Taqman universal pcr master mix reagents kit

Total RNA was isolated from 13 cell lines and 59 gastric cancer patients' tissues, respectively, by using TRIzol (Invitrogen, Carlsbad, CA, USA) as per the manufacturer's protocol. The TaqMan universal PCR master mix reagents kit (Applied Biosystems, Tokyo, Japan) was used. Amplification and detection were performed using a 7900HT Sequence Detection System (Applied Biosystems, Tokyo, Japan) with 40 cycles of denaturation at 95°C (15 seconds) and annealing/extension at 60°C (60 seconds). This was preceded by reverse transcription at 50°C for 30 minutes and denaturation at 95°C for 10 minutes. To quantitate mature miRNAs, TaqMan MicroRNA Assays kits (Applied Biosystems, Tokyo, Japan) were used for detection of miRNA-135a and a control miRNA (RNU6B).

After thawing, PBMCs were stimulated with either a BKPyV LTag peptide pool, a positive control CEF (cytomegalovirus, Epstein-Barr virus and influenza virus) peptide pool, or a negative control HIV peptide pool (JPT Peptide Technology, Berlin, Germany), as previously described [11 (link)]. Quantitative gene amplification was performed by using a Corbett Rotor-Gene 3000 Instrument (Corbett Life Science, Sydney, Australia) with a TaqMan Universal PCR master mix reagents kit and “on demand” sets of primers and probes for cytokine gene expression of IFN-γ, IL-10 and TGF-β1 (Applied Biosystems, Rotkreuz, Switzerland), as previously described [42 (link)]. The negative control was used to compute cytokine gene expression fold changes. Finally, the normalized data were analyzed by the 2^−ΔΔCt method using β-Actin as an endogenous reference gene [43 (link)].