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Need helps with my QPCR result


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#1 Thana

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Posted 13 February 2009 - 06:27 AM

Hello,

I've very little experience with QPCR and I don't know what 's going on with my house keeping gene (RP49) curve.

It used to be fine before but the latest running, the curves of each sample spread all over which was impossible to set the thredshold and the

Stdev is also very high whereas the curve of interest gene was fine.

Could there be something wrong with the gene expression of my cell (RNA extracted from S2 cells) or the primer&probe (Taqman probe)

Can something else go wrong during the process? Does anyone experience the same thing?

Can someone please help me answer this? :)

Thanks

#2 ra_polymerase

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Posted 27 February 2009 - 08:36 AM

It is possible that it is variable expression of your endogenous housekeeping gene, or the housekeeping gene has a shorter half-life than your gene(s) of interest? I don't know anything about RP49 but could you use the more common housekeeping genes like B-actin/GAPDH, etc?

No endogenous housekeeping gene is perfect in qPCR because it is impossible to find one whose expression is stable and constant. Perhaps try using an exogenous housekeeping gene (i.e. spike-in RNA from another organism)?

To be safe I would also re-run with new Taqman and primers, or at least another aliquot of the same probe/primers.




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