Hi, I just stumbled onto an error creating contrasts in limma 2.12 on R 2.6.2, but could not find it in the mailing list archives or google, so I thought I might share it for the next person. Here's what happened: > cont.matrix= makeContrasts(MT_3hvsMT_0h= (M_A3h+M_D3h+M_G3h+M_K3h- (M_A0h+M_D0h+M_G0h+M_K0h))/4, levels=design) Error in makeContrasts(MT_3hvsMT_0h = (M_A3h + M_D3h + M_G3h + M_K3h - : Parameter names must by syntactically valid names in R But my same command had worked on a reduced dataset just a few days earlier. Turns out the issue came from my targets file: > cont.matrix= makeContrasts(MT_3hvsMT_0h= (M_A3h+M_D3h+M_G3h+M_K3h- (M_A0h+M_D0h+M_G0h+M_K0h))/4, levels=colnames(design)[1:186]) #this one worked > cont.matrix= makeContrasts(MT_3hvsMT_0h= (M_A3h+M_D3h+M_G3h+M_K3h- (M_A0h+M_D0h+M_G0h+M_K0h))/4, levels=colnames(design)[1:187]) Error in makeContrasts(MT_3hvsMT_0h = (M_A3h + M_D3h + M_G3h + M_K3h - : Parameter names must by syntactically valid names in R > colnames(design)[187] [1] "W_P48h " One of the sample names had spaces in it... Have a great day, yannick