Abi prism 7700 cycler

ASCs were collected and total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA samples were pre-treated with deoxyribonuclease I (Invitrogen Life Technologies, Carlsbad, CA, USA), and a SuperScript kit (Invitrogen Life Technologies, Carlsbad, CA, USA) was used to synthesize cDNA according to the manufacturer’s recommendations. qRT-PCR was analyzed using miScript SYBR Green PCR Kits (Qiagen). Levels of cell cycle markers (p16, p21, and p53) mRNAs were determined by ABI PRISM 7700 cycler (Applied Biosystems, Foster City, CA). Each sample was analysed in duplicate with ribosomal 18S RNA as an internal control. Using telomeric primers, primers for the reference control gene (mouse 36B4 single copy gene), qPCR settings were performed as previously described [12 (link)]. The relative telomere length was shown as a T/S ratio indicative from the single copy gene. All fold changes in gene expression were determined using the 2 − ΔΔCT method. The values are presented as the mean ± SEM. All primers are listed in Supplemental Table 1.

Total RNA was extracted with Trizol reagent (Life Technologies, USA) using the standard method. cDNA synthesis was performed with 1 μg of total RNA, using the M-MLV Reverse Transcription (Promega, USA) according to the manufacturer’s recommended conditions. The primer sequences of miR-21 specific stem-loop RT can be found in Supplementary Table S1. qRT-PCR was amplified using the miScript SYBR Green PCR Kit (TaKaRa, Dalian, China) and the ABI PRISM 7700 cycler (Applied Biosystems, Foster City, CA). Amplification reactions were performed as 95 °C for 10 s, and followed by 40 cycles at denaturing (95 °C for 5 s), 58 °C −60 °C for 10 s and 72 °C for 10 s, and the dissociation curve analysis of PCR products were determined in the final stage of 55 °C to 95 °C. RNU6B and 18 S rRNA were used as endogenous controls for miR-21 and its target mRNAs expression, respectively. All samples were performed in 3 biological replicates with 3 biological and technical replicates. All ratios-changes were calculated by using the 2^−△△CT mean ± SEM method22 (link). The primers used in this study can be found in Supplementary Table S1.

EAT was isolated and total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). P16, P21, P53, PAI-1, MMP3, CD68, IL-6, MCP-1, and IGF-1 expression were measured using qPCR. RNA samples were pre-treated with deoxyribonuclease I (Invitrogen Life Technologies, Carlsbad, CA, USA), and a PrimeScriptTM RT Reagent Kit (Takara, Japan) was used to synthesize cDNA according to the manufacturer’s recommendations. qPCR was analyzed using SYBR^® RT-qPCR kit (Takara, Japan). Amplification was performed on an ABI PRISM 7700 cycler (Applied Biosystems, Foster City, CA, USA). Fold changes in gene expression were calculated using the 2^−ΔΔCT method. The values are shown as the mean ± SEM. All primers used in this study are listed in Supplemental Table S1.

EAT were collected 10 weeks post-transplantation and total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA samples were pre-treated with deoxyribonuclease I (Invitrogen Life Technologies, Carlsbad, CA, USA), and a SuperScript kit (Invitrogen Life Technologies, Carlsbad, CA, USA) was used to synthesize cDNA according to the manufacturer’s recommendations. qRT-PCR was analyzed using miScript SYBR Green PCR Kits (Qiagen). Levels of macrophage polarization and oxidative stress markers mRNAs were determined by ABI PRISM 7700 cycler (Applied Biosystems, Foster City, CA). Each sample was analyzed in duplicate with ribosomal 18S RNA as an internal control. All fold changes in gene expression were determined using the 2^−ΔΔCT method. The values are presented as the mean ± SEM. All primers are listed in Table S3.