Sensifast probe no rox mastermix

From 8 of the 13 patients, RNA was isolated from bone marrow cells using miRNeasy Mini Kit (Qiagen) according to manufacturer’s protocol. One hundred nanogram RNA was used for complementary DNA (cDNA) synthesis using SensiFAST cDNA synthesis kit (Bioline) in a 20 µl reaction according to manufacturer’s protocol. The expression of CRP was measured with quantitative real-time PCR (RT-PCR) using TaqMan primer/probes (Hs00357041_m1; Thermo Fisher Scientific). Each reaction contained 0.5 μl primer/probe, 6.25 μl SensiFAST Probe no-ROX master mix (Bioline), 4.75 μl RNase-free water, and 1 μl cDNA. After an initial polymerase activation step (2 min, 95°C), 40 cycles (10 s denaturation, 30 s annealing/extension) were run on a Mic RT-PCR system (Labgene Scientific). The expression of hypoxanthine phosphoribosyltransferase 1 (HPRT1; forward primer: 5’-AGAATGTCTTGATTGTGGAAGA-3’, reverse primer: 5’-ACCTTGACCATCTTTGGATTA-3’) was quantified as a reference gene using SensiFast No-ROX Kit and 2.5% input of total cDNA and 40 cycles of 5 s 95°C, 20 s 60°C, 10 s 72°C followed by melting curve analysis. The absence of a CRP amplification signal but the amplification of HRPT1 was considered as the absence of CRP transcription.