Rotor gene q real time pcr equipment

The QuantiNova SYBR Green RT-PCR Kit was used for SYBR Green-based real-time amplification (Qiagen Inc., Venlo, the Netherlands). A 20 mL reaction using 10 mL of QuantiNova SYBR Green RT-PCR Master Mix, 1 mL of each forward and reverse primer (Table 1), 6 mL of nuclease-free water, and 2 mL of cDNA was set up. The thermal profile was 30 min at 50 °C, 15 min at 95 °C, 45 cycles of 15 s at 94 °C, 30 s at Tm, and 30 s at 72 °C, followed by a melting curve from 60 °C to 90 °C. All real-time PCRs were performed using a Rotor-Gene Q Real-Time PCR equipment (Qiagen). For gene expression investigations employing OCT 3/4 and SOX 2, triplicate reactions were conducted, and the mean expression value was determined for the following analyses. The (2-ddct) technique was used to compute the relative expression levels of the genes [23 (link)].

In order to quantify the total amount of bacteria in the samples, an equal concentration (0.1 ng) of total genomic DNA was subjected to real-time PCR amplification with a pair of hypervariable regions 16 S V3 specific primers:^{27 (link)}CCTACGGGAGGCAGCAG
ATTACCGCGGCTGCTGG
10 µM of each of the above primers were added to TB green RT-Master Mix (Cat# RR820L, Takara Bio, Japan) in 20 µl reaction, and samples were analysed in Rotor Gene Q real time PCR equipment (Qiagen, Germany). The following universal amplification condition was used: after an initial denaturation at 95 °C for 10 min, samples were amplified for 40 cycles at 94 °C for 25 s, 51 °C for 25 s, and 72 °C for 25 s.