Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

Using DNA as a reference in qRT-PCR - (Apr/13/2011 )


My plan is to make a qRT-PCR (using RNA) from soil sample to study the activation of one gene in microbes. I just want to know is the gene more expressed in different time points. Because there are a lot of microbes in soil, I can not use any reference gene in qRT-PCR, so I was planning to use an external reference, for example DNA. How does this work?

Thank you a lot for the tips!


You cannot use an external reference DNA because reference genes are used to normalize the qPCR data due to variances in cell number. For example if in one point you take a lot of microbes your qRT-PCR data will tell you there is a lot of your gene in soil. If in another time point you sample much fewer microbes, you will probably also get lower values for your gene. That does not necessarily mean your gene is not expressed in that time point but this is due to a difference in sampling. That's why reference genes are used to normalize sample to sample variations. I would advise you to search for a sequence of a common nucleic acid (e.g. rRNA) that is more or less constantly expressed in soil.