Microarray confirmation and gene isn't present

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Microarray confirmation and gene isn't present

Postby onalisama » Jul 13 2015 11:26 pm

Hi,

We did an initial microarray study and found upregulation of a new gene of interest. But then when we went to do real time PCR to confirm to microarray results, it didn't amplify (never crossed threshold and no convincing gel pictures). After doing a positive control experiment, we showed that the gene did amplify in tissue reported in the literature to have the gene. We got some pretty strange PCR curves from the positive control experiment, but there was a band on the gel, so it seems present.

My question is why the gene might have shown expression on the microarray if it is not even present in our cell culture according to qPCR. Any ideas?

Thanks,
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Re: Microarray confirmation and gene isn't present

Postby mdfenko » Jul 14 2015 6:27 am

maybe the gene is too highly methylated to allow the primer to land properly? (the probes in microarrays are long enough to allow binding of the gene, albeit more loosely than if it wasn't methylated).
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Re: Microarray confirmation and gene isn't present

Postby onalisama » Jul 16 2015 9:35 pm

I should have also said earlier that we used a Taqman primer/probe set pre-designed for our gene. If it is because of high methylation, wouldn't that mean that our positive control would have failed too? We had thick bands on the positive control.
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Re: Microarray confirmation and gene isn't present

Postby mdfenko » Jul 17 2015 6:17 am

not if the positive control gene is not modified.

the gene must have been modified to make it inaccessible to the primers you have. maybe you can prepare a primer pair that set down in sequences unlikely to be modified.
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Re: Microarray confirmation and gene isn't present

Postby onalisama » Jul 19 2015 8:36 am

Thanks for explaining. That's a possibility then.
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Re: Microarray confirmation and gene isn't present

Postby mchlbrmn » Jul 24 2015 5:31 pm

Maybe the array probe detects an expressed splice of the transcript that the qPCR primers cannot detect. In other words, one PCR primer or the other is not present in the splice expressed.
Is it possible that the array primer is not specific for the gene?
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