Isolation of total RNA from different tissues - efficiency? - (Jul/30/2007 )
How can one calculate the efficiency of isolated total RNA from different tissues treated at the same time, when it comes to doing quantitative real-time RT-PCR analysis afterwards? If one compare the real-time expression of a certain gene in different tissues, using housekeeping genes and an external standard reference gene.. How can one be sure that the possible difference in the expression pattern is not due to difference in the isolation efficiency?
well the extraction efficiency of the houskeeping gene is the same as your gene of interest. so it's not, i think, an issue.
Moreover, you should repeat your exp 3times, which will make sure of your results.
If you start the reverse transcription with the same amount of total RNA then you won't need to think about the efficiency of RNA isolation. In addition to that - if possible - make a master mix for reverse transcription and transform all your RNA samples at once using that mix. Then the cDNA efficiencies will also be much more similar.
In fact, I have observed that RNA stability is a more important issue than RNA extraction efficiency. If you RNA samples are not degraded (or at least showing similar degradation patterns) then the real-time data will probably be more consistent.
Repeating the experiment with at least 3 biological replicates (i.e. different RNA extractions from different tissues) and having at least a triplicate for each PCR reaction will help you a lot to reduce both the variance and the pipetting errors.