Dear Gordon,

I have a question about creating the proper design matrix for my data.

I am using Affymetrix GeneChips and the study design is as follow:

_ 40  old male and female exercised during 6 months three times a week. During the 6 months some individuals received protein supplement and some received placebo.
     The design is unbalanced (the protein placebo numbers are not equal , also male and female are not the same number).

At both times( before exercise and after exercise) the Transcriptomics data for all individuals are available.

Subjects        time    gender          supplement    subject-code

1                  0        1                  ?                  A
1                  1        1                  1                  A
2                  0        0                  ?                  H
2                  1        0                  0                  H
3                  0        1                  ?                  J
3                  1        1                  0                  J
..
..
40                0        0                  ?                  K
40                1        0                  1                  K

In the above design time indicates before (0) and after(1) exercise, gender(0=male, 1=female), supplement(0=placebo,1=protein),subject_code is the indication of the same individual.

?  :I wonder that for supplement how I should indicate the protein , placebo at baseline (time=0) because at time=0 ,nobody got any supplement.

Primary questions to be addressed:


1)    Is there any effect of exercise( time)  in old people regarding there gene expression profile? (Which genes are affected based on exercise?)

2)    Is there any effect of protein supplementation ?

3)    Any interaction between protein supplement and exercise effect?

4)    Sex effect ?

So to do so, I need to run paired analysis in limma, but till now the design does not work properly.

May I ask you to give me some help how to design as I should consider several factors( sex, time(before and after exercise), supplement, within individual variation,...interactions)



Thank you so much ion advance

Regards

Pari



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