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

[Wolfgang Huber]

Hi Cei

this paper contains a discussion of this topic.

Antisense artifacts in transcriptome microarray experiments are resolved 
by actinomycin D, by Perocchi et al.:
http://nar.oxfordjournals.org/cgi/content/full/35/19/e128

Fig.1 and Fig.2 show that you can get strand specific measurements, but 
that spurious second-strand synthesis by the reverse transcription step 
needs to be considered / avoided.

Best wishes
      Wolfgang

----------------------------------------------------
Wolfgang Huber, EMBL-EBI, http://www.ebi.ac.uk/huber

Maria Stalteri ha scritto:
> Hi Cei,
> 
> I think you may still be a little confused about what the gene expression 
> arrays are designed to measure.
> 
> The probes on the Affymetrix 3' expression arrays are single-stranded 
> oligos having the sense sequence, i.e. the same sequence as the mRNA they are designed
>  to detect, and the IVT assay used with these arrays produces single 
> stranded cRNAs with the antisense sequence, i.e. the reverse complement
> of the initial mRNA sample.
> 
> The probes on the Affymetrix Exon ST and Gene ST  arrays have the 
> antisense sequence, i.e. the reverse complement of the sequence they are 
> designed to detect, and the WT assay used with these arrays produces a 
> single-stranded cDNA with the sense sequence, i.e. the same sequence
> as the initial mRNA sample.
> 
> The probesets on these arrays are only designed to measure 
> expression from one strand.
> 
> They will only measure expression from both strands in the cases where 
> Affymetrix have tiled probesets on both strands in the same region
> of the genome. This is the case for some probesets that were designed
> based on ESTS, where it wasn't clear which strand the gene was on at the
> time of array design, so probesets were tiled on both strands in the 
> region the EST mapped to.
> 
> 
> As for problems with probeset annotations or discrepancies between one 
> annotation source and another, we have also found that
> the number of annotation errors is probably somewhere close to 10%, 
> and that genes that were close together or had overlapping ends tended
> to cause problems for the annotations.
> 
> Affymetrix grades the reliability of the annotations for the 3' expression 
> arrays as A, B, C or E for each probeset, with A being the most reliable 
> and E being annotations based on EST clusters and generally the least 
> reliable. We have found that although their A and B grade annotations are 
> not always correct either, they are indeed more likely to be correct than 
> the annotations they label as E.
> 
> For the exon arrays, Affymetrix labels its probesets as unique, similar, 
> or mixed depending on whether or to what extent the probes 
> cross-hybridise, so that one can choose to use only those probesets 
> labelled as unique if one wants to avoid cross-hybridising probes.
> (I haven't done any mappings of the probes on the exon arrays yet, so I 
> don't know how true this is.)
> 
> Best wishes,
> Maria
> 
> _______________________________________________