[BioC] Does the strand of a microarray probe matter?

Nick Henriquez n.henriquez at ion.ucl.ac.uk
Thu Nov 20 14:35:13 CET 2008

Dear Cei, Steve,

There are two versions of the correct answer depending on whether we are
talking about an expression or CGH/SNP type array;

If we are using an EXPRESSION array

1) It does not matter on which strand the gene resides.
2) It a not matter of bad probe design. It is either a negative control or a
misnomer derived from genome annotation.

For ANY probe to hybridise it has to be the RC of cDNA and therefore the DNA
homologue of the original RNA sequence. (I'll let you work that one out for

If the probe WAS encoded on "the opposite strand" your labelled target would
not hybridise as it would be the reverse complement of the actual sequence. 

The annotation "opposite strand" stems from the convention that we call one
strand the "coding strand" and the other strand the non-coding or "opposite"
strand. By definition then a gene cannot be encoded by the "opposite"

However, what often happens when sequencing genomes is that we find several
genes encoded on one strand (which we will then call the coding strand) and
then somewhat later also one or more genes on the "opposite" strand. This
annotation is (wrongly in my opinion) retained when genomes are assembled
and thus part of the annotation of the probes.

So an opposite strand probe is at best a kind of negative control, at worst
a misnomer annotation retained when the genome was assembled. Mostly we now
try to use terms like + and - but even that has the drawback that we
generally associate + with coding and - with noncoding. As we all know BOTH
strand encode functional RNAs of various kinds including those coding for

If we are talking about DNA targets, e.g. a SNP array

1) It does not matter on which strand a gene resides, any overlap is a
matter of coincidence- "genes" are rare events on the genome.
2) It is not a matter of bad probe design. Usually it simply does not matter
and this is a sequence that was used historically without knowledge of the
gene (often discovered later). Sometimes the sequence on the coding strand
may have a problem with background or sequence similarity. To get around
this one can try to use the RC (i.e. "opposite strand" sequence) which is
often different enough. Of course if more than 2 similar sequences exist the
problem remains as we can use this trick only once.

Hope this helps,


N.V. Henriquez, Senior Research Associate
Dept. Of Neurodegenerative Diseases
Institute of Neurology, UCL, 
Queen Square House rm 124
Queen Square
London WC1N 3BG

Message: 8
Date: Wed, 19 Nov 2008 10:45:52 -0500
From: Steve Lianoglou <mailinglist.honeypot at gmail.com>
Subject: Re: [BioC] Does the strand of a microarray probe matter?
To: Cei Abreu-Goodger <cei at ebi.ac.uk>
Cc: Bioconductor Newsgroup <bioconductor at stat.math.ethz.ch>
Message-ID: <7710F044-03D5-4572-8EE4-2DB96F4C348C at gmail.com>
Content-Type: text/plain; charset=US-ASCII; format=flowed; delsp=yes

Hi Cei,

On Nov 19, 2008, at 3:51 AM, Cei Abreu-Goodger wrote:

> Hello all,
> Related issues have arisen before, where the probe of a particular  
> array platform was annotated to a gene on the opposite strand. But I  
> was just asked if this even matters, or should it simply be  
> considered a case of bad probe design.
> Does the protocol for different manufacturer's arrays always produce  
> amplified product of both strands for the transcript to be measured?  
> I could imagine that protocols that amplify based on poly-A tails  
> would tend to produce an anti-sense biased amplification product  
> (older Affy arrays?), whereas those based on random priming could  
> produce products of both strands (and so the actual strand that is  
> on the array becomes meaningless).
> Does someone know what is the case in particular for Illumina  
> Beadarrays?

I've never worked on the bench-side of a microarray experiment, but  
for gene expression arrays I was under the impression that most  

(i) extract the the RNA from cell lysate using their poly-A tails as  
(ii) reverse transcribe to cDNA and amplify the cDNA w/ random primers.
(iii) hybridize amplified cDNA to the array

If that's the case, I don't think that the strand of the probe should  
be an issue.

I'd be interested, of course, to hear other people's thoughts on this,  
too (while this info should be easily available from the  
manufacturer's site, or the Methods section of many papers, let's see  
if the lazy-web can help :-).


Steve Lianoglou
Graduate Student: Physiology, Biophysics and Systems Biology
Weill Medical College of Cornell University


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