How do you compare expression in Microarray - (Jun/13/2013 )
How do you compare expression in Microarray?
In qPCR, we use reference genes as control and they should have the same or very similar CT values.
How about Microarray?
do a 2-color hybridization. one will be your control and the other your experimental sample
IF you are talking about single array, for every gene on the array, there is an intensity value which is used for expression analysis. So the intensity value can be used as sort of absolute quantification.
if you perform a 1 color hybridization, then you add use a spike-in at a specific ratio to rna in the starting sample (at least, with agilent microarrays).
Microarrays are ratiometric rather than absolute. They are non-quantitative unlike qPCR. The signal obtained from a probe spot is proportional to the amount of RNA originally present. But due to differences in GC, labelling efficiency and a number of other issues, two genes with the same number of copies in a sample could give a different signal intensity. For that reason - you can only compare a gene to itself.
For example, if you have two samples - A and B and look at the expression of the gene neu, you can say that NEU was more highly expressed in A vs B. However if you are also interested in ESR1 you could not say NEU was more highly expressed that ESR1 just because it had a higher signal.