Realplex2 real time pcr system

JAK2V617F wild-type and mutant alleles were amplified using 2 independent antisense (AS)-quantitative polymerase chain reaction (qPCR) multiplex reactions, 1 specific for the wild-type allele and the other specific for the mutant allele. JAK2 V617F wild-type and mutant primers were as follows: forward primer 5ʹ-CTTTCTTTGAAGCAGCAAGTATGA-3ʹ, probe 6-FAM-TGAGCAAGCTTTCTCACAAGCAT TTGGTTT-TAMRA, wild-type specific reverse primer 5ʹ-GTAGTTTTACTTACTCTCGTCTCCA CATAC-3ʹ, JAK2V617F mutation-specific primer 5ʹ-GTAGTTTTACTTACTCTCGTCTCCAC ATAA-3ʹ. All AS-qPCR reactions were performed in a Realplex2 Real-Time PCR System (Eppendorf, Germany) and consisted of 15 µL of Maxima Probe/ROX qPCR Master Mix (2 × ) (Applied Biosystems, USA), 3 µL of 10 × JAK-2 Prime Time Assay and 1.5 µL 10 × RNAse P Prime Time Assay, and 10 µL of DNA normalized to 2.5 ng/µL in the following thermocycling conditions: denaturation for 10 min at 95 °C, followed by 50 cycles of 95 °C for 15 s and 60 °C for 60 s. Nontemplate control, positive, and negative controls were used in all reactions. The threshold was always set to 0.1 DeltaRn in all qPCR experiments.

Total RNA was extracted by using Qiashredder and RNeasy mini kits (Qiagen, USA) as directed by the manufacturer’s instructions, and the mRNA concentration was measured with a NanoDrop 1000 Spectrophotometer. RNA (1 µg per 20 µL reaction) was reverse transcribed with SuperScript II Reverse Transcriptase (Thermo Scientific, USA) and appropriate primers (Table 1), and quantitative reverse transcription polymerase chain reaction (qRT–PCR) was performed with Maxima SYBR Green Master Mix (Thermo Scientific) on a Realplex2 Real-Time PCR system (Eppendorf, USA). Measurements were normalized to the level of endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) RNA.