Mycbp

Total RNA was purified with miR-Neasy Mini Kit (Qiagen, Valencia, CA, USA), cDNA was transcribed from 10ng RNA using TaqMan^® miRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed using TaqMan^® miRNA assays (Applied Biosystems, Foster City, CA, USA) for miR-22 (#000398) according to the manufacturer's instructions. Expression of U6 (#001973) was used as a reference. For quantification of expression of pri-miR-22 or various mRNAs, RNA extraction was performed with the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). cDNA was subsequently transcribed using the High Capacity cDNA Reverse Transcription Kit followed by PCR analysis using TaqMan^® Gene Expression Assays: pri-miR-22 (#Hs03302632_pri), NCOA1 (#Hs00186661), CDK6 (Hs 01026371_m1), HDAC4 (Hs 01041638_m1), HDAC6 (Hs 00195869_m1), MAX (Hs 00811069_g1), MYCBP (Hs 00429315_g1), PTEN (Hs 02621230_s1) (all from Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. GAPDH (#Hs02758991_g1) was included as a reference. The amplification was performed on an Mx3005P qPCR System (Agilent Technologies, Santa Clara, CA, USA) real-time cycler on standard settings.