**40**wrote:

Hi everyone,

I am trying to sort out how to properly analyze a microarray experiment with multiple groups and "paired" samples and despite my best efforts I am still a bit unsure if I am doing it right.

Here is my set up: I have collected four different cell types from six patients. I want to compare the cell types to each other, but block for "patient effects". Here is an abbreviated list of samples and conditions (*samples*). My arraydata (from affymetrix) is in an expression set I call *exprsset.*

Array | Fraction | Patient |

A1 | CellType1 | P1 |

A2 | CellType2 | P1 |

A3 | CellType3 | P1 |

A4 | CellType4 | P1 |

... | ... | ... |

A24 | CellType4 | P6 |

So, combining the description for paired samples and multiple groups from limmas user's guide gives something like

patient<-factor(samples$Patient) fraction <- factor(samples$Fraction, levels=c("CellType1, "CellType2", "CellType3", "CellType4"))

1. design <- model.matrix(~patient+fraction) fit <- lmfit(exprsset, design) fit <- eBayes(fit)

But from here I don't know how to get at the contrasts I am after (pairwise comparisons of cell types): It seems like R is always contrasting against one sample? If so, how do I get the other comparisons (that does not include this reference sample) and how does R choose this reference?

I couldn't sort this out so after some googling (especially these posts: Question on unbalanced paired design and https://support.bioconductor.org/p/55255/), I instead tried this:

2. design <- model.matrix(~ 0 + fraction + patient ) fit <- lmfit(exprsset, design) contrasts <- makeContrasts(fractionCelltype1 - fractionCellType2, fractionCellType1-fractionCellType3, fractionCellType1 - fractionCelltype4,(all other pairwise comparisons), levels=design) fit2 <- contrasts.fit(fit, contrasts) fit2 <- eBayes(fit2)

topTable with the coef parameter then seems to give me the comparisons I am after so:

**My main question is:** does the nr 2 "script" do what I hope, i.e. that is give me the pairwise celltype comparisons "blocked" for patient effects? If not, how do I correct it?

What I still don't understand here is, if I in the design instead write:

design <- model.matrix(~ 0 + patient + fraction)

and use the same contrasts as in 2., I get: *Warning message:In contrasts.fit(fit, contrast.matrix) : row names of contrasts don't match col names of coefficients. *

Looking at the design matrix: I have 1 less fraction column that I have fractions, so one of the fractions are "missing", which of course makes a discrepancy with my contrasts, hence the error. The same goes for patients if I use the formula for design under 2. (but since I don't make "patient contrasts" in the makeContrasts(), that doesn't seem to matter...). Naively put: Why does one condition "disappear" in the design matrix? Is it again a reference level of some sort and how do I then handle this?

If someone could explain this or point me to some appropriate tutorial about design matrices and how they relate to the linear models, I would greatly appreciate it! I really bugs me that I can't seem to get my head around this!

Best regards,

Anna