Power sybr green qpcr supermix udg

Total RNA from NPC cell lines or tissue specimens was extracted using the TRIzol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 1 μg of the total RNA using a reverse transcriptase kit (Invitrogen). The mRNA level was determined by quantitative Real-time RT-PCR (qRT-PCR) using the Power SYBR Green qPCR SuperMix-UDG (Invitrogen, Grand Island, NY, USA) and was analyzed on an ABIPrism-7500 Sequence Detector System (ABI, applied biosystems, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin was used as an internal control. The primers are listed in Table 2. Relative expression of the indicated gene was normalized to GAPDH or β-actin expression, which yielded a 2^-△△Ct value. All reactions were run in triplicate.

Total cellular RNA was extracted from cultured cells using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was synthesized from 2 μg of the total RNA using a reverse transcriptase kit (Invitrogen). The mRNA level was evaluated by qRT-PCR using the Power SYBR Green qPCR SuperMix-UDG (Invitrogen) and was analysed on Roche Lightcycler 480. All gene expressions were normalized to the housekeeping gene GAPDH, used as an internal standard. The following primers were used: EBV-BamHI-W forward, 5′-CCCAACACTCCACCACACC-3′ and reverse, 5′-TCTTAGGAGCTGTCCGAGGG-3′; GAPDH forward, 5′-CCCCACACACATGCACTTACC-3′ and reverse, 5′-CCTAGTCCCAGGGCTTTGATT-3′. EBV DNA was extracted from EBV-infected cells using Omega tissue DNA Mini Kit (Omega) as recommended by the manufacturer. The copy number of EBV bound to the cell surface or internalized into cells was measured using TaqMan real-time PCR for detection of the BamHI-W fragment region of the EBV genome. Real-time PCR for the GAPDH DNA was used for cell-counting estimation. A calibration curve was performed with each analysis, using DNA extracted from the EBV-positive cell line Namalwa (ATCC CRL-1432), which contains two integrated viral genomes per cell, as a standard. The EBV copy number was expressed as a ratio of the copy number of the EBV genome to the copy number of the GAPDH DNA.

Total cellular RNA was extracted from cultured cells using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was synthesized from 2 μg of the total RNA using a reverse transcriptase kit (Invitrogen). The mRNA level was evaluated by qRT–PCR using the Power SYBR Green qPCR SuperMix-UDG (Invitrogen) and was analysed on Roche Lightcycler 480. All the gene expressions were normalized to the housekeeping gene GAPDH, used as an internal standard. Primers are listed in Supplementary Table 3. EBV DNA was extracted from EBV-infected cells using Omega tissue DNA Mini Kit (Omega) as recommended by the manufacturer. The copy number of EBV bound to the cell surface or internalized into HNE1 cells was measured using TaqMan real time PCR for detection of the BamHI-W fragment region of the EBV genome. Real-time PCR for the GAPDH DNA was used for cell counting estimation. A calibration curve was performed with each analysis, using DNA extracted from the EBV-positive cell line Namalwa, which contains two integrated viral genomes per cell, as a standard. The EBV copy number was expressed as a ratio of the copy number of the EBV genome to the copy number of the GAPDH DNA.

Total RNA was extracted from NPC and nasopharyngeal epithelial cells lines using the Trizol reagent (Invitrogen, USA) according to the manufacturer’s instruction. Reverse transcription of total RNA (2 μg) was performed using SuperScript II reverse transcriptase (GIBCO BRL, Grand Island, NY, USA). The quantification of target and reference glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes was performed using the Power SYBR Green qPCR SuperMix-UDG (Invitrogen, USA) on an iCycler (Bio-Rad, Hercules, CA, USA). CT is defined as the cycle at which the fluorescence is determined to be statistically significant above background. The relative mRNA expression was normalized to the expression of GAPDH, which yielded a 2^-ΔCT value (ΔCT = CT_{(target gene)}–CT_(GAPDH)). All reactions were performed in triplicate in three independent experiments. The primers used for real-time RT-PCR were as follows: IGF-1: forward 5′- GCT CTT CAG TTC GTG TGT GGA −3′ and reverse 5′- GCC TCC TTA GAT CAC AGC TCC −3′; IGFBP-1: forward 5′-CTA TGA TGG CTC GAA GGC TC-3 ′ and reverse 5′-CCC ATT CCA AGG GTA GAC G-3′; GAPDH: forward 5′-GCA CCG TCA AGG CTG AG AAC-3′ and reverse 5 ′-TGG TGA AGA CGC CAG TGG A-3′.