Sybrgreen kits

RNA was isolated from canine lymph node aspirates using RNeasy kits (Qiagen) per the manufacturer’s protocol. cDNA was prepared using iScript kits (BioRad), and qPCR was performed with SYBR green kits (BioRad) in a CFX96 (BioRad) for 40 cycles at 56°C. Copy number was calculated using ΔΔCT in Microsoft Excel.

Liver samples were homogenized in RNAiso Plus (TaKaRa, Dalian, China) for extracting total RNA as described by the manufacturer. RNA concentration and purity were assessed by determining the 260 nm absorbance and 260/280 ratios. For each sample, 1 μg of RNA was reverse transcribed to cDNA using Takara PrimeScript RT reagent kit with gDNA Eraser according to the manufacturer’s protocol. qPCR was actualized using Takara SYBR Green Kits on the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Gene-specific primers were designed using Primer-BLAST (Table S1). The PCR protocol in this study was set as follows: pre-denaturation at 95 °C for 30 s, 40 cycles of denaturation at 95 °C for 10 s, annealing at 58 °C for 20 s, and elongation 72 °C for 20 s. On the basis of the studies by Lin et al. [62 (link)] and Hou et al. [63 (link)], we selected glyceraldehyde-3-phosphate dehydrogenase (gapdh) as the internal control in this study to normalize all data. The relative expression of target genes was calculated using the 2^−ΔΔCt method [65 (link)].

To detect the expression of miRNA and genes associated with adipogenic differentiation, the total RNA was extracted with Trizol reagent (TakaRa, Otsu, Japan). The concentration of total RNA was measured by the NanoDrop 2000 (Thermo, Waltham, MA, USA). Then we used reverse transcription kits (TakaRa, Otsu, Japan) to synthesize cDNA. For miRNA analysis, specific reverse transcription primers and procedures were used, whereas the normal process was performed for mRNA analysis. In real-time quantitative PCR, every reaction performed in triplicate using SYBR green kits on a Bio-Rad iQTM5 system. The expressions of all genes were normalized to GAPDH, but U6 small RNA was internal reference when examined the level of miR-34a. The primer sequences used for qPCR were shown in Table 1.