1. It is absolutely essential to plan
and execute the experiment with utmost care. It is important to begin
the planning of the microarray experiment with a proper question.
This is extremely important as you can end up wasting a lot of your
time otherwise. The experimental model/system should be well
characterized or well defined with an independent experimental verification.
For example, if a growth factor was added which induces differentiation
in 24-48 hours, but you are collecting RNA at three hours post-treatment,
you should still check a parallel culture for verification that differentiation
occurred at the 24-48 hours period.
2. If a drug treatment experiment is
desired, it is important to understand some aspect of biology, e.g.
growth inhibition, stimulation of proliferation, induction of differentiation
is occurring and at what is the effective concentration, time of treatment
that results in the biological parameters. Keep the initial experiment
as simple as possible. This means, don’t plan on using many samples
and x time points. Remember, if you go closer to the end point i.e.
death, growth etc. then you will pull the genes that are late markers
of this state and will not get any hint about the actual finger print
of your drug’s mechanism. If you have a negative control i.e.
a cell does not respond to your drug, then it may be a good idea to
plan to use it, if not in the first experiment, at least in the second
experiment.
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3. Keep in mind you have the option to analyze both
known and novel genes. Plan in advance what you are
going to do with the gene information before deciding whether you want
to pursue known or novel genes.
4. If possible, a quick check for a gene that is known
to be affected by the treatment should be performed. It is recommended
that all experimental treatments be carried out in triplicate to compensate
for biological and experimental variation. In vitro experiments using
cultured cells should be conducted three different times (not three
replicates performed on the same day) strictly following the same experimental
procedures. Tumor specimens should be devoid of adjacent tissues and
if possible, should be micro dissected to obtain as pure a tumor sample
as possible. Cell populations may also be further purified using cell-sorting
techniques such as FACS. Dead cells also should be removed by density
centrifugation. For comparative gene expression analysis, it is essential
that all the experimental conditions such as temperature, CO2, media,
reagents, and sample processing be kept identical for all samples.
5. Spend more time thinking about your question before
planning the chip experiment. The output of genes is still going to
be large, in the hundreds.
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