[BioC] Limma: background correction. Use or ignore?

[J.delasHeras at ed.ac.uk]

Hi Jim,

many thanks for your reply.

> I have never been a big fan of subtracting background, especially if the
> background of the slide is low and relatively consistent. I have two
> main reasons for this.
>
> First, the portion of the slide used to estimate background doesn't have
> any cDNA bound, so you are estimating the background binding of the spot
> by using a portion of the slide that might not be very similar. When we
> were doing more spotted arrays, we would always spot unrelated cDNA on
> the slides as well (e.g., A.thaliana and salmon sperm DNA). These spots
> almost always had a negative intensity if you subtracted the local
> background, which indicates to me that cDNA does a better job of
> blocking the slide than BSA or other blocking agents.

That is true. In the arrays I use at the moment there is no unrelated 
cDNA spotted, but in the previous ones I used there were some bacterial 
spots, and I noticed that effect sometimes. The background I was 
getting was usually very low, but on a few ocassions that it was 
higher, I often *saw* a negative effect on those bacterial spots (and 
indeed on some others that just didn't hybridise).
On the normally clean slides, looking at the data this becomes apparent too.


> Second, you *are* adding more noise to the data. When you subtract, the
> variances are additive. However, if you don't subtract then you take the
> chance that you are biasing your expression values, especially if the
> background from chip to chip isn't relatively consistent. So the
> tradeoff is higher variance vs possible bias. If the background was
> consistent I usually took a chance on the bias in order to reduce the
> variance. As you note, the data usually look 'cleaner' if you don't
> adjust the background.

I just never imagined the effect could be so pronounced. In my new 
arrays I notice that a lot, I think because there are a lot of spots 
that have mid-low intensities...

> Note that these points are directed towards simple subtraction of a
> local background estimate. Other more sophisticated methods may help
> address these shortcomings.

I'm at the moment exploring different methods to estimate the 
background, and also to substract it, before I decide whether I should 
totally ignore background (I think my slides are generally clean 
enough, and I'd rather repeat one experiment than risking my overall 
results if one slide turns out dirtier).

Your comments and those of Naomi and Gordon were very helpful. Thanks a lot.

I was very surprised to learn how background substraction can affect 
the final data so much. I thought I was safe because my slides were 
quite uniform and clean...


> As for references, have you looked at the references that Gordon gives
> on the man page for backgroundCorrect()? That would probably be a good
> place to start.

I have, thanks... the problem I find is that they get too much into the 
actual algorithms and I am looking at a more basic issue... not so much 
"how" to remove background, but "why" do we have to, how it can affect 
our analysis, etc... But I did get some useful info there too, of 
course.

Thanks again!

Jose


-- 
Dr. Jose I. de las Heras                      Email: J.delasHeras at ed.ac.uk
The Wellcome Trust Centre for Cell Biology    Phone: +44 (0)131 6513374
Institute for Cell & Molecular Biology        Fax:   +44 (0)131 6507360
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