Fastking cdna synthesis kit

Gene expression levels of Clec7a in BV2 cells in different groups were detected using quantitative real-time PCR with a TaqMan Gene Expression Assay by CFX96TM Real-Time Systems according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). Total RNA was extracted using TRNzol Universal reagent (TIANGEN, DP424) according to the manufacturer’s protocol. RNA concentration was determined by a spectrophotometer at 260 nm and 280 nm. Identical amounts of RNA (2 µg) were reverse transcribed into complementary DNA (cDNA) using a FastKing cDNA synthesis kit (TIANGEN, KR118) according to the manufacturer’s instructions. Relative mRNA levels were calculated using the 2^−ΔΔCT method [14 (link)]. The sequences of primers are provided in Additional file 2: Table S1.

To verify the transcriptome data, the ef1a gene (GenBank accession no. AB061263) was selected as the endogenous reference gene. There were 17 upregulated DEGs (UR) and six downregulated DEGs (DR) selected for qRT-PCR verification. Total RNA was extracted using the RNA Easy Fast Plant Tissue Kit (TIANGEN, DP452) following the manufacturer’s instructions. The integrity of total RNA was analyzed by 1.0% agarose gel electrophoresis. The information about primer sequences of selected DEGs (Supplementary Table 1) was designed using NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and synthesized by Sangon Biotech (Shanghai, China). First-strand cDNA was synthesized using the FastKing cDNA Synthesis Kit (TIANGEN, KR118-03) following the manufacturer’s instructions. The qRT-PCR gene expression was inspected using a SuperReal PreMix Plus (SYBR Green) (TIANGEN, FP205-03). Reactions were carried out on a LightCycler (LightCycler96 Real-Time PCR, Roche, Switzerland) by the default cycling conditions (15 min at 95°C and 40 cycles of 10 s at 95°C, 20 s at 60°C, 30 s at 72°C and 95°C for 15 s, 60°C for 1 min). The melting curve analysis was used to examine the specificity of each amplification, and the relative expression levels were calculated by the 2^−ΔΔCt quantitative method (Ct, cycle threshold value of target gene) (Willems et al., 2008 (link)).

Total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. Then, 1 μg of total RNA was reverse transcribed into cDNA using the FastKing cDNA synthesis kit (Tiangen Biotech, China). qPCR was performed with TB Green Premix (Takara, Japan). The mRNA expression levels of NK-1R, CGRP, SP, type 1 and 3 collagen were detected. PCR primers were designed based on sequences from the corresponding genes (Table 2). All data were normalized using GAPDH as the internal control by the ΔCT method.