As part of my PhD, I have to give practical courses to the students as well. As a totally new technique, we have done RNA extraction, followed by cDNA synthesis, using the iScript mix by Bio Rad. We tested everything ourselves first, and after cDNA synthesis, the concentration of cDNA was about the same as the starting concentration of RNA (all measurements were done with the Nanodrop). However, when we repeated the experiment in the practical course with the students, suddenly we obtained a lot more cDNA after synthesis. Suppose the concentration of RNA we measured before synthesis was 20 ng/µl, suddenly we obtained 500-1000 ng/µl cDNA.
Does anyone have an explanation about what could have caused this?
Thanks in advance!
Spectrofluorometry (Nanodrop) is unreliable for quantitating cDNA after RT.
Unincorporated dNTPs and input RNA also absorb at 260 nm.
I know that Nanodrop isn't the tool to use (I don't use it for anything I do, but use the Qubit instead). However, since we only have a limited budget for the practical courses, we are obliged to use the Nanodrop. But we also used the Nanodrop when testing out the protocol, and at that time, we did not see the difference between amount of RNA and cDNA.
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