Hello, I have a question regarding the contrasts.matrix. This is design of my experiment: SlideNumber FileName Cy3 Cy5 23 Mo_23.gpr pool con1 24 Mo_24.gpr pool con2 25 Mo_25.gpr pool test 26 Mo_26.gpr pool con1 27 Mo_27.gpr pool con2 28 Mo_28.gpr pool test 15 Mo_15.gpr pool con2 16 Mo_16.gpr pool con2 17 Mo_17.gpr pool con1 18 Mo_18.gpr pool con1 19 Mo_19.gpr pool con2 20 Mo_20.gpr pool con2 21 Mo_21.gpr pool test 22 Mo_22.gpr pool test 29 Mo_29.gpr pool con1 30 Mo_30.gpr pool con1 31 Mo_31.gpr pool con2 32 Mo_32.gpr pool con2 33 Mo_33.gpr pool con1 34 Mo_34.gpr pool con1 35 Mo_35.gpr pool con2 36 Mo_36.gpr pool con2 37 Mo_37.gpr pool test 38 Mo_38.gpr pool test 02 Mo_02.gpr pool test 03 Mo_03.gpr pool test 05 Mo_05.gpr pool con1 06 Mo_06.gpr pool con1 39 Mo_39.gpr pool test 40 Mo_40.gpr pool test It is common reference design, with reference in Cy3 channel, called "pool". I would like to compare the "test" vs. both controls "con1" and "con2". What would be the syntax of makeContrast? design <- modelMatrix(targets, ref="pool") contrast.matrix <- makeContrasts(Test.vs.Controls=(test-(con1+con2)/2),levels=design) contrast.matrix Test.vs.Controls con1 -0.5 con2 -0.5 test 1.0 Is this correct? Thanks for your help -- Robert Ivanek