| C. Choose the reference for microarray s: The main goal in choosing a reference is to ensure significant hybridization signal in every spot on the arrays so that the Ratio of R/G signal in each spot can be quantitated … therefore the identity of the reference is somewhat arbitrary; the data can be subsequently mathematically transformed to reveal the biologically-relevant ratios.
1. Example reference samples
a. Genomic DNA
b. An arbitrary RNA reference pool
c. Time zero RNA, taken just before beginning the experiment
d. A pool of all of the RNA samples recovered from an experiment
RNA taken from the control sample
2. Regardless of which reference is used, be sure to use the identical reference
on all arrays in a given timecourse so as to compare the timepoints to eachother
3. Mathematical transformation example:
a. for a timecourse in which genomic DNA is used as the reference, there will
be one array for each time point INCLUDING the time = 0 sample
b. Each ratio for each spot = Red/Green signal = signal from time point
RNA sample/signal from genomic DNA
c. To transform the data, divide the R/G ratio measured for each gene on the t>0 arrays by the corresponding R/G ratio measured on the t = 0 array to cancel the “genomic DNA” denomenator:
(R/G t > 0 array) / (R/G t = 0 array) = (RNA t > 0 /Genomic DNA) / (RNA t=0 /Genomic
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