14 abi prism 7700 sequence detector system

Gene expression was analyzed using TaqMan gene expression assay (Applied Biosystems). In detail, 0.1 ng cDNA was amplified, in triplicate, in a reaction volume of 25 µl with 10 pmol of each gene-specific primer and the SYBR-green PCR MasterMix (Applied Biosystems). Real-time PCR was performed on the 14 ABI/Prism 7700 Sequence Detector System (PerkinElmer/Applied Biosystems) using a pre-PCR step of 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. Specificity of the amplified products was confirmed by melting curve analysis (Dissociation Curve TM; PerkinElmer/Applied Biosystems) and by 6% PAGE. Samples were amplified with primers for each gene (listed in Table S3). The Ct values were normalized to the GAPDH curve. Results were quantified using the 2^−ΔΔCT method. PCR experiments were performed in triplicate.

Gene expression was analyzed using TaqMan Gene expression assay (Applied Biosystems). Real-time PCR was performed on the 14 ABI/Prism 7700 Sequence Detector System (PerkinElmer/Applied Biosystems), using a pre-PCR step of 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. Specificity of the amplified products was confirmed by melting curve analysis (Dissociation Curve TM; PerkinElmer/Applied Biosystems). Samples were amplified with primers for each gene (GADPH, Hs99999905_m1; RAB2A, Hs00234094_m1; RAB2B, Hs00375685_m1; Thermo Fisher Scientific). The Ct values were normalized to the GAPDH curve. Results were quantified using the 2−ΔΔCT method. PCR experiments were performed in triplicate.