Steponeplus real time pcr system with tower

RT-PCR was used for measuring the mRNA level of CLR in the ACC of rats. To compare the changes of CLR mRNA in ACC of naïve rats without injection of carrageenan (n = 6, as a control) and rats received injection of carrageenan (n = 6, 3 h after injection), an over dose of pentobarbital sodium was administered to the rat and the brain of the rat was removed. The right side ACC tissues, as the inflammatory pain in left hindpaw, were dissected and rapidly stored at −80℃. Total RNA was extracted from the ACC tissues by using the SPARKeasy Improved Tissue RNA kit (SparkJade, Qingdao, China). cDNA synthesis was performed by reverse transcription using the SPARKscript 1st Strand cDNA Synthesis Kit (SparkJade, Qingdao, China). The StepOnePlus™ Real-Time PCR System with Tower (Applied Biosystems) and 2*SYBR Green qPCR Mix (SparkJade, Qingdao, China) were used to amplify CLR. For amplification of both CLR and the reference gene (glyceraldehyde 3-phosphate dehydrogenase, GAPDH), the following PCR protocol was applied: 94°C for 3 min, 94°C for 10 s, 60°C for 34 s, 40 cycles. The primers used were as follows: CLR primer sequences were forward 5′- AGAGCCTAAGTTGCCAACGG-3′, reverse 5′-CCACTGCCGTGAGGTGAATG-3′; GAPDH primer sequences were forward 5′-GACCACCCAGCCCAGCAAGG-3′, reverse 5′-TCCCCAGGCCCCTCCTGTTG-3′ (Zhang et al., 2017b (link)).

Total RNA was extracted using a TRI reagent (Sigma, St. Louis, MO, USA, cat. no. T9424) following the manufacturer’s procedure. The purity and concentration of RNA samples were determined with a Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A reverse-transcriptional reaction was performed with a Hifair III 1st Strand cDNA Synthesis Kit (Yeasen, Shanghai, China. cat. no. 11139ES60) according to the manufacturer’s procedure. qRT-PCR was performed with SYBR green master mix (Yeasen, cat. no. 11203ES08) on a StepOnePlus™ Real-Time PCR System with Tower (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. For primer sequences, see Table S1.

The total RNA of tumour tissues was isolated with TRIzol reagent (Thermo Scientific, Waltham, MA, USA). First-strand cDNA was synthesised from 1 μg total RNA using a HiScript^® II Q RT SuperMix for qPCR Kit (Vazyme, Nanjing, China). The ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) was used for StepOnePlus™ Real-Time PCR System with Tower (Applied Biosystems Inc, Foster City, CA, USA). The cycling conditions were: 90 s at 95 °C, 40 cycles at 95 °C for 5 s, 60 °C for 30 s and 72 °C for 20 s. β-actin (Actb) was used as an internal control. The relative level was calculated by the relative quantification 2-ΔΔCT method. The primer sequences were listed in Table 1.