Hi Naomi, I agree, spike-in can't be relied on for normalization. I had in mind pooled titration controls as in Yang et al (2002), which can be printed on the arrays but which don't need to be spiked into the RNA samples. Best wishes Gordon Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., Ngai, J., and Speed, T. P. (2002). Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Research 30(4):e15. At 11:32 PM 8/09/2006, Naomi Altman wrote: >Spiking in normalization control probes can also be problematic, as >the RNA samples have to be equalized before spiking. >This can, I think, be done when the tissues from which the RNA is >extracted are very similar, and the extraction protocol is very >uniform. Otherwise, spiking variation just gets added to all the >other sources of variation. > >--Naomi > >At 08:07 AM 9/8/2006, Gordon Smyth wrote: >>Dear Dan, >> >>At 08:00 PM 8/09/2006, bioconductor-request at stat.math.ethz.ch wrote: >> >Date: Thu, 7 Sep 2006 13:44:35 +0100 >> >From: "Dan Swan" <bioinformatics.lists at="" gmail.com=""> >> >Subject: [BioC] BioC normalisations for small array 2 colour data? >> >To: bioconductor at stat.math.ethz.ch >> > >> >Hi, >> > >> >I have some data from a small specialised microarray - 200 genes, 1 >> >spiked control, 1 negative control. This is 2 colour data, with dye >> >swaps. I was wondering what an appropriate normalisation for this >> >scenario is within Bioconductor given that Lowess is unreliable for >> ><1000 genes? >> >>Are you quoting someone when you say that lowess is unreliable for >><1000 genes? Who? Print-tip loess normalization is routinely applied >>to arrays with around 200 genes in each print-tip group so 200 genes >>is, far from being unusual, pretty much typical for loess normalization. >> >>The real problem with a specialised microarray is the fact that the >>genes are not randomly chosen, indeed they are typically chosen >>because of their likelihood to be differentially expressed. Hence you >>may be in a situation where the majority of genes may be >>differentially expressed. If that is your case then, in my opinion, >>normalization control probes need to be designed into the array in >>the first place to produce reliable results. >> >>Best wishes >>Gordon >> >> >Any pointers would be gratefully recieved. >> > >> >thanks, >> > >> >Dan >> > >> >-- >> >Senior Research Associate, Bioinformatics Support Unit, >> >Institute for Cell and Molecular Biosciences, >> >Faculty of Medical Sciences, Framlington Place, >> >University of Newcastle upon Tyne, >> >Newcastle, NE2 4HH >> >Tel: +44 (0)191 222 7253 (Leech offices: Rooms M.2046/M.2046A) >> >Tel: +44 (0)191 246 4833 (Devonshire offices: Rooms G.25/G.26) >> >Website: http://bioinf.ncl.ac.uk/support/ >> >>_______________________________________________ >>Bioconductor mailing list >>Bioconductor at stat.math.ethz.ch >>https://stat.ethz.ch/mailman/listinfo/bioconductor >>Search the archives: >>http://news.gmane.org/gmane.science.biology.informatics.conductor > >Naomi S. Altman 814-865-3791 (voice) >Associate Professor >Dept. of Statistics 814-863-7114 (fax) >Penn State University 814-865-1348 (Statistics) >University Park, PA 16802-2111