No ung

Real-time PCR was performed to validate rat and human plasma miRNA using TaqMan^® microRNA reverse transcription kit (ThermoFisher), TaqMan^® Universal Master Mix II, no UNG (ThermoFisher) and TaqMan^® microRNA assay (ThermoFisher) according to the manufacturer’s instructions. The 2^(−ΔΔCt) value was calculated. Cel-mir-39 was used as a spike-in control for plasma miRNA normalization, while U6 was used as the internal control for rat tissue miRNA normalization.

Total RNA was extracted from cells using a PureLink RNA mini kit (Cat number 12183018A; Ambion, USA) according to manufacturer's recommendations and quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Complementary DNA (cDNA) was synthesized from 1 μg RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, USA) and a MultiGene thermal cycler (Labnet) according to manufacturer's instructions. Relative mRNA levels were inferred from the cDNAs using Power SYBR Green master mix (Applied Biosystems, UK) and TaqMan Universal master mix II, no UNG (Applied Biosystems, USA), both according to manufacturer's instructions, and a real-time PCR detection system (Applied Biosystems). Relative mRNA levels were normalized to the reference gene GAPDH, and then gene expression quantification was performed using a comparative Ct method, wherein ΔCT is defined as the difference between the target and reference gene CT values. Primers are listed in Supplementary Tables
S7 and
S8. These primers were either TaqMan primers (Applied Biosystems) or custom primers whose sequences have been published previously.

cDNA from isolated miRNA of myocardium and plasma were synthetized using the cDNA Taqman^® miRNA Reverse Transcription kit or TaqMan^® Advanced miRNA cDNA Synthesis Kit. Synthesized cDNA samples were then subjected to RT-qPCR using the TaqMan^® Universal PCR Master Mix II, No UNG or TaqMan^® Advanced miRNA Assays according to the manufacturer’s instructions (Applied Biosystems). The IDs of the probes are: 478139_mir, miR-499-5p; 465329_mat, miR-669m-5p; 001076, miR-322; 465212_mat, miR-3473b; 479241_mir, miR-10a-5p; 478583_mir, 181b-5p; 002571, miR-155-5p; 478399_mir, miR-146a-5p; 002562, miR-541; 001141, miR-451; 479235_mir, miR30e-5p; 478264_mir, miRNA-19b-3p; 478578_mir, let-7f-5p; 477909_mir, miR-140-5p; 477961_mir, miR-199a-3p; 477994_mir, miR-25-3p; 478293_mir, cel-miR-39-3p; 001973, U6 snRNA; 1232, snoRNA202 and 1234, snoRNA234.
Expression levels between normal and obese mice were quantitatively compared using the ΔΔCt method. The snoRNA202, snoRNA234 and U6 snRNA expression were analysis by NormFinder to choose the most suitable candidate to use it as an endogenous control in myocardium and cel-miR-39 as a spike-in miRNA for miRNA abundance normalization in plasma.

Reverse transcription (RT) was carried out using TaqMan MicroRNA Reverse Transcription Kit in the presence of RNase inhibitor (Applied Biosystems). Briefly, 25 ng of input RNA was reverse transcribed by stem-loop method following manufacturer’s recommendations. To determine the levels of mature miRNA-124a, quantitative Real time PCR (qPCR) was performed following cDNA generation. Briefly, 1 µl of cDNA was used as template in a 10-µl qPCR reaction by adding TaqMan Universal Master Mix II, no UNG (Applied Biosystems), and predesigned TaqMan MicroRNA Assays PCR primers and probes (FAM dye-labeled) for target miRNA-124a (ID: 001182). RNU44 small RNA was used as an endogenous reference gene (ID: 001094). All reactions were performed in triplicate using Biorad CFX 384 or ABI StepOne Plus real-time cyclers following manufacturer’s recommendations.

Briefly, tissues were homogenized, and RNA was isolated using the Nucleospin^® RNA Isolation Kit (Macherey Nagel, GmbH). RNA was quantitated using Nanodrop ND-2000 and cDNA was synthesized from 500 ng of total RNA using a Quantitect^® reverse transcription kit (Qiagen, Germantown, MD, USA). Gene expression analysis of TET 1, 2, and 3 was performed using TaqMan probes (Supplemetary File S4), TaqMan™ Universal Master Mix II, no UNG (Applied Biosystems, Waltham, MA, USA), and the Quant studio 12Kflex system (Applied Biosystems, Waltham, MA, USA).