Hello,

I am currently designing a focused human long-oligo (70 mer) array of 150 genes, and I am wondering which control spots should be added to the array to assist normalization, how many spots will be enough, and then how to do the normalization.

Here is some information I already gathered:

In a paper by van de peppel et al from 2003 (http://www.nature.com/cgi-taf/DynaPage.taf?file=/embor/journal/v4/n4/full/embor798.html) the authors used 9 bacterial control RNAs of different concentrations, and printed at least 2 replicates on each array subgrid. Do you think 9 concentrations are enough?

In the Limma UserGuide p.15 it is recommended, for focused arrays "to include on the arrays a series of non-differentially expressed control spots, such as a titration series of whole-library-pool spots, and to use the up-weighting method". I don't understand what is the meaning of "a titration series of whole-library-pool spots", and will appreciate any further details or references on its preparation so I can deliver it to the experimentalists. Also, will it be OK, in Limma, to give positive weight to the control spots only, and weights of 0 to the 150 genes of study?

I will appreciate any further ideas on which controls to include, how many would be enough, and references, if available. Frankly, I am rather a beginner with spotted arrays and with R, but so far used Limma successfully for several spotted array analyses. Therefore, your advice on normalization of these arrays will also be highly appreciated.

With best regards,
                             Vered
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Vered Caspi, Ph.D.
Bioinformatics Support Unit, Head
National Institute for Biotechnology in the Negev
Building 39, room 214
Ben-Gurion University of the Negev
Beer-Sheva 84105, Israel 

veredcc bgumail.bgu.ac.il
Tel: 08-6479034 054-7915969
Fax: 08-6472983

http://bioinfo.bgu.ac.il
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