Prism 7300 system

Total RNA was isolated from cells or tissues with the help of Trizol reagent (Invitrogen) according to the manufacturer’s protocol. To detect the expression levels of MIR155HG and BRD4, cDNA was reversed from 2 μg of total RNA using a PrimeScript RT Master Mix Kit (Takara, Shiga, Japan). For detecting miR-218-5p level, cDNA was synthesized using miScript II RT kit (Takara). Then, quantitative PCR was performed using SYBR Green PCR Master Mix kit (Takara) on a PRISM® 7300 system (Applied Biosystems, Foster City, CA, U.S.A.). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 was used as an internal inference and the relative expression level was evaluated by 2^−ΔΔCt method. The special primers were listed: MIR155HG, forward 5′-CCCAAATCTAGGTTCAAGTTC-3′, reverse 5′-ATCTAAGCCTCACAACAAC-3′; GAPDH, forward 5′-AGGTGAAGGTCGGAGTCAACG-3′, reverse 5′-AGGGGTCATTGATGGCAACA-3; BRD4, forward 5′-CCCCTCGTGGTGGTGAAG-3′, reverse 5′-GCTCGCTGCGGATGATG-3′; miR-218-5p, forward 5′-AACACGAACTAGATTGGTACA-3′, reverse 5′-AGTCTCAGGGTCCGAGGTATTC-3′; U6, forward 5′-GCTTCGGCAGCACATATACTAAAAT-3′, reverse 5′-CGCTTCACGAATTTGCGTGTCAT-3′.

Lumbar 1–5 (L1-5) DRG ipsilateral and contralateral to injected paws were removed at 0, 4, 8, or 12 weeks for RNA extraction, with DRG from 0 weeks as a control. RNA extraction was performed as described^{45 (link)}. Each DRG pool contained at least 10 DRG from 3 mice. RNA was extracted by using the RNeasy kit (Qiagen, Valencia, CA). Each gene primer (100 nM), derived cDNA, and master mix (SYBR green I and AmpliTaq Gold DNA polymerase [Applied Biosystems, Foster City, CA]) were mixed for PCR reactions and product detection by using the ABI Prism 7300 system. For each assay, preparations were run in triplicate. The thermal cycling conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. The threshold cycle (Ct) values for both targets and the internal reference (mGAPDH) were measured from the same samples, and the expression of the target genes relative to that of mGAPDH was calculated by the comparative Ct method.
The primer sequences were for TDAG8 (197 bp), 5′-atagtcagcgtcccagccaac (forward)/5′-cgcttcctttgcacaaggtg (reverse); ASIC3 (100 bp), 5′-tttcacctgtcttggctcct (forward)/5′-caggatagtggtggggattg (reverse); TRPV1 (151 bp), 5′-tctccactggtgttgagacg (forward)/5′-gggtctttgaactcgctgtc (reverse); and mGAPDH (233 bp), 5′-ggagccaaacgggtcatcatctc (forward)/ 5′-gaggggccatccacagtcttct (reverse).