exogenous control - (Sep/24/2008 )
Hi,
I am looking at doing real time PCR using a large number of primer sets across different developmental samples in shrimp. I have attempted to find a good housekeeper as a reference gene without luck and I am looking at using an exogenous RNA (eg. luciferase) as a reference instead. In most of the papers I have found that have used an exogenous RNA control, they have added the RNA before the RNA extraction based on tissue weight. It is too difficult to get accurate weights for my samples (as they are marine and it is difficult to account for/control water weight, and the samples have extremely varied morphology and size) and I was thinking it would be more accurate for me to add the exogenous RNA at the reverse transcription step. Has anyone ever done this? Is there a reason why I shouldn't add it at the reverse transcription step? Also, how much exogenous RNA should I be adding to my sample RNA.
I thank you in advance for your suggestions.
Hi,
of course you can do it, but then you have to make the assumption that your amount of RNA you reverse transcribe always represents the same amount of cells, which is often done but not quite correct.
Further you assume that the ratio between the different RNA fractions in your total RNA is always the same, which has also been shown to vary a bit.
Be aware of these uncertainties.
good luck!
Jan
of course you can do it, but then you have to make the assumption that your amount of RNA you reverse transcribe always represents the same amount of cells, which is often done but not quite correct.
Further you assume that the ratio between the different RNA fractions in your total RNA is always the same, which has also been shown to vary a bit.
Be aware of these uncertainties.
good luck!
Jan
Thanks Jan,
I hadn't thought of it quite like that - I guess because I have never controlled for this when I have reverse transcribed my total RNA (as I have made these two assumptions) when I have previously used standard curves for qPCR (although I was looking at transcript number per 1000ng of total RNA and wasn't trying to relate it back to a whole organ/individual etc so maybe it doesn't matter?). Is it becoming common practice to include an exogenous RNA extraction control to account for this when performing any type of qPCR now? I am guessing that it is something that is going to have to be accounted for across techniques?
Thanks again