Trizol and rna purification kit

RNA was extracted from livers and adipose tissues by TRIzol and RNA purification kit (Invitrogen, Life Technologies, Carlsbad, CA, USA). All primers and probes for ddPCR were designed by Invitrogen (Invitrogen, Life Technologies, Carlsbad, CA, USA) as per Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines [20 ]. Primer amplification efficiency was over 90% for every RT-PCR assay. cDNA was synthesized by GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CA, USA). The synthesized cDNA was diluted 10 times with water, and 1 μL of diluted cDNA was applied in each RT-PCR using SYBR green super mix (Bio-Rad, Hercules, CA, USA) and analyzed by a Mx3000P instrument (Agilent, Cedar Creek, TX, USA). Cycle conditions were the same as our previous study [21 (link)]. PCR product sizes were analyzed by gel electrophoresis and no primer dimers were observed. Differences in mRNA expression in liver and epidydimal adipose tissues were calculated after normalization to β-actin or 36B4 mRNA expression.

We used a TRIzol and RNA purification kit (Cat no. # 12,183,555, Invitrogen, Thermofisher Scientific, USA) to extract total RNA from knee cartilages. Reverse transcription was performed using Prime Script™ RT Master Mix (Cat no. # RR036B, Takara, USA). This was followed using TB Green Premix Ex Taq II (Cat no. # RR820A, Takara, USA) with forward and reverse primers mentioned in Table 2 for performing real-time PCR. The 2^{− ΔΔCt} method was used to determine miR-373 transcription levels using U6 as the reference gene (Bai et al. 2018 (link)).Table 2

Forward and reverse primers of miR-373 and U6

Gene	Forward sequence	Reverse sequence
miR-373	5′-ATTTTGGTTAATACGGTGAAATTTC-3′	5′-CTATCGCCCAAACTAAAATACGAT-3′
U6	5′-CTCGCTTCGGCAGCACA-3′	5′-ACGCTTCACGAATTTGCGT-3′