《Table 1 Quantitative real time polymerase chain reaction primers》

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《Electroacupuncture promotes peripheral nerve regeneration after facial nerve crush injury and upregulates the expression of glial cell-derived neurotrophic factor》

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GDNF:Glial cell-derived neurotrophic factor.

To identify whether GDNF（NCBI Accession Number:XM_008262127.2）and N-cadherin（NCBI Accession Number:XM_017344097.1）were involved in the neuroprotective effects of EA，their mRNA levels were evaluated by qRT-PCR.All rabbits were sacrificed after their last behavior assessment.Facial neurons in the brainstem tissues were collected.Total RNA was extracted from tissues specimenswith TRIzol（Ambion Inc.，Carlsbad，CA，USA）following the manufacturer’s instructions.Total RNA concentration and quality were determined on a Nanodrop（Thermo Fisher Scientific，Waltham，MA，USA）；cDNA was reverse transcribed with a reverse transcription kit（TAKARA，Japan）.All primers used are shown in Table 1.Each reaction consisted of 10μLSYBR Green Mixture，1μL cDNA，0.5μL（10μmol/μL）of each primer and 8μL PCR-grade water，to yield a final volume of 20μL.PCR was performed on Real-time 7500 PCR detection system（Bio-Rad，USA）under the following conditions:95°C，3 minutes；95°C，5 seconds；56°C，10 seconds；72°C，25seconds，39 cycles；65°C，5 seconds；95°C，50 seconds.β-Actin was used as a reference gene.Relative mRNA levels were determined by the 2–ΔΔCt method（Livak et al.，2001）.