To achieve these ratios, genomic DNA templates equivalent to 3,000, 9,486, and 30,000 haploid cells were combined with cDNA templates equivalent to 3,000, 948, and 300 haploid cells, respectively.
The total volume of the PCR master mixture was 20 μl, to which cDNA template equivalent to 25 ng RNA starting material and 0.5 μM of each primer (Table 1) was added.
The reaction mixture consisted of 0.4 µM of each primer, 12.5 µl of 2X IQ SYBR Green Supermix, and 1 µl of diluted cDNA template (equivalent to 8 ng RNA).
One or two microliters cDNA template (equivalent to 50 and 100 ng RNA) were used for each PCR.
Detection threshold times (T t ) ranged from 10to2020 min using a 1-ng gDNA template (equivalent to 3.22 × 10 genome copies of S. aureus Mu50).
The reaction mixture (20 μl) contained 10 μl of SYBR Green Real-time PCR MixToyobox (Toyobo), 0.5 μM of forward and reverse primers, and cDNA template (equivalent to 70 ng of total RNA).
Each qPCR reaction consisted of 1.5 μl cDNA template (equivalent to 0.30 oocyte), 0.1-0.3 μM of each primer and 50% Power SYBR® GREEN PCR Master mix (Applied Biosystems) in a final 15-μl reaction volume.
PCR reaction was performed in a volume of 20 μl, containing 10 μl of SYBR premix ExTaq (TaKaRa Biotech, Dalian,China), 0.5 μM of forward and reverse primers, and 2 μl cDNA template (equivalent to 0.05 μg of total RNA).
Each reaction was run in triplicate and contained 2.5 μl of cDNA template (equivalent to 25 ng total RNA) along with 900 nM primers in a final volume of 25 μl.
For each miRNA or non-coding RNA qPCR reaction, 2 μl cDNA template equivalent to 3 ng of total RNA was mixed with 10 μl of 2× QuantiTect SYBR Green PCR Master Mix (Qiagen, Germany), 2 μl of 10x miScript Universal Primer (Qiagen, Germany), 2 μl of forward primers (5pmol) and 4 μl if RNase-free water.
For each PCR, 1μl cDNA template equivalent to 100pg total RNA was mixed with 10μl of 2x QuantiTect SYBR Green PCR Master Mix (Qiagen, Germany), 2μl of 10x miScript Universal Primer (Qiagen, Germany) and 5pmol of the forward primers in a final volume of 20μl.
