For quantification of HIF-1α, total RNA was extracted using a high-purity total RNA rapid extraction kit (BioTeke, Beijing, China) and reverse-transcribed into cDNA using
the M-MLV reverse transcriptase. RT-qPCR was then performed on the ExicyclerTM 96 fluorescence quantifier using SYBR Green Master Mix. The relative mRNA expression of HIF-1αwas calculated by the 2-ΔΔCt method and normalized to β-actin. For mitochondrial DNA (mtDNA) content determination, DNA extraction was performed using a tissue genomic DNA
extraction kit (BioTeke). The copy number of mtDNA was evaluated by RT-qPCR, as described in a previous study [23 (link)]. The primers used, synthesized by
GenScript (Nanjing, China), were listed inTable 1 Sequence of primers used in RT-qPCR
the M-MLV reverse transcriptase. RT-qPCR was then performed on the ExicyclerTM 96 fluorescence quantifier using SYBR Green Master Mix. The relative mRNA expression of HIF-1αwas calculated by the 2-ΔΔCt method and normalized to β-actin. For mitochondrial DNA (mtDNA) content determination, DNA extraction was performed using a tissue genomic DNA
extraction kit (BioTeke). The copy number of mtDNA was evaluated by RT-qPCR, as described in a previous study [23 (link)]. The primers used, synthesized by
GenScript (Nanjing, China), were listed in
| Gene | Primers | Sequence (5’-3’) |
|---|---|---|
| mtDNA | Forward | TGAGCCATCCCTTCACTAGG |
| Reverse | TGAGCCGCAAATTTCAGAG | |
| nuclear DNA (β-actin) | Forward | CTGCTCTTTCCCAGATGAGG |
| Reverse | CCACAGCACTGTAGGGGTTT | |
| HIF-1α | Forward | CTACTATGTCGCTTTCTTGG |
| Reverse | GTTTCTGCTGCCTTGTATGG | |
| β-actin | Forward | GGAGATTACTGCCCTGGCTCCTAGC |
| Reverse | GGCCGGACTCATCGTACTCCTGCTT | |
