7900 real time pcr system

Genomic DNA was extracted from 200 μl of a peripheral blood sample of each patient using QIAamp DNA blood mini kit (Qiagen, Hilden, Germany). All patients included in this study were genotyped for CYP2D6^∗2, ^∗4, ^∗6, ^∗10, ^∗17, and ^∗41 alleles by TaqMan^® allelic discrimination assays (Applied Biosystems, Foster City, CA, United States) according to the manufacturer’s instructions. Determination of CYP2D6 gene deletion (CYP2D6^∗5) and duplication was performed using a pre-developed TaqMan^®CYP2D6 Copy Number Assay (Hs00010001_cn, Thermo Fisher Scientific) and as a reference the TaqMan^® Copy Number Reference Assay RNase P (Thermo Fisher Scientific) as previously described (Schroth; Front Pharmacol 2017). Real-time PCR analyses were carried out according to the manufacturer’s instructions on a 7900 Real-Time PCR System (Thermo Fisher Scientific) and data were analyzed with the SDS2.4 software. In the present study, alleles are assigned according to the nomenclature for human CYP2D6 alleles as previously published (Sim and Ingelman-Sundberg, 2010 (link)). Samples with one loss of function or reduced activity allele and a copy number of 3 were assigned EM phenotype. The allele frequencies were tested for deviation from the Hardy Weinberg equilibrium using the exact test¹.

qRT-PCR was performed with TaqMan probes (ThermoFisher Scientific, Waltham, MA, USA) using a 7900 Real Time PCR System. A total of 1 µg of total RNA was transcribed to cDNA using SuperScript^® VILO^TM cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA, USA). cDNA was amplified in the presence of specific TaqMan probes: MBP (UniGene: Hs00921945_m1), MOG (Hs01555268_m1), PLP (Hs00166914_m1), Dicer1 (Hs00229023_m1), Ago2 (Hs01085579_m1), ELL3 (Hs00228559_m1), Exp5 (Hs00382453_m1), β-actin (Hs00921945_g1) using TaqMan^® Fast Advanced Master Mix (ThermoFisher Scientific, Waltham, MA, USA). The PCR reactions were performed on the 96-well plates using TaqMan^® Fast Advanced Master Mix (ThermoFisher Scientific, Waltham, MA, USA). The reaction specificity was checked by melting curve analysis, and relative gene expression was determined by the ΔΔCT method.

Total RNA from treated HASMCs was isolated using TRIzol reagent (Invitrogen). For the quantification of lncRNA and mRNA, the cDNA was obtained by using PrimeScript RT reagent kit (Takara, Dalian, China). The real‐time PCR was performed on the 7900 Real‐time PCR System using SYBR Premix Ex Taq (Takara). For the quantification of miR‐150‐5p, cDNA was obtained by using Taqman MicroRNA Reverse Transcription kit (Thermo Fisher Scientific, Waltham, USA) and real‐time PCR was performed on the 7900 Real‐time PCR System using Taqman microRNA assay kit (Thermo Fisher Scientific). The fold changes for lncRNA, mRNA and miRNA expression were determined using comparative Ct method, and GAPDH was selected as the internal control for lncRNA and mRNA and U6 was selected as the internal control for miRNA.

qRT-PCR was performed with TaqMan probes (ThermoFisher Scientific, Waltham, MA, USA) using a 7900 Real Time PCR System. First, 1 µg of total RNA was transcribed to cDNA using a SuperScript^® VILO™ cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, Massachusetts, USA). cDNA was amplified in the presence of specific TaqMan probes—MBP, MOG, PLP, and β-actin—using TaqMan^® Fast Advanced Master Mix (ThermoFisher Scientific, Waltham, MA, USA). The PCR reactions were performed on the 96-well plates using TaqMan^® Fast Advanced Master Mix (ThermoFisher Scientific, Waltham, MA, USA). The reaction specificity was checked by melting curve analysis, and relative gene expression was determined by the ΔΔCT method.