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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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problems with housekeepings in real-time pcr
Posted by: pmartin (IP Hidden, New member, 7)
Date: November 29, 2005 02:09AM
Hi!!
I´m working with RT-PCR real time and I´m having a lot of problems with my housekeepings. I isolated RNA from human macrophages with RNeasy kit. For the real-time PCR I used 18s like endogenous control in the beginning, and to assure to me, also I used RPLP0 and HPRT1 genes like housekeepings. When analyzing the data I have seen that housekeeping varies, but I do not know if it is by the treatment or the yield of the extraction of the RNA and the retrotranscription. It is possible knowledge it? And to finalize, how you would analyze the data? Would you make the average with the three housekeeping? Would you eliminate the housekeeping that is turned aside with respect to the others? Would you choose of the three the one that seems that it adjusts better? thank you very much to all I wait for your advice a greeting. paula
Re: problems with housekeepings in real-time pcr
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: December 5, 2005 09:07AM
Many so called "housekeeping" genes do have variable expression levels in different tissue and cell type. But if you were using them on the same cell type but under different treatment they would be fine. I would suggest to use 18s rRNA as internal control.
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