My explanation was as follows:
1 μl cDNA template is used in 20 μl reaction mixture subjected to qRT-PCR experiment, for which template concentration must be strictly equal in each sample under study; then we have to determine the number of PCR amplification cycles required for the fluorescent signal to cross the threshold (i.e. exceeds background level) as the actual "raw data" in qPCR are the ct values (cycle threshold). Hence we equalize the concentration of first strand cDNA to 25ng/μl in each sample.
Am I right?
Yes Laurence
and furthermore since all genes are not expressed the same way and amount in cells, you can't correlate quantity of cDNA with ct data.
Bioanalyzer (Agilent) is a good tool (among others) to quantify and qualify DNA and RNA since NA will proceed an electrophoresis and all sizes will be calculated for its concentration.
all the best
fred
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