Mastercycler ep gradient s

The sequences of the primers used for qRT-PCR are listed in Table 5. The mRNA prepared in the above cellular infection was used for cDNA synthesis with RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). qRT-PCR was carried out with Eppendorf Mastercycler epgradient S (Eppendorf, Hamburg, Germany) using TB Green^® Premix Ex Taq™ II (Takara, Dalian, China). The PCR reaction was performed in a 20 μL volume containing 10 μL TB Green Premix Ex Taq II, 0.4 μM specific forward primer and reverse primer, and 2.0 μL diluted cDNA (50 ng/μL). The PCR conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The specificity of qRT-PCR products was examined by melting curve analysis. The expression of each gene was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calculated using the comparative threshold cycle method (2^−ΔΔCT). The experiment was performed in triplicate.

Areas of mitochondrial DNA (mtDNA) that previously have been shown to contain many somatic mutations (12 (link)) were amplified with mitochondrial specific primers, designed to avoid amplification of homology regions in the nuclear DNA (NUMTs). Five sets of mitochondrial specific primer pairs were used, resulting in amplification product between 714 and 928 base pair in length (see Table 1). The PCR reaction mixture contained 0.1 μl of extracted DNA, 0.8 mM dNTPs (0.2 mM of each dNTP) (VWR, Oslo, Norway), 1X Thermopol Buffer, 2 mM MgSO4, 0.075 unit Taq/μl, 0.15 μM of each forward, reverse and fluorescently labeled primer (Integrated DNA Technologies, Leuven, Belgium) in a total reaction volume of 10 μl. The temperature cycling was performed in an Eppendorf Mastercycler ep gradient S (Eppendorf, Hamburg, Germany) with an initial denaturation 94°C for 240 s followed by cycling 38 time under the following conditions, denaturation at 94°C for 15 s, annealing for 40 s with temperature given in Table 1 and elongation at 72°C for 150 s.

Total RNA was extracted from HUVECs by using the Qiagen RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Dnase I was used to eliminate the potential DNA pollutions. and RT and qPCR kits were used to evaluate the expression of target RNAs. RT (20 μl) reactions were performed using the PrimeScript^®RT reagent kit (Takara, Dalian, China) and incubated for 30 min at 37°C followed by 5 s at 85°C. The quality of RT- samples were controlled by testing the density and purity. The 260/280 values should be between 1.8 and 2.0, and the density should be more than 200 μg/ml. For qPCR, 2 μl of diluted RT product was mixed with 23 μl reaction buffer provided by Takara (Takara Inc., Dalian, China) to a final volume of 25 μl. All reactions were carried out using an Eppendorf Mastercycler EP Gradient S (Eppendorf, Germany) under the following conditions: 95°C for 30 s followed by 45 cycles of 95°C for 5 s and 60°C for 30 s. The transcript expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for the normalization of detected RNAs using the comparative 2^−ΔΔCq method (Wagner, 2013 (link)). The primer sequences for qPCR are presented in Table 1.

Total RNAs were extracted from tumorous and adjacent normal tissues using Trizol (Invitrogen) following the manufacturer's protocol. RT and qPCR kits were used to evaluate expression of LncRNA from tissue samples. The 20 μl of RT reactions were performed using a PrimeScript® RT reagent Kit (Takara) and incubated for 30 min at 37°C, 5 s at 85°C and then maintained at 4°C. For RT-PCR, 1 μl of diluted RT products were mixed with 10 μl of 2 × SYBR® PremixEx Taq™ (Takara), 0.6 μl forward and reverse primers (10 μM) and 8.4 μ of Nuclease-free water in a final volume of 20 μl according to manufacturer instructions. All reactions were run on the Eppendorf Mastercycler EP Gradient S (Eppendorf) using the following conditions: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. RT-PCR was done in triplicate, including no-template controls. Amplification of the appropriate product was confirmed by melting curve analysis following amplification. Relative expressions of LncRNAs were calculated using the comparative cycle threshold (C_T; 2^−ΔΔCT) method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous control to normalize the data.