Hsa mir 29b

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.