Dear Gordon & List Members, I've made few hybridizations which I'd like to incorporate to the design described in thread "Strange results in contrasts with dye-swap" (It was reference design, two temperature treatments with seven samples (time points) in both, all compared directly to "time 0" sample). Treatments I'd like to merge with that experiment are direct comparison of: c2 vs k2 c6 vs k6 c8 vs k8 As main experiment this comparisons were repeated four times with dye- swap (two labellings in both directions), using the same biological material as in corresponding replications in the main experiment. Here is my new target frame: FileName Cy3 Cy5 powt _024_1_vs_7ex.gpr k0 k2 1 _228_k0_vs_16ex.gpr k0 c12 1 _229_k0_vs_8ex.gpr k0 c4 1 _230_k0_vs_15ex.gpr k0 k10 1 _235_k0_vs_19ex.gpr k0 k14 1 _237_k0_vs_14ex.gpr k0 c10 1 _238_k0_vs_10ex.gpr k0 c6 1 _239_k0_vs_12ex.gpr k0 c8 1 _240_k0_vs_13ex.gpr k0 k8 1 _248_k0_vs_17ex.gpr k0 k12 1 _249_k0_vs_9ex.gpr k0 k4 1 _253_k0_vs_11ex.gpr k0 k6 1 _254_k0_vs_6ex.gpr k0 c2 1 _256_k0_vs_18ex.gpr k0 c14 1 _189_K8_vs_K0ex.gpr k8 k0 2 _190_K4_vs_K0ex.gpr k4 k0 2 _191_K6_vs_K0ex.gpr k6 k0 2 _192_C8_vs_K0ex.gpr c8 k0 2 _193_C10_vs_K0ex.gpr c10 k0 2 _194_C2_vs_K0ex.gpr c2 k0 2 _198_C14_vs_K0ex.gpr c14 k0 2 _200_K12_vs_K0ex.gpr k12 k0 2 _202_K10_vs_K0ex.gpr k10 k0 2 _206_C6_vs_K0ex.gpr c6 k0 2 _207_C12_vs_K0ex.gpr c12 k0 2 _208_K2_vs_K0ex.gpr k2 k0 2 _209_K14_vs_K0ex.gpr k14 k0 2 _210_C4_vs_K0ex.gpr c4 k0 2 _201_k0_vs_C2ex.gpr k0 c2 3 _203_k0_vs_K6ex.gpr k0 k6 3 _205_k0_vs_C12ex.gpr k0 c12 3 _217_k0_vs_K14ex.gpr k0 k14 3 _219_k0_vs_C4ex.gpr k0 c4 3 _231_k0_vs_C10ex.gpr k0 c10 3 _232_k0_vs_K8ex.gpr k0 k8 3 _233_k0_vs_C8ex.gpr k0 c8 3 _234_k0_vs_C14ex.gpr k0 c14 3 _235_k0_vs_C6ex.gpr k0 c6 3 _236_k0_vs_K10ex.gpr k0 k10 3 _237_k0_vs_K2ex.gpr k0 k2 3 _238_k0_vs_K4ex.gpr k0 k4 3 _239_k0_vs_K12ex.gpr k0 k12 3 _004_8_vs_1ex.gpr c8 k0 4 _005_10_vs_1ex.gpr c10 k0 4 _006_2_vs_1ex.gpr c2 k0 4 _007_15_vs_1ex.gpr k14 k0 4 _008_4_vs_1ex.gpr c4 k0 4 _010_3_vs_1ex.gpr k2 k0 4 _011_9_vs_1ex.gpr k8 k0 4 _012_14_vs_1ex.gpr c14 k0 4 _014_12_vs_1ex.gpr c12 k0 4 _026_13_vs_1ex.gpr k12 k0 4 _036_11_vs_1ex.gpr k10 k0 4 _037_6_vs_1ex.gpr c6 k0 4 _043_7_vs_1ex.gpr k6 k0 4 _044_5_vs_1ex.gpr k4 k0 4 127 k2 c2 1 049 c2 k2 2 133 c2 k2 4 128 k2 c2 3 047 k6 c6 1 050 c6 k6 2 046 c6 k6 4 048 k6 c6 3 130 k8 c8 1 134 c8 k8 2 132 c8 k8 4 045 k8 c8 3 So design matrix will be > xdesign=modelMatrix(xtrg,ref="k0") > xdesign c10 c12 c14 c2 c4 c6 c8 k10 k12 k14 k2 k4 k6 k8 [1,] 0 0 0 0 0 0 0 0 0 0 1 0 0 0 [2,] 0 1 0 0 0 0 0 0 0 0 0 0 0 0 [3,] 0 0 0 0 1 0 0 0 0 0 0 0 0 0 [4,] 0 0 0 0 0 0 0 1 0 0 0 0 0 0 [5,] 0 0 0 0 0 0 0 0 0 1 0 0 0 0 [6,] 1 0 0 0 0 0 0 0 0 0 0 0 0 0 [7,] 0 0 0 0 0 1 0 0 0 0 0 0 0 0 [8,] 0 0 0 0 0 0 1 0 0 0 0 0 0 0 [9,] 0 0 0 0 0 0 0 0 0 0 0 0 0 1 [10,] 0 0 0 0 0 0 0 0 1 0 0 0 0 0 [11,] 0 0 0 0 0 0 0 0 0 0 0 1 0 0 [12,] 0 0 0 0 0 0 0 0 0 0 0 0 1 0 [13,] 0 0 0 1 0 0 0 0 0 0 0 0 0 0 [14,] 0 0 1 0 0 0 0 0 0 0 0 0 0 0 [15,] 0 0 0 0 0 0 0 0 0 0 0 0 0 -1 [16,] 0 0 0 0 0 0 0 0 0 0 0 -1 0 0 [17,] 0 0 0 0 0 0 0 0 0 0 0 0 -1 0 [18,] 0 0 0 0 0 0 -1 0 0 0 0 0 0 0 [19,] -1 0 0 0 0 0 0 0 0 0 0 0 0 0 [20,] 0 0 0 -1 0 0 0 0 0 0 0 0 0 0 [21,] 0 0 -1 0 0 0 0 0 0 0 0 0 0 0 [22,] 0 0 0 0 0 0 0 0 -1 0 0 0 0 0 [23,] 0 0 0 0 0 0 0 -1 0 0 0 0 0 0 [24,] 0 0 0 0 0 -1 0 0 0 0 0 0 0 0 [25,] 0 -1 0 0 0 0 0 0 0 0 0 0 0 0 [26,] 0 0 0 0 0 0 0 0 0 0 -1 0 0 0 [27,] 0 0 0 0 0 0 0 0 0 -1 0 0 0 0 [28,] 0 0 0 0 -1 0 0 0 0 0 0 0 0 0 [29,] 0 0 0 1 0 0 0 0 0 0 0 0 0 0 [30,] 0 0 0 0 0 0 0 0 0 0 0 0 1 0 [31,] 0 1 0 0 0 0 0 0 0 0 0 0 0 0 [32,] 0 0 0 0 0 0 0 0 0 1 0 0 0 0 [33,] 0 0 0 0 1 0 0 0 0 0 0 0 0 0 [34,] 1 0 0 0 0 0 0 0 0 0 0 0 0 0 [35,] 0 0 0 0 0 0 0 0 0 0 0 0 0 1 [36,] 0 0 0 0 0 0 1 0 0 0 0 0 0 0 [37,] 0 0 1 0 0 0 0 0 0 0 0 0 0 0 [38,] 0 0 0 0 0 1 0 0 0 0 0 0 0 0 [39,] 0 0 0 0 0 0 0 1 0 0 0 0 0 0 [40,] 0 0 0 0 0 0 0 0 0 0 1 0 0 0 [41,] 0 0 0 0 0 0 0 0 0 0 0 1 0 0 [42,] 0 0 0 0 0 0 0 0 1 0 0 0 0 0 [43,] 0 0 0 0 0 0 -1 0 0 0 0 0 0 0 [44,] -1 0 0 0 0 0 0 0 0 0 0 0 0 0 [45,] 0 0 0 -1 0 0 0 0 0 0 0 0 0 0 [46,] 0 0 0 0 0 0 0 0 0 -1 0 0 0 0 [47,] 0 0 0 0 -1 0 0 0 0 0 0 0 0 0 [48,] 0 0 0 0 0 0 0 0 0 0 -1 0 0 0 [49,] 0 0 0 0 0 0 0 0 0 0 0 0 0 -1 [50,] 0 0 -1 0 0 0 0 0 0 0 0 0 0 0 [51,] 0 -1 0 0 0 0 0 0 0 0 0 0 0 0 [52,] 0 0 0 0 0 0 0 0 -1 0 0 0 0 0 [53,] 0 0 0 0 0 0 0 -1 0 0 0 0 0 0 [54,] 0 0 0 0 0 -1 0 0 0 0 0 0 0 0 [55,] 0 0 0 0 0 0 0 0 0 0 0 0 -1 0 [56,] 0 0 0 0 0 0 0 0 0 0 0 -1 0 0 [57,] 0 0 0 1 0 0 0 0 0 0 -1 0 0 0 [58,] 0 0 0 -1 0 0 0 0 0 0 1 0 0 0 [59,] 0 0 0 -1 0 0 0 0 0 0 1 0 0 0 [60,] 0 0 0 1 0 0 0 0 0 0 -1 0 0 0 [61,] 0 0 0 0 0 1 0 0 0 0 0 0 -1 0 [62,] 0 0 0 0 0 -1 0 0 0 0 0 0 1 0 [63,] 0 0 0 0 0 -1 0 0 0 0 0 0 1 0 [64,] 0 0 0 0 0 1 0 0 0 0 0 0 -1 0 [65,] 0 0 0 0 0 0 1 0 0 0 0 0 0 -1 [66,] 0 0 0 0 0 0 -1 0 0 0 0 0 0 1 [67,] 0 0 0 0 0 0 -1 0 0 0 0 0 0 1 [68,] 0 0 0 0 0 0 1 0 0 0 0 0 0 -1 *Is* it correct? I.e. is it make correct use of direct (cx vs kx) comparisons? *Can* I use the same code as previously > xdesign_dye=cbind(DyeEffect=1,xdesign) > fit.userguide.dye=lmFit(img_lA_ncav,xdesign_dye) > contrast.matrix.d=makeContrasts(p2="c2-k2",p4="c4-k4",p6="c6-k6",p8="c 8-k8",p10="c10-k10",p12="c12-k12",p14="c14-k14",levels=xdesign_dye) > fit2.ug.d=contrasts.fit(fit.userguide.dye,contrast.matrix.d) > fit2.ug.d=eBayes(fit2.ug.d) > test.ug.d=decideTests(fit2.ug.d,method="global",adjust.method="BH",p .value=0.05) Thanks in advance, Best Regards, Maciej Jończyk, MSc Department of Plant Molecular Ecophysiology Institute of Plant Experimental Biology Faculty of Biology, University of Warsaw 02-096 Warszawa, Miecznikowa 1