Hello everyone,

My goal is to verify the change in gene expression in 4 maize varieties under cold stress.

I have designed an interwoven loop design with 8 samples and 2 treatments. The 8 samples are the 4 maize varieties in control conditions and in cold stress.

The design is in the file attached.

A, C, E, G are the four varieties under control conditions; B, D, F, H are the same four varieties under cold stress. For every sample I have used 3 biological replicates.

According to the design the dyes are balanced without the need for a dye swap.

I was not able to specify to Robin the interwoven loop design, therefore I have considered the three loops separately. When it comes to assign the dyes to the samples on the slides, I am asked for a reference sample, which by default is the first name I insert. This is a concern to me, since in a loop design there is no reference sample. The issue with assigning a sample as a reference is that the results differ significantly according to reference sample chosen. I have no single variety that can be considered as a reference.

Moreover, when I run the quality checks I get warning for all the MA-plots which I suppose it is normal when a treatment like cold is applied or when different varieties are considered.

Thanks for paying attention!

Mauro