Statistician here. Biology know-nothing.
I'm working with microarray data (specifically Affymetrix HGU133A however I'm generally interested in this stuff). I assume that the processing of these microarrays follows three major steps (1) extract sample tissue (2) extract mRNA/cDNA or whatever from a sample into solution (3) put solution on chip and cook it and then look at the chip's intensity measurements. My question is as follows: can I expect that the same total amount of my labeled target nucleic acid is going onto each chip? That is, do the people preparing the microarray experiment try to fix the amount of labeled target that goes into the solution put on each chip? Or maybe the concentration of the labeled target nucleic acid is attempted to be held constant from one run to the next?
I've tried to look into the operating procedures for some of these microarray preparations but its too dense for me to understand exactly what is going on in these experiments and I can't find anyone talking about this anywhere.
Thanks!
Yes. For each experimental sample, the bench would i) quantify the amount of RNA isolated from the sample (and perform some quality checks); ii) generate cDNA from some amount X ng of RNA (X being constant across all samples); iii) and then an equal amount of cDNA would be loaded onto the array.