[BioC] dye swap vs. control channel

Fri Oct 24 11:32:49 MEST 2003

Totally agree with Ramon. The use of a reference sample is a very good
practice when you want to compare more than 3 different samples: there
are not so many missing values, every pairwise comparison is straight
forward and the different properties of the dyes are not a problem any
longer. However, the problem is to find this reference. For a time
course experiment, we tried using a pool of RNA extracted at the
different time points. In theory, the idea was good,specially because we
would then obtain a signal in the reference channel for all the
interesting genes, those lightening up at any time point, but in
practice the number of missing values didn't improve with respect to
previous experiments where we used RNA extracted at t0 as reference. Has
anyone used a pool of RNA at different time points? For prokaryotes we
used gDNA as reference and it worked pretty well...(see Taalat et al.
for reference)

About the dye swap when we need to balance the different properties of
the dyes I always wonder why to use it is such a problem. Unfortunately,
for two-color microarrays we usually need one technical repeat to
validate the results. Since we have to do it, anyway, why not to swap
the dyes and have some extra information that might be also useful for
the normalization of the data?

Best regards,

Fatima

-----Original Message-----
From: bioconductor-bounces at stat.math.ethz.ch
[mailto:bioconductor-bounces at stat.math.ethz.ch] On Behalf Of Ramon
Diaz-Uriarte
Sent: Friday, October 24, 2003 11:05 AM
To: rossini at u.washington.edu; A.J. Rossini; Isaac Mehl; bioconductor
Subject: Re: [BioC] dye swap vs. control channel

> I think it boils down to whether you are interested in a direct
> head-to-head comparison (then dye swapping is essential), or in more
> general, less direct statements about groups of samples, with the
> possibility of generalizing past the experiment (then choice of
> reference sample is critical).

Along these linese, if the researcher is certain that she is interested
in 
comparing two (and just these two) groups, then I prefer a dye-swap
design. 
If the question is not as well specified, if more groups could be of
interest 
in the future ---recall connected design issues---, or if they want to
try to 
find predictors (say, discrminant analysis) or, (in spite of admonitions

against it) use clustering, I prefer to use a standard (a pool --below),

always in the same channel. I think with this approach we tend to view
the 
standard or reference as something we need to live with with cDNA
arrays, not 
something we really care about or wish we had to use ---I guess we wish
we 
had affy arrays.

>
> in, given the associated costs.  Right now, if you have a good
> reference or reference prototype for your situation, and can afford
> biological replications,  I'd probably do reference comparisons.

When a reference is used, and seen as a necessary experimental evil, I
often 
want it to be as representative as possible, but at the same time as
little 
variable as possible (i.e., for the reference use aliquots from the pool
so 
that variation from pool to pool sample is nearly inexistent). This is 
something we have done: form the pool using samples from, say, 30 normal

subjects; then, "validate" the pool by using another set of normal
samples 
(not used in the construction of the pool); if the pool is OK, then we
should 
see no genes differentially expressed. (The size of the set of normal
samples 
for "validation" is choosen so that it is at least a little bit larger
than 
the sample size of the experimental conditions).

Best,

Ramón

>
> So all of a sudden, we've moved beyond statistical design, to
> cost-function based decision theory and risk analysis.
>
> Sorry, no easy answers this morning.
>
> I'd be interested in hearing others opinions on the topic as well.
>
> best,
> -tony
>
> Isaac Mehl <imehl at ucsd.edu> writes:
> > this design brings up a question i always have.  is it "better" to
do
> > all experiments in one channel (Cy5) and compare every sample to a
> > standard (cy3)?  this way you can use less arrays or do more
biological
> > replicates.  IMHO getting repeated measurements of biological
variation
> > is more important than dye swap.
> >
> > very interested to hear what people think about this topic since it
is
> > integral to experimental design.
> >
> > -isaac
> >
> >
> >
> > DIF1,2 and 3 are different but similar drugs...............
> >
> >>> Slides 1-6 are treatment 1 (DIF1) Vs No treatment
> >>> Slide1 Cy5/Cy3 (DIF1/no treatment)
> >>> Slide2 Cy3/Cy5 (DIF1/no treatment)
> >>> Slide3 Cy5/Cy3 (DIF1/no treatment)
> >>> Slide4 Cy3/Cy5 (DIF1/no treatment)
> >>> Slide5 Cy3/Cy5 (DIF1/no treatment)
> >>> Slide6 Cy5/Cy3 (DIF1/no treatment)
> >>>
> >>> Slides 7-12 are treatment 2 (DIF2) Vs No treatment
> >>> Slide7 Cy5/Cy3 (DIF2/no treatment)
> >>> Slide8 Cy3/Cy5 (DIF2/no treatment)
> >>> Slide9 Cy5/Cy3 (DIF2/no treatment)
> >>> Slide10 Cy3/Cy5 (DIF2/no treatment)
> >>> Slide11 Cy3/Cy5 (DIF2/no treatment)
> >>> Slide12 Cy5/Cy3 (DIF2/no treatment)
> >>>
> >>> Slides 13-18 are treatment 3(DIF3) Vs No treatment
> >>> Slide13 Cy5/Cy3 (DIF3/no treatment)
> >>> Slide14 Cy3/Cy5 (DIF3/no treatment)
> >>> Slide15 Cy5/Cy3 (DIF3/no treatment)
> >>> Slide16 Cy3/Cy5 (DIF3/no treatment)
> >>> Slide17 Cy3/Cy5 (DIF3/no treatment)
> >>> Slide18 Cy5/Cy3 (DIF3/no treatment)
> >>>
> >>> I'd obviously like to compare across the different treatments
DIF1,2
> >>> and 3
> >
> > --
> > -isaac mehl
> > gene expression lab (gele)
> > salk institute
> > 10010 n. torrey pines rd.
> > la jolla ca. 92037
> > http://genex.salk.edu
> >
> > _______________________________________________
> > Bioconductor mailing list
> > Bioconductor at stat.math.ethz.ch
> > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor

-- 
Ramón Díaz-Uriarte
Bioinformatics Unit
Centro Nacional de Investigaciones Oncológicas (CNIO)
(Spanish National Cancer Center)
Melchor Fernández Almagro, 3
28029 Madrid (Spain)
Fax: +-34-91-224-6972
Phone: +-34-91-224-6900

http://bioinfo.cnio.es/~rdiaz
PGP KeyID: 0xE89B3462
(http://bioinfo.cnio.es/~rdiaz/0xE89B3462.asc)

_______________________________________________
Bioconductor mailing list
Bioconductor at stat.math.ethz.ch
https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor