Hello all, I would be very grateful for any suggestions or advise regarding the following. We are about to run some Illumina Mouse-6 BeadChips. We want to compare a mutant vs wild type tissue. The problem is that we only have the following: 2 male mutant 2 female mutant (and enough wild types of both genders) Since the Illumina beadchips have 6 lanes, what would be the best combination(s) to set up? Previously we have seen a rather strong "chip" effect, so ideally we would like to keep as much as possible on a single chip. We have also seen quite a strong gender effect. Would something like the following be the best option? 2 male mutant + 1 female mutant vs 2 male WT + 1 female WT Or would pooling the male/female samples help to avoid the gender effect, since we're only interested in the mutant vs WT effect? Say something like: pool (Male Mutant 1, Female Mutant 1) + pool (Male Mutant 1, Female Mutant 2) + pool (Male Mutant 2 + Female Mutant 1) vs equivalent pools of wild types The other option would be simply to have 1 technical replicate for the mutants and test each gender in a separate chip, but I don't believe technical replicates are the best option. Again, any suggestions are more than welcome... Thanks! Cei -- The Wellcome Trust Sanger Institute is operated by Genome Research Limited, a charity registered in England with number 1021457 and a company registered in England with number 2742969, whose registered office is 215 Euston Road, London, NW1 2BE.

Hello Cei, One cause of the chip effect that you have observed in the past might be the hybridization buffer. Illumina tech reps suggested that the buffer contents might not all be dissolved so we agitated the buffer longer and the chip effect went away. We have seen quite nice results since then. If you are still concerned, you can use two chips but run a technical replicate on each one. I would avoid pooling. Lynn Amon Cei Abreu-Goodger wrote: > Hello all, > > I would be very grateful for any suggestions or advise regarding the > following. > > We are about to run some Illumina Mouse-6 BeadChips. We want to compare > a mutant vs wild type tissue. The problem is that we only have the > following: > 2 male mutant > 2 female mutant > (and enough wild types of both genders) > > Since the Illumina beadchips have 6 lanes, what would be the best > combination(s) to set up? > Previously we have seen a rather strong "chip" effect, so ideally we > would like to keep as much as possible on a single chip. We have also > seen quite a strong gender effect. > > Would something like the following be the best option? > > 2 male mutant + 1 female mutant > vs > 2 male WT + 1 female WT > > Or would pooling the male/female samples help to avoid the gender > effect, since we're only interested in the mutant vs WT effect? Say > something like: > > pool (Male Mutant 1, Female Mutant 1) + pool (Male Mutant 1, Female > Mutant 2) + pool (Male Mutant 2 + Female Mutant 1) > vs > equivalent pools of wild types > > The other option would be simply to have 1 technical replicate for the > mutants and test each gender in a separate chip, but I don't believe > technical replicates are the best option. > > Again, any suggestions are more than welcome... Thanks! > > Cei > > > >