7300 sds software

qPCR was conducted using an ABI7300 RT-qPCR System with the following protocol: 95°C dissociation step for 15 sec, 60°C amplification step for 1 min, repeated for 40 cycles. The Applied Biosystems 7300 SDS software was used to calculate Cq values. Each sample was done in triplicate and averaged. DNA and RNA extractions were done as previously described.^{87 (link)} Mouse and viral DNA were isolated from mouse tissue using the Qiagen DNeasy Blood and Tissue Kit (Qiagen). iTAQ universal Syber Green supermix (Invitrogen) and 300uM of MCMV Gb or GAPDH primers were mixed with isolated DNA for qPCR analysis. Absolute viral copy numbers were extrapolated using a standard curve of known quantities of purified MCMV BAC. RNA was extracted from cells using Trizol (Invitrogen). Genomic DNA was removed with 0.002 U of DNase I (Thermo). cDNA was synthesized using 1 ug of RNA reverse transcribed for 50 min at 42°C using oligo(dT) primer (IDT) and SuperScript II RT (Invitrogen). For qPCR analysis, 2 ul of prepared cDNA was mixed with iTAQ universal Syber Green supermix (Invitrogen) and 300uM of ERAAP primers. ERAAP mRNA levels were compared by ΔΔCT using average Cq values normalized to GAPDH.

Peripheral venous blood (3 mL) was collected from NPC survivors into EDTA-containing tubes and centrifuged at 3,000 × g for 5 min. Total plasma DNA was extracted using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Fluorescence polymerase chain reaction (PCR) was carried out using an EBV PCR quantitative diagnostic kit (Da-An Genetic Diagnostic Center, Guangzhou, China) targeting the BamHI-W region of the EBV genome. Data were analyzed using the Applied Biosystems 7300 SDS software (Beijing, China).

Peripheral venous blood (3 mL) was collected before treatment from each patient into EDTA-containing tubes and centrifuged at 3000 rpm for 5 min. Total plasma DNA was extracted using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Fluorescence polymerase chain reaction (PCR) was carried out using EBV PCR quantitative diagnostic kit (Da-An Genetic Diagnostic Center, Guangzhou, China). EBV-DNA levels were measured using real-time quantitative toward the BamHI-W region of the EBV genome. The experimental data were analyzed using Applied Biosystems 7300 SDS software for statistics and an EBV-DNA level of <100 copies/mL was defined as negative.

Before the start of treatment, peripheral venous blood (3 mL) was collected from each patient into EDTA-containing tubes and centrifuged at 3,000 g for 5 min. Total plasma DNA was extracted using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Fluorescence polymerase chain reaction (PCR) was carried out using an EBV PCR quantitative diagnostic kit (Da-An Genetic Diagnostic Center, Guangzhou, China) targeting the BamHI-W region of the EBV genome. Data were analyzed using Applied Biosystems 7300 SDS software (Beijing, China).