Lightcycler faststar dna master sybr green 1 kit

An aliquot of 20 ng of DNA was subjected to quantitative PCR by Applied Biosystems QPCR 7300 instrument (Foster City, CA, USA) using LightCycler-FastStar DNA Master SYBR Green I kit (Roche Applied Sciences) referred to48 (link). The primers were as following: for mtDNA-ND1: Forward 5′-AACA TACCCATGGCCAACCT-3′; Reverse 5′-AGCGAAGGGTTGTAGTAGCCC-3′. For β-globin: Forward 5′-GAAGAGCCAAGGACAGGTAC-3′; Reverse 5′-CAACTTCATCCACGTTCACC-3′. The relative mtDNA copy number was measured by normalization of the crossing points in quantitative PCR curves between ND1 and β-globin genes using the RelQuant software (Roche Applied Sciences).

Real-time quantitative PCR was performed on a DNA Engine Opticon 2 machine (MJ Research, Bio-Rad, Hercules, CA, USA) using the Light Cycler-Fast Star DNA master SYBR Green I kit (Roche, Basel, Switzerland). The PtoXET16A-specific and internal control (Actin) primer pairs were designed using Primer Express 3.0 software (Applied Biosystems, Life Technologies, New York, NY, USA). The PCR program included an initial denaturation at 94 °C for 5 min, and 40 cycles of 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C, and a final melt-curve of 70–95 °C. The specificity of the amplified fragments was checked by melting curve. All reactions were carried out in triplicate, and the data were analyzed using the Opticon Monitor Analysis Software 3.1 tool (MJ Research, Bio-Rad, Hercules, CA, USA).

Quantitative PCR was performed on a DNA Engine Opticon 2 machine (MJ Research) using the LightCycler-FastStar DNA master SYBR Green I kit (Roche). The PCR program was described in Zhang et al. [90 ]. The melting curve was used to check the specificity of the amplified fragments. All reactions were carried out in triplicate for biological repeats, and the real-time data were analyzed using the Opticon Monitor Analysis Software 3.1 tool. Specific primer sets were designed for each gene using Primer Express 3.0 software (Applied Biosystems) (Additional file 6: Table S3). The efficiency of the primer sets was calculated by performing real-time PCR on several dilutions of first-strand cDNAs. Efficiencies of the different primer sets were similar. The specificity of each primer set was checked by sequencing PCR products. The results obtained for the different samples were standardized to the levels of Actin.