[BioC] technical vs biological replication in real-time

[Juan Pedro Steibel]

Hello Niki,
You should not treat biological and technical replicates in the same 
way. The standard deviations provided by the termocycler software only 
pertain to the within sample (purely technical) variability. Moreover, 
to infer upon treatment effects in a broad inference context you should 
refer to the sample-to-sample variability (so-called biological 
variability). For this last purpose you have 4 reps (not 12).

You can use a hierarchical model to account for different levels of 
replication. This can be done using a linear mixed model including both 
the test and control gene and computing the (corrected) contrasts of 
interest.

Please follow the link to a short paper with a linear model (model [3]) 
that we have proposed for this type of analysis. We did not use R for 
the computations, but perhaps the lme4 package could be used.

http://www.wcgalp8.org.br/wcgalp8/articles/paper/23_528-1915.pdf

Best,
Juan Pedro





r.athanasiadou wrote:
> Hi everyone,
> My question is of statistical nature.
> I am trying to finalize the analysis of real-time PCR experiments.
> I have 4 technical replicates (pipeting errors) and three biological
> replicates (repeat treatment).
>
> I use gapdh as an external reference gene.
>
> 1) Can I average all replication regardless of technical/biological?
> 2) Similarily can I use the standard error from the combined standard
> deviations and what would the population size be in that case? (would it be
> 4 technical x 3 biological?)
>
>  If I can not do the above how does someone proceed with these two different
> types of replication?
>
>
> I can imagine that the technical replication reflects only on the threshold
> cycles and the biological replication at the actual fold
> difference[2^-(threshold cycle)] but how can I combine the two differently
> calculated standard errors?
>
>
>
> I apologize that the e-mail is not directly linking to some BioC package but
> there are no packages for real-time and I had nowhere else to turn!
>
> Thanks
> Niki
>
>
>
>
> Swann Building
> The King's Buildings
> University of Edinburgh
> Edinburgh, EH9 3JR
> Scotland
> UK
>
> tel:(0044)0131-6507072
> fax:(0044)0131-6505379
> R.Athanasiadou at sms.ed.ac.uk
>
>
>   

-- 
=============================
Juan Pedro Steibel
Postdoctoral researcher
Statistical Genetics
Department of Animal Science 
Michigan State University
1205-I Anthony Hall
East Lansing, MI
48823 USA 
Phone: 1-517-353-5102
E-mail: steibelj at msu.edu