What to use for Standard Curve - (May/11/2011 )
I have been purchasing high quality RNA from Ambion to construct my standard curves with. (I am running relative standard curve to test for gene expression.) The main problem is that I am using a ton of this very expensive RNA. Is there a better alternative? Can I use my reference sample for the standard curve?
You can use any RNA that is concentrated enough to fill range of your samples when serially diluted. But if you use efficiency corrected relative quantification formula instead of standard curve method, you only need to run the standard curve once to get the efficiency.
could you please suggest any papers that using the efficiency corrected relative quantification formula? Or could you please explain a bit about that?
I've read the qiagen protocol for real-time pcr kit (critical factors for successful real time pcr), it stated that if there are differences in PCR efficiency between the HK gene and GOI gene, the Livak and Pfaffl methods cannot be used, so we have to generate the standard curve. So if the pcr efficiency is not comparable, can I use that "efficiency corrected relative quantification formula"? Or is that formula is one of Livak or Pfaffl methods?