Equalization of hybridization probes

Brian Fristensky frist at cc.umanitoba.ca
Fri Aug 10 15:06:45 EST 2001


With regards to equalization of probes prior to adding to a
hybridization:

Say that you're doing timecourse experiments, measuring gene 
expression at different times after some treatment. 
For each timepoint, you want to add a fixed amount of probe
for hybridization on duplicate arrays.

One approach is to simply add 'equal' amounts of RNA to each
labeling reaction, and then add equal volumes of the final
reaction to mix to each hybridization. The sources of variation are
error in initial quantitation of the RNA, and differences in
labeling efficiencies among RNA samples.

In the days of radioactive probes, it was common practice to equalize
probes by adding equal COUNTS to each filter.

Could an analogous thing be done to equalize fluorescently-labeled
samples? After labeling the RNA population, quantitate by spotting
a dilution of each sample, induplicate, onto a filter and measure 
the amount of signal. Take the mean for each labeled cDNA population
and add equal 'counts' to each hybridization.

This should be better, because it controls for 2 different
sources of variation. Can anyone think of a reason that this 
is not valid? 

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Brian Fristensky                | 
Department of Plant Science     | Too much of the people's music
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Office phone:   204-474-6085    | 
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