Single tube taqman assays

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 were assessed through NanoDrop^® ND-1000 Spectrophotometer (Waltham, MA, USA). To evaluate transcript changes, 1000 ng of total RNA was reverse-transcribed to cDNA using the “High Capacity cDNA Reverse Transcription Kit” (Applied Biosystems, Carlsbad, CA, USA). The following single-tube TaqMan assays (Applied Biosystems, Carlsbad, CA, USA) were used to detect and quantify genes using the Viia7 DX real time PCR instrument (Life Technologies, Waltham, MA, USA): DNMT1 (Hs00154749_m1), DNMT3a (Hs01027166_m1), DNMT3b (Hs00171876_m1), HDAC1 (Hs02621185_s1), HDAC2 (Hs00231032_m1), HDAC3 (Hs00187320_m1), HDAC4 (Hs01041638_m1), HDAC6 (Hs00195869_m1), and GAPDH (Hs02786624 g1). miRNA expression levels were determined by TaqMan RT-PCR, using the single-tube TaqMan miRNA assays (hsa-miR-29b, assay ID 000413; hsa-miR-22, assay ID 000398, Applied Biosystems) to quantify mature miRNAs, by the use of the StepOne Thermocycler (Thermo Fisher Scientific, Waltham, MA, USA) and the sequence detection system, as previously reported [51 (link)]; miRNAs expression levels were normalized on RNU44 (assay ID 001094). Comparative real-time polymerase chain reaction (RT-PCR) was performed in triplicate.

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.