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Schott, Johanna
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@schott-johanna-5433
Last seen 8.3 years ago

Dear list,
I am pre-processing Affymetrix Mouse Gene 1.0 ST Arrays and use the
oligo package. I do not want to quantile normalize them all together,
because my samples come from different polysome fractions or
compartments of the cell, and therefore show consistent and
biologically meaningful differences in signal distribution.
For seperate probe summary by median polish, however, the groups are
too small:
The smallest groups have only 3 microarrays, which leads to identical
values within many probe sets across the three samples.
My idea is to perform quantile normalization for the individual
groups, but probe summary for all microarrays (30) together, to have a
more reliable estimate of the probe effect
and to avoid that I lose the variability of my samples when a group
consists of only 3 microarrays.
Is this reasonable, or is anyone aware of artifacts that I would
introduce by performing median polish for probe summary on microarrays
that have not been quantile normalized together?
Here is some code to illustrate what I am doing:
# I load the required packages:
library("oligo")
library("pd.mogene.1.0.st.v1")
# the CEL files are opened twice, once in groups (here only group 1 as
an example), and once all together:
list_cel <- list.celfiles("group1")
group1 <- read.celfiles(list_cel)
list_cel <- list.celfiles("all_groups")
all_groups <- read.celfiles(list_cel)
# I perform background correction and quantile normalization for the
pm values of the individual groups (here only group1):
pms_group1 <- pm(group1)
bg_group1 <- backgroundCorrect(pms_group1)
norm_group1 <- normalize(bg_group1)
# I replace the pm values in the GeneFeatureSet all_groups by the
normalized values of group 1:
exprs(all_groups)[pmindex(all_groups), 1] <- norm_group1[,1]
exprs(all_groups)[pmindex(all_groups), 2] <- norm_group1[,2]
exprs(all_groups)[pmindex(all_groups), 3] <- norm_group1[,3]
# after having done this for ALL the groups, I perform only the probe
summary on all_groups:
pp_all <- rma(all_groups, background = F, normalize = F, target =
"core")
I guess that fRMA together with fRMAtools would be an alternative for
pre-processing my microarrays in small groups? As long as my way of
doing it in oligo is appropriate,
I would like to stick to it.
Thank you very much in advance for warning me if my idea is wrong!
Johanna Schott
Johanna Schott, PhD candidate
AG Stoecklin
Posttranscriptional control of gene expression
DKFZ-A200
INF280
69120 Heidelberg
Tel.: 06221/54-6855