Artificial RNA as normalizer (HK gene) - (May/27/2013 )
I want to ask: has anyone of you ever used artificial RNA as normalizer (housekeeping gene) in real-time PCR? Because I once assayed my samples (with HK gene: GAPDH), the GAPDH Ct from pre-treatment samples and post-treatment samples are too way different. It isn't good, right? Since the HK gene should give you the same or just a bit different Ct if you run the samples with different treatment. So, someone suggested me to use the artificial RNA, but I don't know the advantages of that.
I really need some help on this problem, any advices I'll be very thankful.
For housekeeping genes you should be testing multiple genes to find the one(s) that are most stable with your treatments, not just assuming that GAPDH will work.
The use of spiked RNA defeats the purpose of using a housekeeper gene and should not be used in the place of housekeepers! - having said that, spiked RNA of a known concentration can be used to act as an inter-plate control if you want to ensure that different plates are running the same.