Superscript first strand synthesis supermix

Total RNA of human islets and cells was extracted using RNeasy Plus Micro Kit (Qiagen), and cDNA was synthesized using 1 µg RNA of each sample and reverse transcribed using SuperScript First-Strand Synthesis SuperMix (Invitrogen) according to the manufacturer's protocol. Quantitative RT-PCR (RT-qPCR) was performed using PowerUp SYBR Green Master Mix (Applied Biosystems) on QuantStudio 5 Real-Time PCR Systems (Applied Biosystems). GAPDH and ACTB were used as endogenous controls. Relative gene expression levels of CLEC11A and ITGA11 in human islets and EndoC-βH1 cells were normalized by subtracting the geometric average C_t value of these two endogenous controls (Vandesompele et al. 2002 (link)) and further calculated with the 2^–△Ct method. Relative gene expressions in rhCLEC11 treatment experiments in human islets and EndoC-βH1 cells were normalized by subtracting the C_tvalue of ACTB and GAPDH, respectively, from the C_t value for the gene studied. Relative mRNA expression of the gene studied was calculated with the 2^–△△Ct method. Primers were synthesized by Integrated DNA Technologies, and the sequences are listed in Supplementary Table 1.

Total RNA was extracted from normal and gastric cancer tissues using TRIZOL reagent (Invitrogen, Carlsbad, CA). Agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) were employed to assess the quality and concentration of RNA. A total of 1 μg of high-quality RNA was reverse-transcribed to first-strand cDNA with Superscript First-Strand Synthesis SuperMix (Invitrogen). Real-time PCR was conducted with SYBR Green Master mix (Applied Biosystems, Foster City, CA). The reaction (95°C for 15 s and 60°C for 1 min) was run using the ABI PRISM 7500 Real-Time PCR System (Applied Biosystems) with relative expression analyzed by the 2^−ΔΔCt method. The sequences of the primers used to amplify Nell-1 and the control gene GAPDH are listed in Table 2.