Minimal residual disease measurement
Total RNA was isolated from bone marrow samples using the RNeasy Mini Kit (Qiagen, Valencia, CA); concentrations of the extracted RNA were evaluated by spectrophotometry. The reverse transcription (RT) step was performed using a Thermoscript RT-PCR system (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. Real-time quantitative RT-PCR (RQ-PCR) reactions were performed on an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Branchburg, NJ). Ready-made primers and TaqMan probes (Assays-on-Demand Gene Expression Product) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) Hs99999905_m1, tyrosine hydroxylase (TH ) Hs01002188_g1, and paired-like homeobox 2B (PHOX2B ) Hs00243679_m1 were purchased from Applied Biosystems. Each mRNA expression was quantified using TaqMan Universal PCR Master Mix II (Applied Biosystems, Foster City, CA, USA, cat no.4440040). PCR reactions were performed in a total volume of 15 μL, and the thermal reaction conditions were as follows: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 1 min, in which fluorescence was acquired and detected by StepOne Real-Time PCR System (Applied Biosystems).