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).