Quantitative real-time PCR

For real-time quantification of target gene expression, one-step real-time Polymerase Chain Reaction (RT-PCR) was performed using FastStart Universal SYBR Green Master (ROX) in a StepOnePlus^™ Real-Time PCR System (Life Technologies-Invitrogen, California, U.S.A.). A fragment of the NOMO1 gene was amplified from the DNA of patients and controls using the following primers: F: 5′-agctccatgtggatggagtc-3′ and R: 5`-acggatgaagtacagagttc-3. As internal control, the 36b4 gene was amplified from the same DNA using the primers: F: 5′-cagcaagtgggaaggtgtaatcc-3′ and R: 5′-cccattctatcatcaacgggtacaa-3.

Ten μl RT-PCR of a mix containing 15 ng of total DNA, 1 μl of the primer dilution, 4 μl FastStart Universal SYBR Green Master and 4 μl H₂O were used for amplification. One-step RT-PCR reactions were carried out in 96-well optical reaction plates, covered with MicroAmp^® Optical Adhesive Film (Life Technologies-Invitrogen, California, U.S.A). Cycling was as follows: 10 minutes at 95°C followed by 40 cycles of 95°C for 15 seconds, 58°C for 45 seconds and 72°C for 15 seconds. RQ Manager software was used to analyse the values.

The comparative Ct method (2^−ΔΔCt) was used to calculate the relative expression levels of each amplicon. RT-PCR specificity of each PCR reaction was verified by melting curve analysis.