7500 fast real time pcr instrument

First, RNA was reverse-transcribed using the High-Capacity cDNA Archive kit according to the manufacturer’s protocol (Life Technologies, USA). Complementary DNA was then amplified by real-time reverse transcription quantitative PCR (RT-qPCR) using TaqMan probes purchased as Assays-on-demand Products for Gene Expression (Life Technologies, USA) and a 7500 Fast Real-Time PCR instrument (Life Technologies, USA). The GAPDH gene was selected as an internal control for RNA input and reverse-transcription efficiency. All RT-qPCRs were performed in triplicate for both the target genes (SRC: Hs01082246_m1; LYN: Hs00176719_m1; CKB: Hs00176484_m1) and the internal control (GAPDH: NM_002046.3).
The relative quantification of gene expression was calculated according to Livak and Schmittgen [50 (link)]. The corresponding control sample was designated as a calibrator from each tumor.

RNA was reverse transcribed using the Reverse Transcription System according to the manufacturer's protocol (A3500; Promega, USA). Complementary DNA was then amplified by real-time reverse transcription quantitative PCR (RT-qPCR) using TaqMan probes purchased as Assays-on-Demand Products for Gene Expression (Life Technologies, USA) and a 7500 Fast Real-Time PCR instrument (Life Technologies, USA). The ACTB gene was selected as an internal control [63 (link)]. All RT-qPCRs were performed in triplicate for both the target genes (YWHAE: Hs00356749_g1; CDC25B: Hs00244740_m1; MYC: Hs00153408_m1) and the internal control (ACTB: 4333762F).
The relative quantification (RQ) of gene expression was calculated according to Livak and Schmittgen [64 (link)]. In tissue sample analyses, the corresponding control sample was designated as a calibrator from each tumor. In the cell line analysis, the siRNA control-transfected cells were used as a calibrator. The gene expression in the MNP01 was also designated as a calibrator from all GC cell lines.

SARS-CoV-2 viral load was measured in the liquid collected in the SKC biosampler, in the BLAM nebulizer after bio-aerosolization, and in the initial virus suspension.
Briefly, 15 μL of the liquid were mixed with 45 μL of distilled water and submitted to thermolysis for 3′ at 98 °C, followed by 5′ at 4 °C.
Then, SARS-CoV-2 was quantified by Real Time PCR using for target the N (nucleocapsid) gene (CDC primers and probe: 500 nM forward primer GGG AGC CTT GAA TAC ACC AAA A, 500 nM reverse primer TGT AGC ACG ATT GCA GCA TTG, 125 nM probe FAM-AYC ACA TTG GCA CCC GCA ATC CTG-BHQ1, Eurofins, Luxembourg and Luna Universal Probe One-Step RT-qPCR Kit; New England Biolabs, Ipswich, MA, USA) on the 7500 Fast Real-Time PCR instrument (Thermo Fisher Scientific, Waltham, MA, USA, protocol: 50 °C for 10′, 95 °C for 1′, and then 40 cycles at 95°for 10″, 60° for 30″). The nCoV-CDC-Control Plasmid (Eurofins) was used to produce the standard curve for the quantification. The N gene was selected based on previous environmental sampling recovering infectious particles from air samples [40 (link),41 (link),42 (link)].

After the total mRNA was isolated, it was reverse-transcribed using the High-Capacity cDNA kit according to the manufacturer’s protocol (Thermo Fisher Scientific, USA). The mRNA and cDNA concentration and quality were determined using a NanoDrop spectrophotometer (Kisker, Germany) and 1% agarose gels, respectively. Samples were stored at -80 ° C until use. cDNA was then amplified by real-time quantitative PCR (qPCR) using TaqMan probes: TTLL12: Hs00209450_m1, CDKN3: Hs00193192_m1, CDC16: Hs00187430_m1, PTPRA: Hs00160751_m1, MZT2B: Hs01117110_sH, UBE2T: Hs00928040_m1 and ACTB (4333762F; Thermo Fisher Scientific, USA) gene was selected as an internal control [27 (link)]. All qPCRs were performed in triplicate in 7500 Fast Real-Time PCR instrument (Thermo Fisher Scientific, USA).
The relative quantification of gene expression was calculated according to the method of Livak and Schmittgen [71 (link)]. We used a control sample of non-neoplastic gastric mucosa cells MNP01 (Normal gastric mucosa cell Line 01) pooled from 10 healthy patients [16 (link)], which was used as a calibrator for each tumoral sample. The mRNA and protein data are expressed as the median and interquartile range (IQR) of fold change in gene expression level in the gastric tumors normalized to the ACTB gene and relative to levels in the adjacent non-neoplastic control sample.

The presence of human internal control gene RNAse P and SARS-CoV-2 RNA (N1 N2 and N3) in the samples was evaluated through quantitative reverse transcription-polymerase chain reaction (qRT-PCR), as described in CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel [7 ]. RNA was isolated and purified from 400 uL of nasal fluid specimens using MagCore Viral Nucleic Acid Extraction Kit (RBC Bioscience, Taiwan) according to manufacturer’s instructions. RNA is reverse transcribed to cDNA and subsequently amplified in the Applied Biosystems 7500 Fast Real-Time PCR Instrument using TaqPath™ 1-Step RT-qPCR Master Mix (Thermo Fisher scientific, USA) and N1, N2 and N3 primer and probe set [7 ]. Fluorescence intensity is monitored at each PCR cycle by Applied Biosystems 7500 Fast Real-Time PCR System with SDS version 1.4 software. The cycle threshold (CT) values of qRT-PCR are inversely related to the copy number of human or viral RNA.
The cycle threshold values of RT-PCR were used as indicators of the copy number of SARS-CoV-2 RNA. A cycle threshold value less than 40 is interpreted as positive for SARS-CoV-2 RNA and gene RNase P. If no increase in fluorescent signal is observed after 40 cycles, the sample is assumed to be negative.

The 3 × 10⁶ HepG2 cells seeded in 10 cm dish were transfected with 18 μg plasmids containing 1.05-mer HBV genome under the control of CMV promoter. Three days post transfection, culture media were harvested and centrifugated at 5000 × g for 30 min to remove cell debris, followed by precipitation with 8% polyethylene glycol (PEG) 8000 at 4°C for overnight. The precipitates were resolved in DMEM and stored at −80°C. The HBVcc DNA was quantified by quantitative PCR (qPCR) using the 7500 Fast real-time PCR instrument (Thermo Fisher Scientific). The 5 × 10⁴ HepG2-NTCP cells were seeded in 24-well plate at day 1 and were infected by HBVcc at multiplicity of infection (MOI) of 5000 genome equivalents per cell (geq/cell) in DMEM with 3% FBS, 4% PEG8000, and 2% dimethyl sulfoxide (DMSO) at day 2. After 24 h infection, the cells were washed five times with PBS to remove residual HBV and HBsAg as much as possible, then cultured for 7 days in DMEM with 3% FBS and 2% DMSO. The culture media at 7 days post infection (dpi) were used for quantification of HBsAg and HBeAg by chemiluminescence immunoassay (CLIA) kits (Autobio diagnostics Co., Zhengzhou, China) and the cells were fixed for immunofluorescence assay.