Hi all, I'm having some trouble in understanding how to ste the Error() term in the aov() function when fitting a hierarchical ANOVA. I have data concerning the expression of 2 miRNAs in 3 different cell lines, with 2 different extraction methods. The data is organized as follows : Line Extraction Target Expression 1 BC54 miRNA RNU48 22.48 2 BC54 miRNA RNU48 22.36 3 BC54 miRNA 221 26.49 4 BC54 miRNA 221 26.42 5 BC54 RNAtot RNU48 22.95 6 BC54 RNAtot RNU48 22.98 7 BC54 RNAtot 221 26.35 8 BC54 RNAtot 221 26.43 9 BC108 miRNA RNU48 24.19 10 BC108 miRNA RNU48 24.01 11 BC108 miRNA 221 29.94 12 BC108 miRNA 221 29.61 13 BC108 RNAtot RNU48 22.42 14 BC108 RNAtot RNU48 22.29 15 BC108 RNAtot 221 26.79 16 BC108 RNAtot 221 26.73 17 BC233 miRNA RNU48 21.84 18 BC233 miRNA RNU48 21.69 19 BC233 miRNA 221 25.95 20 BC233 miRNA 221 25.98 21 BC233 RNAtot RNU48 22.55 22 BC233 RNAtot RNU48 22.58 23 BC233 RNAtot 221 25.11 24 BC233 RNAtot 221 25.45 For the two miRNA and for each cell line I want to compare the expression value between the two extraction methods. As there are no random effects I tried to use the aov() function to do so. I don't know which of the two following models is the "good" one : This one : > mir221.aov=aov(Ct~Extraction+Error(Target/Line/Extraction),data=mir221) with this model the extraction has no significant effect : > summary(mir221.aov) Error: Target Df Sum Sq Mean Sq F value Pr(>F) Residuals 1 99.675 99.675 Error: Target:Line Df Sum Sq Mean Sq F value Pr(>F) Residuals 4 16.9779 4.2445 Error: Target:Line:Extraction Df Sum Sq Mean Sq F value Pr(>F) Extraction 1 2.8912 2.8912 1.3568 0.2966 Residuals 5 10.6545 2.1309 Error: Within Df Sum Sq Mean Sq F value Pr(>F) Residuals 12 0.164150 0.013679 or this one : > mir221.aov=aov(Ct~Extraction+Error(Target/Line),data=mir221) with this model the extraction has a significant effect: > summary(mir221.lm) Error: Target Df Sum Sq Mean Sq F value Pr(>F) Residuals 1 99.675 99.675 Error: Target:Line Df Sum Sq Mean Sq F value Pr(>F) Residuals 4 16.9779 4.2445 Error: Within Df Sum Sq Mean Sq F value Pr(>F) Extraction 1 2.8912 2.8912 4.5431 0.04794 * Residuals 17 10.8187 0.6364 Could you please help me understanding which is the right model for my question ? Thanks by advance Jonas Mandel U900 - Bioinformatics unit Institut Curie Paris