Quantstudio 5 real time pcr

The total RNA from the cultured BMMs was extracted with the TRIzol reagent (Invitrogen) according to the manufacturer's recommended protocol. All experiments were conducted only with high-quality RNA (A260/A280 ratio of 1.8–2.0 and A260/A230 ratio of 2.0–2.2). Reverse transcription was performed with 1 μg of RNA and RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer's protocol. The cDNA was amplified by using Applied Biosystems Power-Up SYBR green PCR master mix (Thermo Fisher Scientific) and quantified by using Quantstudio®5 Real-Time PCR (Thermo Fisher Scientific). Primers were used as previously reported [17 (link)], and the genes were normalized to encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All real-time PCR reactions were performed at least three times, and all data were analyzed by using the 2^−ΔΔCt method [22 (link)].

Real‐time polymerase chain reaction (RT‐PCR) was carried out differentially (tumour compared to paired normal) with SYBR® Premix Ex Taq II (TaKaRa) and target gene‐specific primers (VEGFA, VEGFB, KDR, CXCR1, CXCR2 and PTGS2) (Table 1) using QuantStudio 5 Real‐Time PCR (Thermo Fisher). Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used as the internal control. Samples were run in triplicates for 40 cycles at 95°C for 30 s and 60°C for 30 s. 2^ΔΔCT method was employed to calculate the fold quantification among paired samples. All experiments were repeated three times. Negative controls with Milli‐Q water instead of template DNA were included.

Total RNA was extracted from Sw.71, Jurkat, MOLT4, Ramos, and Raji cells using the RNeasy Mini Kit according to the manufacturer's instructions (Qiagen). To analyze gene expression levels, SYBR Green-based one-step RT-PCR was performed as described previously (74 (link)). Human glyceraldehyde-3-phosphate (GAPDH) was used as an internal control gene. The sequences of the forward and reverse primers were as follows: Gag-pol and env, 5′-TAGCAAACCGTCAAGCACAG-3′ and 5′-TGGACGGGCTATTATCCTTG-3′; Tax/Rex, 5′-ATCCCGTGGAGACTCCTCAA-3′ and 5′-AACACGTAGACTGGGTATCC-3′; HBZ, 5′-AGAACGCGACTCAACCGG-3′ and 5′-TGGCACAGGCAGGCATCG-3′; GAPDH, 5′-TTCTTTTGCGTCGCCAGCCGA-3′ and 5′-GTGACCAGGCGCCCAATACGA-3′. Data were collected using QuantStudio 5 Real-Time PCR (Thermo Fisher Scientific), and relative expression levels were calculated using the 2^–ΔΔCt method.

RNA extraction was performed from 200 μl of plasma samples. miRNAs were isolated using Total RNA Purification Plus Kit (Norgen Biotek, Thorold, ON, Canada) according to the manufacturer's protocol, as previously described [39 (link)]. As an internal control, 10 ftmoles of cel-miR-39a was spiked into each plasma sample after lysis.
RNA from skin biopsies was extracted with an RNeasy tissue lipid kit (Qiagen, Valencia, CA, USA), as previously described [40 (link)]. miRNA expression levels in skin samples were normalized to housekeeping RNA Z30 expression.
miRNA levels were analysed using the TaqMan quantitative real-time PCR (qRT-PCR) and quantified with the QuantStudio5 real-time PCR (Thermo Fisher Scientific, Massachusetts, United States). Primers for miR-200c, miR-200a, miR-200b, miR-429, miR-141, Z30, and cel-miR-39a and the reagents for reverse transcriptase and qPCR reactions were all obtained from Applied Biosystems. miRNA expression levels in each sample were normalized to cel-miR-39a. Relative expression in fold was calculated using the comparative Ct method (2^–ΔΔCt) [41 (link)].
Given the logarithmic nature of the q-PCR CT, a decrease in ΔCT corresponds to an increase in miRNA levels in the analysed samples. Therefore, data were expressed as −ΔCT, in order to obtain a positive correlation between miRNA levels and clinical parameter values.

To determine the specificity of the assay, we performed interference tests involving the cross-reactivity of pathogens which produce similar symptoms as SARS-CoV-2 and also involving potential endogenous and exogenous interfering substances. Samples in the cross-reactivity assay were prepared by adding cultured isolates or nucleic acid of the cross-pathogens (as shown in Table S3) into 37 low-concentration VLPs-simulated samples (3 × LOD) and 37 negative samples, respectively. Samples in interference testing were prepared by adding 4 potential endogenous and 6 exogenous interfering substances (as shown in Table S4) into 10 low-concentration VLPs-simulated samples (3 × LOD) and 10 negative samples, respectively. Each cross-pathogen or interfering substance was tested three times in low-concentration and negative samples. The experiments were performed on a QuantStudio™ 5 real-time PCR (Thermo Fisher Scientific, USA) instrument with one lot of reagents.

Total RNA was separated from HCC cell lines with an RNA isolater (Vazyme, #R401-01-AA), and its concentration was determined using NanoDrop ONE (Thermo Fisher Scientific). The RNA was then reverse-transcribed into cDNA using a reverse transcription kit (Vazyme, #R323-01). qRT-PCR detection was performed using the AceQ Universal SYBR qPCR Master Mix kit (Vazyme, #Q511-02) in the QuantStudio™ 5 Real-Time PCR (Thermo Fisher Scientific) (PRC1: forward primer: CGC​CAT​GAG​GAG​AAG​TGA​GG, reverse primer: TTG​TAA​CCG​CTG​GTC​CTC​TG; RACGAP1: forward primer: ACG​TTG​AAT​AGG​ATG​AGT​CAT​GGA, reverse primer: AAA​GTC​CTT​CGC​CAA​CTG​GA). All procedures were conducted in accordance with the manufacturer’s instructions. The thermal cycling parameters for amplification were as follows: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 10 s, and annealing and extension at 60°C for 1 min for a total of 40 cycles.