It turns out that the house-keeping genes were spotted at much higher
density than the rest of the spots.
I guess this may have been to help with spot-finding or something.....
 
Anyway, I think this gives me the right to treat the two populations of
spots separately during normalisation and analysis.
 
Many thanks
Mick

  _____  

From: Kasper Daniel Hansen [mailto:khansen@stat.Berkeley.EDU] 
Sent: 27 September 2005 18:50
To: michael watson (IAH-C)
Cc: bioconductor@stat.math.ethz.ch
Subject: Re: [BioC] Normalisation knocks my "housekeeping genes" off!


On Sep 27, 2005, at 6:54 AM, michael watson ((IAH-C)) wrote:


	Hi

	I have a small array that contains lots of spots for genes that
*should*
	show no differential expression, and guess what?  They don't!
The raw
	data for these spots looks great, they all appear in a really
tight
	cluster on an M vs A plot and have a log2 ratio of just above
zero,
	which can easily be explained by dye bias.

	HOWEVER, the general trend on the M vs A plot, when a loess line
is
	fitted, is not so good.  In fact, M shows a negative
relationship with
	A.  Therefore, when I loess normalise, it pushes all my log2
ratios for
	the house-keeping genes up to around 1!  

	I'm at a bit of a loss as to what to do - if I assume my
house-keeping
	genes should all be zero, I will push the trend of M decreasing
with A
	even further down.  If I correct for this trend using loess
	normalisation, my house keeping genes will all display a
two-fold
	up-regulation.... :-(

	Hmmmm.



If your housekeeping genes are spread out across the intensity range
(which it does not really sound like from your description of a "tight
cluster"), then I would trust the housekeeping genes. If not, you have
no way of knowing how to normalize genes with an intensity far away from
your housekeeping genes. Are all your housekeeping genes from probes
with really low or high signal. If so, I guess the phenomena you see
could be due to some extrapolation at the boundary of the intensity
range, but I am raving here.


Kasper

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