Hello Group, I am analyzing individual patient arrays and need some help on the following questions. 1. If I have a dataset with four types of patients (disease1, disease2, disease3, healthy) and want to get the differential genes between disease and healthy. I can get the contrasts between healthy and disease comparisons using contrasts in LIMMA. But here each disease has 2-3 arrays from different patient blood samples. These are not true replicates, per se but thats all we have. My question is how do we treat them? Can I consider them as replicates? (I understand LIMMA will combine the replicate data.) here is the design........ 1. disease1 patient1 2. disease1 patient2 3. disease1 patient3 4. disease2 patient4 5. disease2 patient5 6. disease3 patient6 7. disease3 patient7 8. disease3 patient8 9. healthy patient1 10. healthy patient2 11. healthy patient4 2. In the above scenario, If I want differentially expressed genes in disease1 and disease2 combined w.r.t to healthy, is the following is correct way in LIMMA? makeContrats(dis1&dis2 <- "(disease1 + disease2) /2 - healthy", levels = design); If I want differentially expressed genes in all three diseases combined w.r.t to healthy makeContrats(dis <- "(disease1 + disease2 + disease3) /3 - healthy", levels = design); please let me know is it the right approach. Greatly greatly appreciate your help, Sincerely Prasad