Luna universal qpcr master mix

To confirm the differential gene expression identified from RNA-seq data, six candidate genes were selected to be performed with quantitative reverse transcription PCR, including cfap206, rnf8, naa38, saxo2, tdo2b, and tlr5. The gapdh gene was used as a reference. The reverse transcription was conducted using LunaScript^® RT SuperMix Kit (New England BioLabs, Ipswich, MA, USA) with 400 ng input of total RNA, following the manufacturer’s procedure. The Oligo 7.0 software (Molecular Biology Insights Inc., Cascade, CO, USA) was used to design qPCR primers for these genes (Table S3). The primers were synthesized by Eurofins (Eurofins Genomics LLC., Louisville, KY, USA), and the target size of PCR products and amplification efficiency were evaluated by 1.5% agarose gel electrophoresis. The Bio-Rad C1000 Touch Thermal Cycler with CFX96 Real-Time PCR Detection Systems (Bio-Rad Laboratories, Hercules, CA, USA) was employed to perform qRT-PCR experiments. The PCR reaction was conducted in a 20 µL mixture consisting of 8 µL of nuclease-free water, 0.5 µL of each primer (10 µmol/L), 1 µL of cDNA template, and 10 µL of Luna Universal qPCR Master Mix, using Luna^® Universal qPCR Master Mix (New England BioLabs, Ipswich, MA, USA). All qRT-PCR assays were conducted by incubation in 96-well plates, followed by 40 cycles at 95 °C for 15 s and 60 °C for 30 s with two technical replicates.

Each stool sample was tested for SARS-CoV-2 presence using both a probe-based and dye-based qPCR. The probe-based qPCR was performed using the Luna Universal Probe qPCR Master Mix (New England BioLabs, Cat. No. M3004L) and the CDC primers. Each reaction consisted of a total volume of 20 μl with 1.5 μl of 6.7 μM primer/probe mix, 4 μl of cDNA, 4.5 μl of water, and 10 μl of Luna Universal Probe qPCR Master Mix. The qPCR was carried out following manufacturer recommendations on a Viia7 Real-Time PCR system (Thermofisher). The dye-based qPCR was performed using the Luna Universal qPCR Master Mix (New England BioLabs, Cat. No. M3003L) and the remaining primers: nsp14, 16S, HPRT, β-actin, GAPDH. Each reaction consisted of a total volume of 20 μl with 1 μl of 10 μM of forward and reverse primers, 4 μl of cDNA, 5 μl of water, and 10 μl of Luna Universal qPCR Master Mix (NEB). The qPCR was carried out following manufacturer recommendation on a Viia7 Real-Time PCR system (Thermofisher).

Each stool sample was tested for SARS-CoV-2 presence using both a probe-based and dye-based qPCR. The probe-based qPCR was performed using the Luna Universal Probe qPCR Master Mix (New England BioLabs, Cat. No. M3004L) and the CDC primers. Each reaction consisted of a total volume of 20 μl with 1.5 μl of 6.7 μM primer/probe mix, 4 μl of cDNA, 4.5 μl of water, and 10 μl of Luna Universal Probe qPCR Master Mix. The qPCR was carried out following manufacturer recommendations on a Viia7 Real-Time PCR system (Thermofisher). The dye-based qPCR was performed using the Luna Universal qPCR Master Mix (New England BioLabs, Cat. No. M3003L) and the remaining primers: nsp14, 16S, HPRT, β-actin, GAPDH. Each reaction consisted of a total volume of 20 μl with 1 μl of 10 μM of forward and reverse primers, 4 μl of cDNA, 5 μl of water, and 10 μl of Luna Universal qPCR Master Mix (NEB). The qPCR was carried out following manufacturer recommendation on a Viia7 Real-Time PCR system (Thermofisher).

Left auricles in Protocol 2 were stored in an RNAlater™ Stabilization Solution (Thermo Fisher Scientific) at − 30 °C until analysis. Total RNA was extracted from the left auricles using a Monarch Total RNA Miniprep Kit (New England BioLabs, MA, USA), according to the manufacturer’s instructions. Subsequently, the RNA was reverse-transcribed to cDNA using a LunaScript RT SuperMix Kit (New England BioLabs). Next, following the manufacturer’s guidelines, quantitative real-time PCR was performed using a Luna Universal qPCR Master Mix (New England BioLabs). Two microliter of cDNA (40 ng) was amplified with 500 nM each of the specific primer combinations for the target genes and 10 μL Luna Universal qPCR Master Mix (New England BioLabs) in a total volume of 20 μL. PCR conditions were as follows: initial denaturation of 95 °C for 60 s, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. Then, real-time PCR was conducted for each sample according to a relative standard curve method, using a StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific). Standard curves were obtained from serially diluted sample mixtures, after which the expression levels of samples were measured using these standard curves. Primers for the respective target genes (listed in Supplementary Table S2 online) were designed to anneal at 60 °C.

Forward and reverse primer pairs for SYBR-Green-based RT-qPCR analysis were designed in-house and validated under stringent conditions (efficiency between 90–110% and R² > 0.99), as described previously [55 (link),69 (link),70 (link),71 (link)]. Table 1 lists the validated RT-qPCR primer sequences utilized in this study. RT-qPCR reactions were performed using the QuantStudio 5 Real-Time PCR instrument (ThermoScientific) in 96-well plates. The RT-qPCR reaction mix was completed in 15 μL reaction volume consisting of 5 ng cDNA, 600 nm forward and reverse primers, and 7.5 μL 2× Luna Universal qPCR Master Mix (NEB, M3003). The RT-qPCR cycling parameters consisted of 95 °C for one min followed by a two-step denaturation and extension cycle of 95 °C for 15 s and 60 °C for 30 s for a total of 40 cycles. Plate reading was performed at the end of the extension phase. A DNA melt curve was performed at the completion of each RT-qPCR experiment to assess the amplification specificity. The RT-qPCR data was analyzed using the 2^−∆∆Ct analysis method, as described previously [72 (link),73 (link)]. All samples were normalized to two independent housekeeping genes (RSP18 and RPL4). The relative mRNA expression of each gene was reported as the mRNA fold change ± standard error of means (SEM) relative to the CGL1^dCas9 control cells.

RNA was isolated from Ewing sarcoma cells using the Trizol reagent (Invitrogen, Thermos Fisher Scientific, Waltham, MA, USA). After DNase digestion, 1 μg of total RNA was reverse transcribed by Reverse Transcriptase M-MLV (Promega, Madison, WI, USA) following the manufacturer’s instructions. PCR reactions were run in triplicate on a QuantStudio1 Real Time qPCR instrument (Thermos Fisher Scientific, Waltham, MA, USA). For all experiments, no-RT controls were performed. PCR amplification was carried out with 1 μL of the 1:10 diluted reverse transcription sample with 10 μL of 2× Luna Universal qPCR Master Mix (New England Biolabs -NEB) and 4 pmol of specific gene primer pairs in a 20-μL total volume in 96-well microtiter plates.
Primers: (pncCCND1_B Fw: 5′-TGAGATTCTTGGCCGTCTGT-3′; pncCCND1_B Rev 5′-CCATATCCAAGCCGGCAGA-3′; CCND1-Fw 5′-GTGCAAGGCCTGAACCTG-3′; CCND1-Rev 5′-CGGGTCACACTGATCACTC-3′; GAPDH Fw: 5′-TGGTCACCAGGGCTGCTT-3′; GAPDH Rev 5′-CATGTAGTTGAGGTCAATGAAGG-3′).

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.