One step rt pcr master mix reagents

TaqMan real-time qRT-PCR was used to quantify viral RNA levels and for confirmation of the microarray results for the mRNA levels of selected genes. Primers (Sigma) and probe (PE Applied Biosystems, Warrington, United Kingdom) (Table 1) were designed using Primer Express (PE Applied Biosystems). Briefly, the assays were performed using 2 μl of total RNA and the TaqMan FAST Universal PCR master mix and one-step RT-PCR Mastermix reagents (PE Applied Biosystems) in a 10-μl reaction. Amplification and detection of specific products were performed using the Applied Biosystems 7500 Fast real-time PCR system with the following cycle profile: one cycle at 48°C for 30 min and 95°C for 20 s, followed by 40 cycles at 95°C for 3 s and 60°C for 30 s. The data are expressed in terms of the cycle threshold (C_T) value, normalized for each sample using the C_T value of 28S rRNA product for the same sample, as described previously (19 (link)– (link)21 (link)). The final results are shown as 40-C_T using the normalized value, or as fold change from uninfected controls.

The cDNA samples were also screened for the expression of 84 pathway-focused cytokines and by means of RT² Profiler PCR Array Human Common Cytokines (PAHS-021A; Superarray, Frederick, MD, USA) on an ABI 7500 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA), according to the manufacturer’s protocols. Experiments were performed once between Ad-G-AT2R-EGFP-transduced cells and Ad-CMV-EGFP-induced cells to screen cytokines generally.
Real-time RT-PCR was used to validate the expression profiling. Genes were chosen based on their possible physiological roles and on the extent to which they were regulated by the overexpression of AT2R. Oligonucleotide primers and Taqman probes specific for Bcl-2, IL6 and IL8 were obtained from Applied Biosystems. Isolation of total RNA was done as described above. Purified RNA (25 ng) was used to do RT-PCR in an Applied Biosystems Prism 7000 Sequence Detection System, with the use of One-Step RT-PCR Master Mix Reagents. Expression of the housekeeping gene GAPDH was used to normalize mRNA expression. Quantitation and analysis of gene expression was determined using the comparative cycle threshold (C_T) method as described in Applied Biosystems User Bulletin #2. Primers used to detect the levels of other cytokines are shown in Table 1 and real-time PCR was performed using SYBR Premix Ex Taq (TAKARA) regents as described above.