Confirmed gene knockdown not showing on array - (Jul/22/2011 )
Hi,
I've microarrayed samples after a gene knock-down (KD confirmed by qRT-PCR to be 95%) with Affymetrix Gene 1.0 ST arrays and Biotique XRAY software. The problem is, that this well expressed gene does not show up among the differentially expressed genes. My suspicion is that the expression in the KD samples drops below the background and is therefore not recorded by the software. Absolute, unmodified intensitiy of the probe for this gene can be found and is significantly lower in the knock-down samples. Could anyone give me some tips on this or e.g. tell me how to get the results from genes which are expressed in one control and completely silenced in the other?
Thanks
you shouldn't be able to detect it in knock down. since you knocked it down the gene won't be well expressed. the methods you are using to detect are for expression of the gene, not for the presence of the gene.
you can try a cgh (comparative genomics) array to show the presence or absence of the gene in the knock down compared to the control.
mdfenko on Fri Jul 22 13:59:47 2011 said:
you shouldn't be able to detect it in knock down. since you knocked it down the gene won't be well expressed. the methods you are using to detect are for expression of the gene, not for the presence of the gene.
you can try a cgh (comparative genomics) array to show the presence or absence of the gene in the knock down compared to the control.
Thanks for your reply. Would there be no way of telling/showing that the probes on the arrays hybridised with Negative Control RNA are binding to the gene in question and the probes on the array hybridised with Knock-down RNA aren't? Background correction is done on a per-chip basis with the Affy system. Normalisation is performed on the whole set of chips.