> My question is, if I need to compare between 5 time points, should I do 
> comparison pairwise?

No, do them all at once with Cuffdiff: 

(a) set 'Perform Replicate Analysis' to 'Yes';
(b) create 5 replicate conditions, one for each time point;
(c) add your replicates for each time point. 

There's a Cuffdiff flag to do time series analysis, but it isn't implemented 
yet in Galaxy, so you'll get pairwise comparisons for all conditions. You can 
use the filtering tool to reduce Cuffdiff outputs to only the timepoint 

> I will use cuffmerge to merge 0hour-1, 0hour-2, 0hour-3, 
> 1hour-1,1hour-2.1hour-3 to generate one cuffmerge file.


> Then I will run cuffdiff using the merged file, include two groups, group 1 
> is 0 hour (add 0hour 1-3 in group 1) and group 2 is 1hour (add 1hour1-3 in 
> group 2).

Use the process I described above to do all pairwise comparisons in one run.

Good luck,

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