Are these 2 scenarios the same?
Setting up a "No RT" control in a 2-step qPCR is done by:
1. including all components in the reaction mixture but omitting the reverse transcriptase enzyme when doing a cDNA synthesis in the 1st step.
2. using RNA in the place of cDNA when doing the second step of the qPCR.
They are the same.
I use the first scenario though, as it keeps the control sample in similar conditions to the other samples incase I am carrying over PCR inhibitors etc. But, essentially, at the end of the first scenario you are left with RNA to use in the subsequent PCR reaction.
One of the "no RT" objectives is to check whether there is DNA contamination in the mRNA preparation or not. If there is DNA contamination, then the result of qPCR will probably coming from DNA eventhough there is not any target mRNA (cDNA) template that you want to study. In such case, you will always get PCR positive result either with or without the present of target mRNA at the RT stage.
Therefore, the set up of your "no RT" control should contain:
1. Your RNA prep plus the ingredient of your RT step, but without RT enzyme as the control template (the first step)
2. All the ingredient of your PCR step (the second step)
If you have positive result after the second step for the "no RT" control, then most probably you have DNA contamination in your RNA preparation. I hope this can help...
I just wondered about the usefulness of this -RT control. As Ubanako suggests, one can simply use the isolated RNA to check for cDNA contamination. The only difference is, that you do not treat the samples in the same manner, when you want to compare them with +RT cDNA samples. But the gDNA content should be the same, right? So if the sole purpose is to check for gDNA contamination, taking the RNA should work similarly
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