Polytron powergen 500 s1

RNA was isolated from 20 mg of cardiac, lung, and liver tissue or from isolated cardiac mitochondria, using the miRNeasy Mini Kit (product no.: 217004, Qiagen, Hilden, Germany ) per manufacturer’s instructions, with minor modifications. For tissue, 700 mL QIAzol was used for homogenization with a Polytron PowerGen 500 S1 tissue homogenizer (Fisher Scientific, Hampton, NH). Total RNA was isolated from both the whole tissue and mitochondrial samples and used in subsequent analyses. RNA was converted to cDNA with the First-strand cDNA Synthesis kit for miRNA (product no.: HP100042, Origene, Rockville, MD), per manufacturer’s instructions. Differential expression of miRNA-378a-3p, miRNA-378a-5p, PGC-1β, mitofusin 1 (Mfn1), and mediator complex subunit 19 (Med19) were measured. Primer design for qPCR is provided (Table S1). Briefly, primers for mRNA transcripts were derived using Primer-BLAST through NCBI, while miRNA forward primers were designed to span the 5’ region of the sequence with a standard reverse to flank the poly-A tail following cDNA synthesis. Expression was normalized to GAPDH in whole tissue and U6 in isolated mitochondria. Experiments were performed on the Applied Biosystems 7900HT Fast Real-Time PCR system (Applied Biosystems, Foster City, CA), using 2X SYBR Green Master Mix. Quantification was achieved using the 2^−ΔΔC_T method (Livak and Schmittgen, 2001 (link)).