[BioC] two channel data normalization limma

[Maite Iriondo [guest]]

Dear Bioconductor Community,

I am Maite Iriondo and I am currently finishing a MSc programme, doing my
thesis in microarray data analysis even though I lack of bioinformatic
background as I come from Food Science.
I am trying to understand the different options for normalizing two channel
microarray data, and I have very basic questions that I would appreciate if
they could be answered.

1- I have read in the literature that two channel data can be analized by
single channel and two channel normalization approaches (using log
intensities and log ratios respectively) (Yang and Thorne, 2003). However,
I do not understand how these approaches are applied in the limma package
(Bioconductor). For two channel normalization you use a within array and
between array normalization and for single channel analysis you use just
the options of the between array normalization function?

2- My data was partially analised  with one scanner and then changed to
another, which gives huge differences on the widths of the boxplots. I read
in Smyth and Speed (2003) that scale normalization between arrays is
recommended. Which function in limma does this calculation? is it the
normalizeBetweenArrays (method= scale)?

3- My data consists of a series of microarrays from P.putida growth
experiments at different temperatures, taking samples at different time
points for each temperature. Also, a second experiment was carried doing
microarrays using adapted and non adapted cells to low temperatures of P.
putida at different temperatures and again different time points for each
temperature. As a summary:

A) Microarrays at 30, 10 and 5ºC with 5 time point samples (eg. At 30ºC I
have data from 5 microarrays that correspond to 5 time points in which the
cells were taken)
B) Microarrays at 5 and 10ºC using adapted and non adapted cells with 5
time point samples

In the beginning I normalized all the data together using different options
of normalizeWithinArrays() and normalizeBetweenArrays(), but the resulting
MAplots and boxplots dont look too good. I was wondering if I should
separate experiments A and B. Also, I read that for time series experiments
you should do single channel analysis. But for comparing gene expression
between different temperatures I should use two channel analysis. This
means that I should analyse each temperature separately using a single
channel approach and then use a two channel approach for all the data to
compare gene expression between temperatures?

Thank you for your time and effort in advanced. I know that these questions
are very basic but I am trying to put my concepts in order.

Maite

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