Primetime qpcr assay

Expression of YAP target genes and genes associated with the naïve and primed state of pluripotency was assessed using qPCR. RNA was isolated using a Qiagen RNAeasy Plus Mini Kit (Cat. # 74134; Qiagen), and the RNA concentration and purity determined using a NanoPhotometer (Implen, Munich, Germany). cDNA was synthesised using a high-capacity reverse transcription kit (Cat. # 4368814; Thermo Fisher Scientific). qPCR reactions were set up in triplicate, with each 10μl PCR reaction containing 1X TaqMan Fast Universal Master Mix (Cat. # 4352042; Thermo Fisher Scientific), 1X PrimeTime® qPCR Assay (Integrated DNA Technologies) or TaqMan Gene Expression Assay (Thermo Fisher Scientific) and 10ng of cDNA. PCR reactions were run on a QuantStudio 12K Flex Thermocycler (Cat. # 4471087; Life Technologies). Following the first two steps of heating the samples to 50°C for 2 min and denaturing them at 95°C for 10 min, reactions were subjected to 40 cycles of 95°C for 15 s and 60°C for 1 min. The Ct values were obtained from the QuantStudio 12K Flex Software with auto baseline settings and were then exported to the ExpressionSuite Software (Thermo Fisher Scientific) for analysis.

Total RNA was isolated using a GeneJET RNA Purification kit (Thermo scientific K0732) and then quantitative real-time PCR (qRT-PCR) was carried out using a LightCycler®; 480 RNA Master Hydrolysis Probes kit (Roche 04991885001) according to the manufacturer’s instructions. The TaqMan-gene-specific primers were designed using Integrated DNA Technologies’ PrimeTime qPCR Assay web application. TaqMan-gene-specific primers were designed using an Integrated DNA Technologies proprietary software program. All experiments were normalized for PCR sample loading and reaction, and data represent one of three independent experiments for ct (threshold cycle) plots. All gene expression values were normalized to total levels of the control housekeeping gene, GAPDH, whose primers are presented in Table 1.

qPCR was performed using PrimeTime qPCR Assay (Integrated DNA Technologies) and the primers/probes indicated in Supplemental Table 9. PCR was performed on a CFX96/C1000 qPCR machine (Bio-Rad). Cycling conditions were as follows: one cycle of 95 ºC for 3 min, 39 cycles of 95 ºC for 15 s, 60 ºC for 1 min. Comparative Ct method was used to determine relative reads. Total mtDNA levels were determined by comparing mtDNA MT-ND1 or MT-COX1 to nDNA ACTIN.

Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for microarray data validation. Four key Treg transcripts were selected: FOXP3 (Hs.PT.58.3671186), IL2RA (Hs.PT.58.2187899), CTLA4 (Hs.PT.58.3907580) and IKZF2 (Hs.PT.58.2960172) (Integrated DNA Technologies Inc.). GAPDH (Hs.PT.39a.22214836) was used as a reference gene as, from microarray data, its expression was stable across all time points, with no variations amongst patient groups. All the 2MIU-IL-2 and placebo patient samples from the four time points were screened. Three technical replicates were completed for each condition.
200 ng of total RNA were retrotranscribed to cDNA using 5× qScript^TM DNA Supermix (Quantobio) by incubating samples at 25°C for 5 min, at 42°C for 30 min and at 85°C for 5 min. Subsequently, cDNA was mixed with 20× predesigned PrimeTime^® qPCR Assay (Integrated DNA Technologies Inc.) and 2× Luna^® Universal qPCR Master Mix (New England BioLabs^® Inc.). The mixture was incubated at 95°C for 3 min and 40 cycles of amplification at 95°C for 10 s and 60°C for 30 s were performed using C1000 Touch^TM Thermal cycler (Bio-Rad). Raw Ct values were retrieved using CFX Maestro^TM software (Bio-Rad). ΔCt, ΔΔCt and relative concentration (R) values were computed as follows:
ΔCt=Ct gene of interest – Ct reference gene ΔΔCT=ΔCT – (average ΔCT placebo sample) R=2−ΔΔCt.

Both AAV and Ad vector genomes in all sampling sites were quantified by qPCR. Tissue DNA was extracted using NucleoSpin® DNA RapidLyse kit (Machery-Nagel), according to the manufacturer’s instructions. The total amount of vector was quantitated using AAV2 ITR or LacZ specific PrimeTime® qPCR assay (Integrated DNA Technologies) and TaqMan Fast Advanced Master Mix (Applied Biosystems) and measured in StepOnePlus™ Real-Time PCR instrument (Applied Biosystems)¹⁹