[BioC] marray and gathering additional information fromGenePixGPR file

Marcus Davy mdavy at hortresearch.co.nz
Thu Jul 20 07:08:13 CEST 2006

Yes the spot intensities (labeled "Empty") which are recorded in a GenePix
gpr file should be distributed similarly to the background intensities on a
16 bit GenePix scanner (between 0 and several hundred). Essentially they are
measures of background in the foreground spot area.

Yes I mean A values, and I should have mentioned that the grouping on an MA
scale I described is without background correction. If you background
correct I would expect a shift in the MA plot distribution to the left for
the A scale. Depending on the method chosen a lot of NA's will be evaluated
when foreground is less than or equal to background.

I think that due to the distribution of background intensities, there is a
probability that for some empty or buffer only spots the foreground measure
will be higher than the background in both the Red and Green channel
simultaneously so that these small positive numbers do not get converted to
NA's when calculating M.
In the case of buffer only spots this could be due to the physical contact
between the metal printtip and the glass slide, the buffer deposited, or
even high PMT scanner settings adding noise during scanning.


On 7/19/06 10:06 PM, "J.delasHeras at ed.ac.uk" <J.delasHeras at ed.ac.uk> wrote:

> Quoting Marcus Davy <mdavy at hortresearch.co.nz>:
>> Just to add further comment to this discussion about empty spots in GenePix.
>> Empty spots could be due to the physical printing not taking place at that
>> spot position or buffer which contains no physical cDNA/oligo probe could be
>> deposited by the printing quill at that spot position.
>> In gpr files I've looked at, GenePix image analysis provides a non zero
>> value (but close to zero) to these intensities.
> close to "background", rather.
>> On an MA scale, you should
>> see them distributed around 0 on the M scale and have Abundance values
>> slightly lower than other non expressing spots.
> By Abundance you're referring to the A values, right?
> What I tend to see is a spread for those spots, it can be quite broad,
> but as you say, they're always distinctly "clustering" at lower A
> values.
> If you substract background, then you do get zero values (or 0.5,
> actually, if you use the method "half" to make sure you don't get
> negative intensities, which can happen for those "empty" spots easily)
>> They are usually labelled "" or "Empty" in the annotation.
> one word of warning: check that empty spots are really empty.
> On my latest arrays, I noticed quite a few spots labelled as empty,
> when I could clearly see a spot. I got worried I was using the wrong
> annotation, so I contacted the people who made the arrays. It turned
> out that after printing, they found that they couldn't trust the
> identity of some cDNAs, and they simply marked those spots as "empty"
> in the annotation. It can be very bad, if you're using those spots to
> get an estimate of background, as some people do.
> Jose


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