Gene Expression Analysis of DRG Neurons

In accordance with our previous study [10 (link)], rat L1 to L3 DRGs were harvested to analyze the gene expression of substance P (SP) and calcitonin gene-related peptide (CGRP), because bilateral L1–L3 DRGs innervate the lower IVDs [14 (link)]. In brief, the TRIzol reagent (Invitrogen, Life Technologies Corporation, CA, USA) was used to extract the total RNA, and the cDNA was produced with reverse transcriptase kit (TaKaRa, Shiga, Japan). The next qRT-PCR was conducted using SYBR Premix Ex Taq Kit (TaKaRa, Shiga, Japan) in the ABI 7500 Sequencing Detection System (Applied Biosystems, CA, USA). In this process, the reaction condition was set as follows: 40 cycles of denaturation at 95°C for 5 s and amplification at 60°C for 24 s. In this study, the primer sequences were designed and used: rat GAPDH: forward 5′-ATGACTCTACCCACGGCAAG-3′ and reverse 5′-TACTCAGCACCAGCATCACC-3′; rat SP: forward 5′-TGGTCAGATCTCTCACAAAGG-3′ and reverse 5′-TGCATTGCGCTTCTTTCATA-3′; rat CGRP: forward 5′-TCTAGTGTCACTGCCCAGAAGAGA-3′ and reverse 5′-GGCACAAAGTTGTCCTTCACCACA-3′; human GAPDH: forward 5′-CAGGAGGCATTGCTGATGAT-3′ and reverse 5′-GAAGGCTGGGGCTCATTT-3′; and human NGF: forward 5′-GCAAGCGGTCATCATCCCATCC-3′ and reverse 5′-TCTGTGGCGGTGGTCTTATCCC-3′.
All reactions had triplicate repeat and the housekeeping gene was GAPDH. To normalize the targeted gene expression, the 2^-△△Ct method was used when compared with the gene expression of GAPDH.