*2.9. qRT-PCR*

Total RNA was isolated with the TRIzol reagen<sup>t</sup> (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instruction. The RNA quality was verified by spectrophotometry. Later on, the cDNAs were synthesized using the ReverTra Ace qPCR RT kit (Toyobo, Tokyo, Japan). For mitochondrial DNA analyses, total DNA was extracted by a DNA extraction kit (NEP002-1, Dingguo, China) according to the manufacturer's instructions. qRT-PCR was performed using the Light Cycler®Nano SYBR Green I Master on a Light Cycler® Nano System. The PCR conditions were as follows: 10 min at 95 ◦C, followed by 40 cycles of 95 ◦C for 30 s, 56 ◦C for 30 s and 72 ◦C for 30 s. The mRNA levels were calculated using the 2−ΔΔCT method normalized to ACTB mRNA.

The Primers used in this study: Drp1, 5-TAGTGGGCAGGGACCTTCTT-3 (F) and 5-TGCTTCAACTCCATTTTCTTCTCC-3 (R); ACTB, 5-CACCAGGGCGTGATGGT-3 (F) and 5-CTCAAACATGATCTGG GTCAT-3 (R); NADH dehydrogenase subunit I (ND1), 5-TACGCAAAGGTTCCCAACG-3 (F) and 5-GGTGATGGTGGATGTGGC-3 (R); cytochrome C Oxidase Subunit IV Isoform 1 (COX4I1), 5-TAGAAACCGTCTGAACTATCC-3 (F) and 5- ATGATTATGAGGGCGTGA-3 (R); β-globin, 5-GTTACTGCCTG TGGGGCAA-3 (F) and 5-CAAAGGTGCCCTTGAGGTT-3 (R). The qRT-PCR assay was designed and performed in accordance with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines.
