Viiatm7 real time pcr system

Overall RNA was isolated from pulp samples via TRIzol® Reagent (Takara, Japan) as per the protocol provided by the supplier. PrimeScript™ RT Master Mix (Takara, Japan) was applied to convert RNA into cDNA via reverse transcription. a ViiATM7 RealTime PCR System (Applied Biosystem, America) was applied to perform Quantitative RT-PCR assays with SYBR® Premix Ex TaqTM Tool (TaKaRa Biotechnology, Kusatsu, Japan) following the provided instructions. As HMOX1, LOX, ACTG1, STAT3, GNB5 were considered as hub genes. The detailed sequences of the primers for each gene are listed in Additional file 2: Table S1. The gene gapdh was applied as the inner reference. The ΔΔCT method was applied to measure the comparative genetic expression levels. Experimental data were expressed as the mean ± SD and assessed by Wilcoxon test. The threshold for statistical significance was set at the level of P being 0.05. Therefore, in all cases, P < 0.05 was considered statistically significant.

Real-Time PCR was used to assess CXCR4 and CXCL12 mRNA regulation after CCD. L4 and L5 lumbar DRG were dissected in control mice and CCD mice. Total RNAs of DRG were extracted using the RNeasy Plus Micro Kit (Qiagen, Hannover GmbH, Germany) according to the manufacturer’s protocol. 0.3 μg of total RNA was reversely transcribed to cDNA using maxima H minus First strand cDNA synthesis kit (Thermo Scientific, Rockford, IL), according to the manufacturer’s instructions. Real-time quantitative PCR was performed with the above prepared cDNA and SYBR Green Master Mix (Invitrogen, Carlsbad, CA). The primers for CXCR4, CXCL12 and β-actin were as follows: Forward Primer (CXCR4) 5′-AGGAAACTGCTGGCTGAAAAGG-3′, Reverse primer (CXCR4) 5′-GGAATTGAAACACCACCATCCA-3′; Forward Primer (CXCL12) 5′-GTCTAAGCAGCGATGGGTTC-3′, Reverse primer (CXCL12) 5′-GAATAAGAAAGCACACGCTGC-3′; Forward Primer (β-actin) 5′-GCATTGCTGACAGGATGCAG-3′, Reverse primer (β-actin) 5′-CCTGCTTGCTGATCCACATC-3′. The amplification conditions were 10 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, 30 s at 60 °C and extension at 72 °C for 30 sec. Quantitation of mRNA was performed by using Applied Biosystem ViiATM 7 Real-time PCR System (Applied Biosystem, Foster city, CA). The gene β-actin was used to normalize the mRNA levels of each sample.