Taqman microrna human assays

We performed individual miRNAs assay by Taqman quantitative real-time PCR (QRT-PCR) for quantification of abnormally expressed miRNAs in PMF and control granulocytes and in CD34+ cells. cDNA was synthesized from total RNA using microRNA-specific RT primers contained in the TaqMan microRNA Human Assays (Applied Biosystems). Briefly, single-stranded cDNA was synthesized from 10 ng total RNA in 15-μL reaction volume with the High-Capacity cDNA Archive Kit (Applied Biosystems) using 1 mM deoxyribonucleoside triphosphates, 50 U Multiscribe reverse transcriptase, 3.8 U RNase Inhibitor, and 50 nM of miR-specific RT primers. The reaction was incubated at 16°C for 30 minutes followed by 30 minutes at 42°C, and inactivation at 85°C for 5 minutes. Each generated cDNA was amplified by QRT-PCR with sequence-specific primers from the TaqMan microRNA Assays on an ABI Prism 7300 real-time PCR system (Applied Biosystems). PCR reactions included 10 μL 2× Universal PCR Master Mix (No AmpErase UNG), 2 μL each 10× TaqMan MicroRNA Assay Mix and 1.5 μL reverse-transcribed product; they were incubated in a 96-well plate at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Expression variations were calculated using the RQ method and a t-test p-value of 0.05 was used as threshold to identify differentially expressed elements.