Abi prism 7900 sds 2

For confirming differences in expression of genes of interest found on gene chip, quantitative real-time PCR (qRT-PCR) analysis was used. For that purpose, the ABI PRISM 7900HT Fast Real-Time PCR System equipment (PE Applied Biosystems, USA) and the ABI PRISM 7900 SDS 2.2.2 Software were used. In all gene expression experiments, cytoplasmic β-actin (Actb) (VIC/MGB Probe, Primer Limited) was used as the endogenous reference gene (PE Applied Biosystems, USA), All reactions were performed using the TaqMan Gene Expression Master Mix (PE Applied Biosystems, USA) and the TaqMan Gene Expression Assays (FAM) according to the instructions of the equipment and reagent manufacturers. All samples to be compared were run in the same experiment and every reaction was run in quadruplicate. The amount of the target gene was compared to the housekeeper gene by means of the 2^−ΔCT method [39 (link)]. The following TaqMan Gene Expression Assays (FAM) were used: Ppard (Mm00803184_m1); Fmo2 (Mm0049019_m1); Sult3a1 (Mm00491057_m1); Lepr (Mm0040181_m1); Wfs1 (Mm01220326_m1).

Blood samples of 37 patients and 33 healthy controls were obtained. Total RNA from skin biopsies was converted to cDNA using random primers and High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems). For duplex qRT-PCR analysis TaqMan Gene Expression Assays were used: VIC (housekeeping gene ActinB) and FAM (gene of interest) probes and TaqMan Gene^® Expression Master Mix (Applied Biosystems). The TaqMan^® Gene Assay IDs were the following: Hs01060665_g1 (ActinB), Hs00761940_s1 (Saa1), Hs00754237_s1 (Saa2). qRT-PCR was performed using ABI PRISM 7900HT Fast Real-Time PCR System equipment (Applied Biosystems) and the ABI PRISM 7900 SDS 2.2.2 Software. Each reaction was performed in quadruplicate to minimize technical errors. Real-time PCR data for gene of interest was expressed as mean ΔCT value relative to housekeeping gene. ΔCT of controls was subtracted from ΔCT of PD to yield ΔΔCT. Relative expression i.e. fold change was calculated using 2^-ΔCT-function. The data of studied genes following normal distribution were parametrically tested by unpaired t-test.