[BioC] OFF TOPIC How did we control for or remove background hybridization to the vector backbone ?

[Marcelo Laia]

Hi,

I apologizes for send this off topic. But, I think that it could be
interesting for Normalization procedures on arrays.

We make an array with non-redundant ESTs clones from shotgun libraries
containing fragments range from 100 to 1,000 bp. Plasmid DNA diluted
was spotted on positively charged slides. The clones were immobilized
in duplicate. Plus, that array has a lot of blank spots (without DNA),
for control.

After others lab procedures (DNA fixation, Cy3 and Cy5 hybridization,
etc), images were scanned and the raw values were background corrected
(subtraction) and normalized by the Variance Stabilization and
Normalization Method (VSN). Subsequently, limma package was used to
compute and identify the differentially expressed genes.

Now, we have a question about vector backbone:

Although we have doing a local background subtraction, how did we
control for or remove background hybridization to the vector backbone?

Have you any ideas?

All comments/criticisms are very welcome!

Thank you very much.

-- 
Marcelo Luiz de Laia
Universidade Estadual Paulista - www.unesp.br
Jaboticabal - SP - Brazil

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