[BioC] Which method best fit nylon single channel technology ?

[TEXTORIS Julien]

Hi all,

I'm using a nylon technology, with a radioactive labelling of the
sample, and with cDNA probes spotted on the membrane.

We do first an hybridisation with a vector oligonucleotide, that match
all spot on the chip. This is to adjust for the different quantities
spotted on the chip.
Then we hybridize with the complex set issued by RTPCR from the sample.

I can't figure out really what it implies when i use the different
methods in Bioc. As it is a single channel procedure i'd think it's
closer to affy chips for example. But in the other hand, as we do the
first hybridization, and as it is a cDNA technology, i wrote a read
function to load my data in a RGlist object, and then use it in limma.
In this function, R channel correspond to the complex set, and G channel
to the vector set. (two successive hybridisations on the same membrane).

But in fact my ratio "complex / vector" has not really the same meaning
as "sample labelled with Cy5 / reference sample labelled with Cy3".

These questions raised as i was reading the monograph on bioconductor.
The methods used to identify differentially expressed genes are not the
same for an affybatch, and for a batch using limma for example. As i'm a
wet-lab biologist, and as my skills in statistics are low, i don't
really understand if it's the same or not, but i would say it's not ?
So did you use different approaches because in one case your are using
a signal from a single channel, oligo chip, and on the other one, a dual
channel with a ratio of expression of a cDNA chip ?

If it is true, the fact that my ratio "doesn't really represent the same
thing as a dual channel cDNA chip" would mean that i have to use a
specific method, and then which is the best.


I don't know if what i say is clear, but i would appreciate your
comments on this. Do you think in this case i should consider my ratio
as a normalised (a first step of) value, and then think as a single
channel ?

Thanks for any help,

Julien