Do I need to pool the base line samples together - (Mar/30/2011 )
I am testing gene expression levels for inflammatory factors with real time PCR. I got RNA samples ofr a week, and test them for the purpose of baseline. The results showed the gene expression levels varied very much. I think I should pooled those RNA samples together as one control. Any opinions or suggestions? Thanks,
By a "baseline" you probably mean calibrator for your samples. Usualy when needed to normalise to a calibrator group of "normals" (like deseased patients expression vs healthy controls) RNA from healthy controls is pooled and the pool used as a calibrator in qPCR equations.
But you need to keep in mind, that if your controls varies a lot, you have to calculate the range for healthy controls and compare it to the samples you have.
Like if the controls varies from 1,5 fold to 0,7 fold from your pooled calibrator, and your sample has expression 1,4 or 0,75 fold, it's still within the "normal" group and not significant. Only differences bigger than the normal range can be called upregulation or downregulation from normal state.
And, if your controls varies a lot, you have to have a large enough group of controls to pool and analyse (like say 10).
Trof on Fri Apr 1 13:19:32 2011 said:
By a "baseline" you probably mean calibrator for your samples. Usualy when needed to normalise to a calibrator group of "normals" (like deseased patients expression vs healthy controls) RNA from healthy controls is pooled and the pool used as a calibrator in qPCR equations.
But you need to keep in mind, that if your controls varies a lot, you have to calculate the range for healthy controls and compare it to the samples you have.
Like if the controls varies from 1,5 fold to 0,7 fold from your pooled calibrator, and your sample has expression 1,4 or 0,75 fold, it's still within the "normal" group and not significant. Only differences bigger than the normal range can be called upregulation or downregulation from normal state.
And, if your controls varies a lot, you have to have a large enough group of controls to pool and analyse (like say 10).
Thanks for your answer, and it is really helpful.
I am doing a time series study. I samples before treatment and series time pints post treatment, and want to check gene expresssion changes over time 0. I also sampled continuously for 8 time points before treatment. I found the gene expression level varied a little much at those 8 time points. I am still wondering if I should pool those samples for 8 time points as a reference, or treat them as calibrator?