Cfx96 c1000 thermal cycler

Total cellular RNA was prepared from cell cultures using TRI®Reagent according to the manufacturer’s instructions (Merck, 93,289). One microgram of total cellular RNA was used for cDNA synthesis with a SuperScript III First-Strand Synthesis Supermix cDNA synthesis kit (Invitrogen, 18,080), and 1.5% of the cDNA was used for quantitative real-time polymerase chain reaction with SYBR Green Rox mix (ThermoFisher, 11873913) and gene-specific oligonucleotide primers in a CFX96 C1000 thermal cycler (BIO-RAD Laboratories Ltd., Hemel Hempstead, UK). Analysis of relative expression levels between target and calibrator genes used the 2^−∆∆Ct method [17 (link)].

The SARS-CoV-2 nucleic acid standards were diluted to 100 copies per microliter (copies/μL) in nuclease-free sterile water. The Complementary DNA (cDNA) of SARS-CoV-2 standards and clinical samples were synthesized using a CFX96 C1000 thermal cycler (Bio-Rad Laboratories) and HiScript III 1st Strand cDNA Synthesis kits (+gDNA wiper) (R312, Vazyme Biotech Co. Ltd., Nanjing, China). Dilations of the SARS-CoV-2 standards of 1, 10, and 100 copies/μL were prepared through dilution of the cDNA libraries of the SARS-CoV-2 standards according to the original concentration, and then confirmed by detecting the N and ORF1ab regions of the SARS-CoV-2 genome using qRT-PCR. The qPCR tests were performed at our laboratory using the AceQ qPCR Probe Master Mix (Q112, Vazyme Biotech Co. Ltd., Nanjing, China) on an ABI StepOnePlus real-time PCR system. qRT-PCR tests of clinical specimens were routinely performed at the hospital using 2019-nCoV nucleic test kits (Bojie Ltd., Shanghai, China).

RNA was prepared via the RNAprep Pure Kit (Magen) with DNase I for reverse transcription using the PrimeScript RT kit (Vazyme). Quantitative PCR was performed with SYBR qPCR Mix (Tsingke) in the CFX 96 C1000 Thermal Cycler (Bio-Rad).

For analysis of the gene expression profile of nonelectroporated and electroporated tolDCs and control DCs, total RNA was isolated. The cells were disrupted and homogenized using guanidine-thiocyanate-containing lysis buffer. Total RNA was isolated using an RNeasy microkit (Qiagen, Antwerp, Belgium). The RNA concentration was determined by measuring absorbance at 260 nm using a Nanodrop spectrophotometer (Wilmington, DE, USA). Reverse transcription of the obtained RNA into cDNA was performed using an iScript™ Advanced cDNA Synthesis Kit (Bio-Rad). Subsequently, SYBR^® Green technology was used for relative mRNA quantification by qPCR in a CFX96 C1000 thermal cycler (Bio-Rad). qPCR reactions were conducted at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s, and at 60°C for 30 s. All primer sets were obtained from Bio-Rad; validation data are shown in Table S1 in Supplementary Material. qPCR was performed in triplicate, and resulting mRNA levels were normalized to levels of the reference genes beta-actin and phosphoglycerate kinase 1. Melt curve analysis was performed to confirm the specificity of the amplified product. Bio-Rad CFX manager v3.1 was used for data processing and analysis.

RNA was extracted from cells using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and then reverse-transcribed into cDNA using the PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio, Kusatsu, Japan). Quantitative PCR was performed with a Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan) using CFX96 C1000 Thermal Cycler (Bio-Rad). mRNA levels were calculated using the comparative C_T method [14 (link)] and normalized to the values of the GAPDH gene. cDNA was amplified by gene-specific primers (IL1B: forward 5′-AACAGGCTGCTCTGGGATTC-3′, reverse 5′-AGTCATCCTCATTGCCACTGT-3′; CXCL8: forward 5′-AAGAAACCACCGGAAGGAAC-3′, reverse 5′-ACTCCTTGGCAAAACTGCAC-3′; CCL2: forward 5′-CCCAGTCACCTGCTGTTATAAC-3′, reverse 5′-AGATCTCCTTGGCCACAATG-3′; CXCL2: forward 5′-GCAGGGAATTCACCTCAAGAAC-3′, reverse 5′-AGCTTCCTCCTTCCTTCTGG-3′; GAPDH: forward 5′-ACCATCTTCCAGGAGCGAGAT-3′, reverse 5′-ATGACGAACATGGGGGCATC-3′).