Trizol extracted total RNA was used for cDNA synthesis employing a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). SYBR green based Real-time quantitative PCR (qPCR) was performed in ABI 7500 Cycler detection system (Applied Biosystems) using gene- and species- specific primers (Table 2 ). β-actin was employed as a housekeeping gene, and a delta Ct method was employed to calculate relative mRNA expression14 (link),17 (link).
Primer sequences.
| Primer | Sequence |
|---|---|
| Mfn2 | F-ATGCAGACGGAAAAGCACTT |
| R-ACAACGCTCCATGTGCTGCC | |
| Mfn2 promoter | F-TGCCCGATGAGTCACTTCAC |
| R-CAAGGGGCGAAAAACCAAGG | |
| Mlh1 | F-CCTGCTCCCCGCGCTTTCTT |
| R-CGGGGAGGCTGTGCTTCTGC | |
| Mlh1 promoter | F-GTCATCCACATTCTGCGGGA |
| R-CTCTGCTGAGGTGATCTGGC | |
| CytB | F-ATGGTAGATGTGGCGGGTTT |
| R-TCTCCGATCCGTCCCTAACA | |
| β-Actin | F-AGCCTCGCCTTTGCCGATCCG |
| R-TCTCTTGCTCTGGGCCTCGTCG | |
| Mfn2 | F-ACAAAGTTCTGCCATCTGGG |
| R-TGCTCATCCTGATGGAGGGC | |
| Mfn2 promoter | F-GTCTGCCCGATGAGTCACTT |
| R-AAACCAAGGGCGTGGAGTA | |
| Mlh1 | F-AAGCATAAGCCATGTGGCCC |
| R-TTCCCGTACTCTTCACTGGG | |
| Mlh1 promoter | F- TTCCCGAGTAGAGGCGACC |
| R- AACCCAGGGGGGTGCTTGG | |
| Dnmt1 | F-ACCTACCACGCCGACAT |
| R-AGGTCCTCTCCGTACTCCA | |
| CytB | F-CCCATTCATTATCGCCGCCC |
| R-GGTCTCCTAGTAGGTCTGGG | |
| β-Actin | F-CCTCTATGCCAACACAGTGC |
| R-CATCGTACTCCTGCTTGCTG | |