Dear list Members. I'd like to ask for advice. I have 2x2 factorial experiment conducted in a loop-design (two-colour data). There are two maize lines (dh7, dl3) and two temperature treatments (cold=c and control=k). Following hybridizations were conducted : dh_c vs dh_k; dh_k vs dl_k; dl_k vs dl_c; dl_c vs dh_c (forming a square on a diagram) and two (diagonal on a diagram), namely: dl_k vs dh_c and dh_k vs dl_c. I have four biological replications of this design, including dye-swap (i.e. two hyb. cy3-cy5 and two cy5-cy3). In each biological replication I have also three technical replications of each RNA source (i.e. in dh_c vs dh_k; dl_c vs dh_c and dl_k vs dh_c, sample dh_c is from the same RNA pool). I don't know how analyse it in limma. I want to get following contrasts: dh_c vs dh_k; dh_k vs dl_k; dl_k vs dl_c; dl_c vs dh_c and include effect of temperature, maize line and interaction. My targets frame is: SlideNumber FileName Cy3 Cy5 1 93 c_093_DH_K_vs_DH_CHex.gpr dh7_k_1 dh7_c_1 2 104 c_104_DH_CH_vs_DH_Kex.gpr dh7_c_2 dh7_k_2 3 116 c_116_DHK_vs_DHCHex.gpr dh7_k_3 dh7_c_3 4 16 c_016_DH_C_vs_DH_Kex.gpr dh7_c_4 dh7_k_4 5 94 c_094_DH_K_vs_DL_Kex.gpr dh7_k_1 dl3_k_1 6 105 c_105_DL_K_vs_DH_Kex.gpr dl3_k_2 dh7_k_2 7 117 c_117_DHK_vs_DLKex.gpr dh7_k_3 dl3_k_3 8 139 c_139_DL_K_vs_DH_Kex.gpr dl3_k_4 dh7_k_4 9 92 c_092_DL_CH_vs_DL_Kex.gpr dl3_c_1 dl3_k_1 10 106 c_106_DL_K_vs_DL_CHex.gpr dl3_k_2 dl3_c_2 11 118 c_118_DLCH_vs_DLKex.gpr dl3_c_3 dl3_k_3 12 23 c_023_DL_K_vs_DL_Cex.gpr dl3_k_4 dl3_c_4 13 95 c_095_DL_CH_vs_DH_CHex.gpr dl3_c_1 dh7_c_1 14 107 c_107_DH_CH_vs_DL_CHex.gpr dh7_c_2 dl3_c_2 15 119 c_119_DLCH_vs_DHCHex.gpr dl3_c_3 dh7_c_3 16 136 c_136_DH_C_vs_DL_Cex.gpr dh7_c_4 dl3_c_4 17 101 c_101_DL_K_vs_DH_CHex.gpr dl3_k_1 dh7_c_1 18 103 c_103_DH_CH_vs_DL_Kex.gpr dh7_c_2 dl3_k_2 19 121 c_121_DLK_vs_DHCHex.gpr dl3_k_3 dh7_c_3 20 15 c_015_DH_C_vs_DL_Kex.gpr dh7_c_4 dl3_k_4 21 100 c_100_DH_K_vs_DL_CHex.gpr dh7_k_1 dl3_c_1 22 102 c_102_DL_CH_vs_DH_Kex.gpr dl3_c_2 dh7_k_2 23 120 c_120_DHK_vs_DLCHex.gpr dh7_k_3 dl3_c_3 24 140 c_140_DL_C_vs_DH_Kex.gpr dl3_c_4 dh7_k_4 In "Cy3" and "Cy5" columns last number denotes biological replication. I've read in limma documentation, that this desing can be analysed using single channel analysis. So I created following contrast matrix: FileName dye dhc dhk dlc c_093_DH_K_vs_DH_CHex.gpr 1 -1 1 0 c_104_DH_CH_vs_DH_Kex.gpr 1 1 -1 0 c_116_DHK_vs_DHCHex.gpr 1 -1 1 0 c_016_DH_C_vs_DH_Kex.gpr 1 1 -1 0 c_094_DH_K_vs_DL_Kex.gpr 1 0 1 0 c_105_DL_K_vs_DH_Kex.gpr 1 0 -1 0 c_117_DHK_vs_DLKex.gpr 1 0 1 0 c_139_DL_K_vs_DH_Kex.gpr 1 0 -1 0 c_092_DL_CH_vs_DL_Kex.gpr 1 0 0 1 c_106_DL_K_vs_DL_CHex.gpr 1 0 0 -1 c_118_DLCH_vs_DLKex.gpr 1 0 0 1 c_023_DL_K_vs_DL_Cex.gpr 1 0 0 -1 c_095_DL_CH_vs_DH_CHex.gpr 1 -1 0 1 c_107_DH_CH_vs_DL_CHex.gpr 1 1 0 -1 c_119_DLCH_vs_DHCHex.gpr 1 -1 0 1 c_136_DH_C_vs_DL_Cex.gpr 1 1 0 -1 c_101_DL_K_vs_DH_CHex.gpr 1 -1 0 0 c_103_DH_CH_vs_DL_Kex.gpr 1 1 0 0 c_121_DLK_vs_DHCHex.gpr 1 -1 0 0 c_015_DH_C_vs_DL_Kex.gpr 1 1 0 0 c_100_DH_K_vs_DL_CHex.gpr 1 0 1 -1 c_102_DL_CH_vs_DH_Kex.gpr 1 0 -1 1 c_120_DHK_vs_DLCHex.gpr 1 0 1 -1 c_140_DL_C_vs_DH_Kex.gpr 1 0 -1 1 As a reference sample I choosen "dlk", I've included also dye effect. Now, I don't know how can I include information about technical replication. I assume that if I don't do it, it will be treated as biological replication. I read about command "duplicateCorrelation" is it the right one to deal with these problem? I also have problem in contrasts definition. Is it simply dhc-dhk, dhk-dlk, dlk-dlc, dlc-dhc and for main effect (maize line): (dhc+dhk-dlc-dlk)/4; for effect of temperature (dhc+dlc-dhk-dlk)/4 and for interaction (dhc-dlc)-(dhk-dlk)? Thank You in advance for help. Yours sincerely Maciej Jo?czyk Department of Plant Molecular Ecophysiology Institute of Plant Experimental Biology Faculty of Biology, University of Warsaw 02-096 Warszawa, Miecznikowa 1 ___________________________________ NOCC, http://nocc.sourceforge.net -- This email was Anti Virus checked by Astaro Security Gateway. http://www.astaro.com