Kit” (Applied Biosystems, Carlsbad, CA). The single-tube TaqMan assay (Applied Biosystems, Carlsbad, CA, USA) was used to detect and quantify MYC (Hs00153408_m1), according to the manufacturer’s instructions, using Viia 7 Dx multicolor detection system (Applied Biosystems, Carlsbad, CA, USA). The obtained threshold cycle (CT) values were normalized on GAPDH (Hs03929097_g1). Comparative real-time polymerase chain reaction (RT-PCR) was carried out in triplicate, including no-template controls. Relative expression was calculated using the comparative cross threshold (Ct) method. Taq-Man^® MicroRNA assays (Life Technologies) were used to detect and quantify mature mir-22-3p (assay ID 000398), according to the manufacturer’s guidelines on a ViiA7 System (Thermo Fisher Scientific, Waltham, Massachusetts, USA). MiR-22-3p expression was normalized on RNU44 (assay ID 001094).