Genomic DNA for the analysis of mtDNA content was isolated from human BMNC cells or mouse LSK cells using a TaKaRa MiniBEST Universal Genomic DNA Extraction kit (TaKaRa Bio Inc.). The relative mtDNA copy number was determined by qPCR with primers for the mitochondrial 16S rRNA gene and the nuclear Actin gene as previously described19 (link). All PCRs were performed in triplicate. The primers used to amplify 16S rRNA were 5′-GGTGCAGCCGCTATTAAAGG-3′ (16S rRNA, forward) and 5′-ATCATTTACGGGGGAAGGCG-3′ (16S rRNA, reverse).
For the measurement of Bcl-2 and Bax mRNA levels, the primers used were 5′-GACTGAGTACCTGAACCGGCATC-3′(Bcl-2, forward), 5′-CTGAGCAGCGTCTTCAGAGACA-3′(Bcl-2, reverse), 5′-ATGCGTCCACCAAGAAGC -3′(BAX, forward), and 5′-CAGTTGAAGTTGCCATCAGC-3′(BAX, reverse), according to a previous study20 (link). The relative mRNA levels of Bcl-2 and Bax were normalized to ACTB, the primers used for ACTB were 5′-TGACGTGGACATCCGCAAAG-3′ (ACTB, forward); and 5′-CTGGAAGGTGGACAGCGAGG-3′ (ACTB, reverse).
For the measurement of Bcl-2 and Bax mRNA levels, the primers used were 5′-GACTGAGTACCTGAACCGGCATC-3′(Bcl-2, forward), 5′-CTGAGCAGCGTCTTCAGAGACA-3′(Bcl-2, reverse), 5′-ATGCGTCCACCAAGAAGC -3′(BAX, forward), and 5′-CAGTTGAAGTTGCCATCAGC-3′(BAX, reverse), according to a previous study20 (link). The relative mRNA levels of Bcl-2 and Bax were normalized to ACTB, the primers used for ACTB were 5′-TGACGTGGACATCCGCAAAG-3′ (ACTB, forward); and 5′-CTGGAAGGTGGACAGCGAGG-3′ (ACTB, reverse).
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