Express sybr greener qpcr supermix kit

Analysis of mitochondrial DNA content was performed by quantitative PCR (qPCR) on a 7500 Fast real-time cycler (Applied Biosystems, Thermo Fisher Scientific) using the EXPRESS SYBR GreenER qPCR Supermix kit (ThermoFisher Scientific). Specific primers for mitochondrial DNA (mtDNA; Fw primer: 5′-CAACCATTCATTCCAGCCTT-3′; Rev primer: 5′-GAAAATTTTAAATGGCCGCA-3′) were designed as previously reported25 (link). The relative levels of mitochondrial DNA were normalized to nuclear DNA using primers for actin (Dm_Act79B, QuantiTect Primer Assay, Qiagen). For reverse transcription (RT)-qPCR analyses, the isolation of total RNA was performed using Sepasol-RNA I Super G (Nacalai Tesque). RT was performed using a SuperScript VILO cDNA Synthesis Kit (ThermoFisher Scientific), and subsequent qPCR was performed using the EXPRESS SYBR GreenER qPCR Supermix kit with the following primers (dCHCHD2 Fw primer: 5′-TCCACTCGTCGCACCGCACCTGTG-3′; dCHCHD2 Rev primer: 5′-ACGGCACTGGGAGGAGCGCTCATG-3′; dMICS1 Fw primer: 5′-GCTCAACATCTTTATCCGCATC-3′; dMICS1 Rev primer: 5′-CACCTTAAACCAACTCAGTTCTTC-3′; RP49 Fw primer: 5′-ACTTCATCCGCCACCAGTCG-3′; RP49 Rev primer: 5′-CGGGTGCGCTTGTTCGATCC-3′). The relative transcript levels of the target genes were normalized against RP49 mRNA levels. Quantification was performed using the comparative Ct method.

RT-PCR was performed with Express SYBR GreenER qPCR Supermix kit (Invitrogen) according to the manufacturer's instructions. The reaction was carried out with a 7900 HT Fast RT – PCR system (Applied Biosystems, Foster City, CA, USA). The primers presented in Table 5 were designed with Primer3Plus online software from consensus sequences provided by Affymetrix for each gene of interest. All experiments were analyzed in duplicate. β₂ microglobulin mRNA expression served to normalize and compare the expression values of genes of interest. The results were quantified by the ΔΔCt method in Microsoft Excel.