Quantitative PCR for Gene Expression

PCR experiments were performed on total RNA isolated from tissues, treated as described above, using the Triazol reagent (Invitrogen). RNase-free DNase (Ambion, Austin, TX, USA) was used for RNA extraction and cDNA was purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the respective manufacturer’s instructions. Single-strand cDNA samples were obtained using the iScriptcDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) from at least 20 ng of purified RNA. To determine mRNA expression, the following primers were used: (1) Cx36: forward, 5′-ATACAGGTGTGAATGAGGGAGGATG-3′, reverse, 5′-TGGAGGGTGTTACAGATGAAAGAGG-3′⁷² (link) (2) Actb: forward, 5′-GTGGGGCGCCCCGGCACCA-3′, reverse, 5′-CTCCTTAATGTCACGCACGATTT-3′⁷³ (link) (3) Hprt: forward, 5′-TCCTCATGGACTGATTATGGACA-3′, reverse, 5′-TAATCCAGCAGGTCAGCAAAGA-3′⁷⁴ (link) (4) Rpl13A: forward, 5′-TCCTCATGGACTGATTATGGACA-3′, reverse, 5′-TAATCCAGCAGGTCAGCAAAGA-3′.⁷⁴ (link) The new synthesized cDNA was amplified using the oligonucleotide primer listed above, the nucleic acid stain iQ SYBER Green Supermix (Bio-Rad) and an iCycler IQ Real-time PCR System (Bio-Rad).