Chamq sybr color qpcr

To further verify the reliability and accuracy of the transcriptomic data, we used quantitative real-time PCR (qRT-PCR) for verification. Alkaline phosphatase-like (ALP-like), trypsin-1-like, C-type lectin domain family 4 member G-like, transcript variant X1 (Clec4g-like, transcript variant X1), crustacyanin-C1 subunit-like, phosphoenolpyruvate carboxykinase, cytosolic [GTP]-like, transcript variant X1 (PCK, cytosolic [GTP]-like, transcript variant X1), protein argonaute-3-like (AGO-3-like) and chymotrypsin BI-like(CHT BI-like) were selected for qRT-PCR analysis. Table 1 shows the primers designed for the seven genes. The cDNA was synthesized with HiScript Q RT SuperMix for qPCR with gDNA wiper (Vazyme, China). qRT-PCR was performed on an ABI7300 fluorescence quantitative PCR instrument (Bio-Rad, USA) with ChamQ SYBR Color qPCR (Vazyme, China) according to the manufacturer’s recommendations. All reactions were performed in triplicate. The expression level of the target gene in each sample was quantified by the relative fold change normalized to β-actin with the 2^−ΔΔCT method.

Total RNA was extracted from cells using Trizol (Takara, Otsu, Shiga, Japan) according to the manufacturer’s instructions, and cDNA was synthesized using the HiScript II Q RT Supermix for quantitative polymerase chain reaction (qPCR) (Vazyme, Nanjing, China). Next, qPCR was performed with ChamQ SYBR Color qPCR (Vazyme, Nanjing, China). All the data were normalized to GAPDH. The primer sequences used are listed below: GAPDH, 5′-ACGGGAAGCTCACTGGCATGGCCTT-3′ (sense), 5′-CATGAGGTCCACCACCCTGTTGCTG-3′ (antisense); IL-1ß, 5′-GAAATGCCACCTTTTGACAGTG-3′ (sense), 5′-TGGATGCTCTCATCAGGACAG-3′ (antisense); IL-6, 5′-TTCACAAGTCGGAGGCTT-3′ (sense), 5′-CAGTTTGGTAGCATCCAT-3′ (antisense); TNF-α, 5′- CAGGCGGTGCCTATGTCTC-3′ (sense), 5′- CGATCACCCCGAAGTTCAGTAG-3′ (antisense); INOS, 5′-CAGGAGGAGAGAGATCCGATTTA-3′ (sense), 5′-GCATTAGCATGGAAGCAAAGA-3′ (antisense); CD86, 5′-TCAATGGGACTGCATATCTGCC-3′ (sense), 5′-GCCAAAATACTACCAGCTCACT-3′ (antisense); Col2a1, 5′-CTTCACAGCGGTAGATCCCAG-3′ (sense), 5′-ACCAGGGGAACCACTCTCAC (antisense); MMP3, 5′-ACTCCCTGGGACTCTACCAC-3′ (sense), 5′-GGTACCACGAGGA CATCAGG-3′ (antisense); MMP13, 5′-TGATGGACCTTCT GGTCTTCTGG-3′ (sense), 5′-CATCCACATGGTTGGGAAGTTCT-3′ (antisense); Sox9, 5′-CAGCCCCTTCAACCTTCCTC-3′ (sense), 5′-TGATGGTCAGCGTAGTCGTATT-3′ (antisense). The 2^−ΔΔCq method was used to determine the relative mRNA levels of the target genes.