Hi, Please bear with me. The design model (common reference and dye-swap) is as follows: Array 1: Wild type cells at 0h (Cy5) vs Pooled RNA (Cy3) i.e. WT.0h.Cy5 vs PR.Cy3 Array 2: Pooled RNA (Cy5) vs Wild type cells at 0h (Cy3) i.e. PR.Cy5 vs WT.0h.Cy3 Array 3: Wild type cells transfected with empty plasmid at 8h (Cy5) vs Pooled RNA (Cy3) i.e. WT.EP.8h.Cy5 vs PR.Cy3 Array 4: Pooled RNA (Cy5) vs Wild type cells transfected with empty plasmid at 8h (Cy3) i.e. PR.Cy5 vs WT.EP.8h.Cy3 Array 5: Wild type cells transfected with plasmid expressing P53 at 8h (Cy5) vs Pooled RNA (Cy3) i.e. WT.EP+P53.8h.Cy5 vs PR.Cy3 Array 6: Pooled RNA (Cy5) vs Wild type cells transfected with plasmid expressing P53 at 8h (Cy3) i.e. PR.Cy5 vs WT.EP+P53.8h.Cy3 Array 7: Wild type cells transfected with empty plasmid at 16h (Cy5) vs Pooled RNA (Cy3) i.e. WT.EP.16h.Cy5 vs PR.Cy3 Array 8: Pooled RNA (Cy5) vs Wild type cells transfected with empty plasmid at 16h (Cy3) i.e. PR.Cy5 vs WT.EP.16h.Cy3 Array 9: Wild type cells transfected with plasmid expressing P53 at 16h (Cy5) vs Pooled RNA (Cy3) i.e. WT.EP+P53.16h.Cy5 vs PR.Cy3 Array 10: Pooled RNA (Cy5) vs Wild type cells transfected with plasmid expressing P53 at 16h (Cy3) i.e. PR.Cy5 vs WT.EP+P53.16h.Cy3 I need to identify two sets of differentially expressed genes induced solely by the over-expression of P53 at 8h, and at 16h. I took the following steps using the limma package: 1) Background correct each array 2) Normalize each array 3) Normalize array 1,3,5,7,9 (array 2,4,6,8,10) to have identical Cy3 (Cy5) distribution; Pooled RNA serves as baseline 4) To eliminate the effect of empty plasmid at 8h and 16, WT.P53.8h.Cy5 = WT.EP+P53.8h.Cy5 - WT.EP.8h.Cy5 + WT.0h.Cy5; similarly for Cy3 at 8h and 16h 5) Create the following RGlist: WT.P53.8h.Cy5 vs WT.0h.Cy3 WT.0h.Cy5 vs WT.P53.8h.Cy3 WT.P53.16h.Cy5 vs WT.0h.Cy3 WT.0h.Cy5 vs WT.P53.16h.Cy3 6) Create the design matrix given WT.0h.Cy5 and WT.0h.Cy3 as the reference 7) Create contrast matrix for WT.P53.8h - WT.0h and WT.P53.16h - WT.0h 8) Use lmfit, and ebayes. Couple of questions: A) Are my steps reasonably sound ? B) Should log2 transformed be used for step 4 ? In my perspective, I think not to use the log2 transformed values, as the difference in intensities is to be computed. Please comment C) Better alternative you may have to what I have ? Thanks Stanley