Quantstudio real time pcr software

Total RNA was isolated from cells or precision-cut liver slices using a Maxwell^® LEV simply RNA Cells/Tissue kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. RNA concentrations were determined using a NanoDrop One spectrophotometer (Thermo Fisher Scientific). Conversion of RNA to cDNA was performed using MLV (murine leukemia virus) reverse transcriptase (Promega) in an Eppendorf Mastercyler gradient device, with the gradient at 20 °C for 10 min, 42 °C for 30 min, 20 °C for 12 min, 99 °C for 5 min and 20 °C for 5 min. The transcription levels were measured in 10 ng cDNA by quantitative real-time PCR (SensiMix™ SYBR^® Low-ROX Kit, Bioline, Taunton, MA, USA) using a QuantStudio 7 Flex Real-Time PCR system (hold stage: 95 °C for 10 min; PCR stage: 40 cycles of 95 °C for 15 s and 60 °C for 25 s; melt curve stage: 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s). Data was analyzed using QuantStudio Real-Time PCR software (Thermo Fisher Scientific). For each model, mRNA expression was normalized to housekeeping genes (either ACTB for primary HSC and LX-2, or RNA18S5 for HHSteC, TWNT-4 and PCLS), and expressed as 2^−ΔΔCt (fold induction). Differences between treatment groups and untreated controls are in the main text referred to as percentage increase or decrease. The used primer sequences are listed in Table 1.

Total RNA was isolated from human cells using Quick‐RNA Microprep kit (Zymo Research) or from murine SVC using Direct‐zol RNA Miniprep Plus kit (Zymo Research) according to the manufacturer's instructions. cDNA synthesis was performed on 100–200 ng of total RNAs with Superscript III (Thermofisher) and random hexamer primers. Relative gene expression levels were assessed using Taqman® Probes and TaqMan Fast Advanced Applied mastermix (Thermofisher) on a QuantStudio™ 5 (QS5; Thermofisher). Human and murine Assay‐On‐Demand are listed in Table S1. Each of the samples was run in duplicate, and the relative amount was normalized to 18 S or PPIB housekeeping gene. Data were analyzed using QuantStudio Real‐Time PCR software (Thermofisher).

DNA was extracted from whole blood using a PAXgene Blood DNA kit (Qiagen, Hillden, Germany) as previously described [40 (link)]. SNPs rs2266782 (G>A, p.Glu158Lys, assay ID c_2461179_30) and rs2266780 (A>G, p.Glu308Gly, assay ID C_2220257_30) were genotyped using TaqMan Drug Metabolism Genotyping Assays and real time PCR using an Applied Biosystems QuantStudio 7 Flex Real-Time PCR System following the manufacturer’s instructions (ThermoFisher Scientific, Waltham MA, USA). QuantStudio Real-Time PCR software was used to determine the genotypes (ThermoFisher Scientific). Hardy-Weinberg equilibrium was tested using the HWChisq function in the R package HardyWeinberg (v1.7.5). Relationships between genotype and continuous variables were tested without assuming an inheritance pattern (i.e., categorically coded) unless otherwise stated.

Raw PCR Ct values were generated using QuantStudio™ Real-Time PCR Software with automatic baseline and threshold (Thermo Fisher). The data set was uploaded to Thermo Fisher Data Cloud and further analyzed using Thermo Fisher Cloud Software-Relative Quantification. PCR Ct values ≥ 35 were considered as undetectable and excluded from further analysis. An appropriate internal normalizer could not be identified due the composition of the different subcellular sources of RNA. Therefore, the Global Mean Normalization method [54 (link)] was used to normalize the TLDA or single-tube data. The relative miRNA levels or Gapdh mRNA quantity (RQ) in each fraction were expressed as 2^−∆Ct value (2 to the power of negative deltaCt).

Total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA). cDNA was prepared using cDNA reverse transcription kit (Invitrogen). Quantitative reverse transcription PCR was performed using the following TaqMan^™ Gene Expression Assays were used: IMPDH1 = Hs00992210_m1, IMPDH2 = Hs00168418_m1, and ACTB = Hs99999903_m1. PCR data were analyzed using QuantStudio^™ Real-Time PCR Software (Thermo fisher).