Dear list,

I have one question regarding microarray data comparisons using Affymatrix
yeast whole genome chips.
Here are my samples: strains-- WT vs mut, each of them has Total RNA (named
as T), translating mRNA (named as P), non-translating mRNA (named as NP) and
goes to each chip. Therefore, the final numbers of chips are 2 (strains) X 3
(3 different RNAs) X 2 (repeat) = 12.

I have done analyses of WT vs mut with three different RNAs separately.
However, my boss wanted me to compare the values of T-P in WT vs mut. *
Theoretically*, the outcome should be similar as results from NP
comparisons.

So, I tricked those normalized dataset in the following steps:
  1. import .cel files ( only T and P, no NP)
  2. Normalize by RMA
  3. convert logged values to unlogged values
  4. execute T-P for each strain
  5. "shift" values by addition of a fixed values. ( to avoid negative
values)
  6. log values obtained from step5
  7. Scale the dataset (now the values are named as NP2)
  8. execute limma to analyze dataset (WT_NP2 vs mut_NP2)
(ps. step 3-7 are trying to subtract P signals from Total-RNA signals)

Unfortunately, even I do get DEGs, but they are not existing in my previous
results.
I know this analysis may not make sense in statistic, but at least I want to
know if there is any explanation for these data.

Any comments or suggestions are really appreciated!!!
Hui-Yi

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