Hello Heidi,

thanks for you reply, problem solved! no error anymore. I do not have
replicates of each miRNA in the card, so after setting ndups=2 I am getting
no error and a result that makes sense. I had a typo in my previous message
I have to treatments but the replicates is 2x3 and not 2x2, but that is ok
for the test right? It would be better to have a balanced design, but that
is what I have available.

Thanks again,

with kind regards,
Andreia

On Thu, May 20, 2010 at 10:38 PM, Heidi Dvinge <heidi@ebi.ac.uk> wrote:

> Hello Andreia,
>
> > Dear all,
> >
> > I am using HTqPCR to analyse different expression of miRNAs, where I am
> > comparing two treatments, 1 with 2 biological replicates and the second
> > with
> > 2 biological replicates
> >
> Okay, so you have two samples with two replicates each.
>
> > I have followed the vignete, read the files, made raw.cat and filtered
> out
> > IQR<1.5 and category= undetermined and type=endogenous
> >  and everything went well until the fit test:
> >
> > qDE.limma<-limmaCtData(iqr.filt, design=design, contrasts=contrasts,
> > ndups=2, spacing=1)
> > Error in dim(M) <- c(spacing, ndups, ngroups, nslides) :
> >   dims [product 396] do not match the length of object [402]
> >
> What's the dimensions of your data, i.e. number of genes on each card?
> show(iqr.filt) What does traceback() say?
>
> Using ndups=2 and spacing=1 means that there's a replicate of each gene,
> and they're located right next to each other. Is this the case you your
> array?
>
> > can someone tell me what this means? it still performed the test but I
> > would
> > like to clear out this.
> >
> > The other weird thing is the result of the test:
> >
> >    genes feature.pos     t.test      p.value  adj.p.value        ddCt
> > meanTarget meanCalibrator categoryTarget
> > 39   miR-122a       H7;H8  6.7414852 6.141932e-06 0.0002456773
>  14.8264063
> > 34.833906      20.007500   Undetermined
> > 1     miR-18a       A1;A2  5.5408137 5.366491e-05 0.0010732983
>  13.5365104
> > 30.431094      16.894583   Undetermined
> > 10    miR-368     B11;B12 -4.7841570 2.320152e-04 0.0030935366
> -23.1045833
> > 7.119063      30.223646   Undetermined
> >
> > the feature.pos is different in both treatments but it is not, I have
> > checked and re-checked the files and miR-18a is in pos. A1 in all cards.
> >
> This is because of ndups=2, spacing=1 that you've specified when you did
> the test. It then says that miR-18a is in position A1, but since it should
> be replicated and the replicates are next to each other, it must also be
> in A2. Ditto for H7;H8 and B11:B12.
>
> If your spots on the plate are not replicated, you need to set ndups=1. If
> they're replicated but not next to each other, you either need to adjust
> the spacing parameter accordingly, or sort your file so they are.
> ?limmaCtdata gives an example of how to reorder data to get the genes in
> consecutive rows.
>
> When you filter your data, have you then got the same number of replicates
> of each spot afterward? What does table(featureNames(iqr.filt)) say?
>
> HTH
> \Heidi
>
> > Thanks for your help.
> > With kind regards
> >
> > Andreia
> > --
> > --------------------------------------------
> > Andreia J. Amaral
> > Unidade de Imunologia Clínica
> > Instituto de Medicina Molecular
> > Universidade de Lisboa
> > email: andreiaamaral@fm.ul.pt
> >          andreia.fonseca@gmail.com
> >
>
>
>


-- 
--------------------------------------------
Andreia J. Amaral
Unidade de Imunologia Clínica
Instituto de Medicina Molecular
Universidade de Lisboa
email: andreiaamaral@fm.ul.pt
         andreia.fonseca@gmail.com

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