I am trying to use limma for the analysis of 2DGE proteomic data and am having great difficulty constructing the design and contrast matrices for a 2x2 factorial experiment. A small section of the data spreadsheet is below. I have extracted all sorts of objects from this spreadsheet including a matrix of only the data, a matrix of the factors, factor names, etc. What I have not been able to do is figure out how to translate those into the required design and contrast matrices. I am interested in the main effects of the 2 factors as well as the interaction between them. Any help, including code samples, is greatly appreciated! Thanks, Mark W. Kimpel MD Indiana U. School of Medicine, Indianapolis, IN Experiment Keratinocytes: Nutrition, JP8 2x2 Design Factor Nutrition High High Low High Low High High Factor JP8.vs.H2O H2O H2O H2O Jp8 Jp8 H2O JP8 Sample.ID may1501g-3390 may1501e-3391 may1501h-3392 may1501l- 3393v1 may1501n-3394 may1501q-3395 may1501b-3396 Spot.Number Data.start 14 776.39 1136.96 847.86 1211.93 1366.17 1247.95 627.66 17 765.35 720.84 705.24 624.39 668.91 759.81 639.89 19 244.53 274.41 188.65 251.76 287.13 -1 168.21 21 140.24 214.14 116.44 181.11 264.72 166.28 175.97 22 647.23 802.11 256.37 630.47 1046.78 717.15 1196.63 27 274.84 389.64 459.78 701.53 722.04 720.45 191.88 103 493.72 412.51 400.92 295.2 432.94 390.06 450.54 105 271.05 189.73 413.47 266.26 -1 -1 -1 106 1451.17 1390.25 1215.72 1506.79 1427.72 1321.7 1876.38 107 -1 215.85 -1 113.77 -1 151.19 -1 108 145.56 304.96 244.99 621.74 525.55 663.87 225.03 109 6422.49 7251.3 6418.58 5977.25 6685.45 6098.35 6482.68 [[alternative HTML version deleted]]