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The cycle threshold (Ct) value of each reaction on the chip was obtained with the Fluidigm RT-PCR analysis software.
The qRT PCR was performed according to the manufacturer's protocol, and the total volume of each reaction was 15 μl.
The volume of each reaction was 20 μl, and appropriate dye and protein controls were included.
The "relevance" or importance of each reaction was also taken into account.
Afterward, 20 μl of each reaction mixture was separated on a 1.2% agarose gel.
Meanwhile, the values of each reaction order, m and n, are slightly decreased.
The volume of each reaction was 100 µl.
Then 10% of each reaction was loaded onto 15% PAGE.
Three technical replicates of each reaction were performed.
A fraction of each reaction mixture was saved as a control (total fraction).
The specificity of each reaction was evaluated based on the melting temperatures of the PCR products.
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