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I don't find housekeeping genes: what should I do?

qRT-PCR housekeeping genes

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6 replies to this topic

#1 Renato Ivan de Ávila

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Posted 26 September 2013 - 06:36 PM

Hello all,

 

I am trying to normalize my experiment using SYBR Green qRT-PCR kit for RotorGene. I used 8 endougenous controls (b-actin, GAPDH, HSP90, PGK1, b2M,18SrRNA, HPRT and GUSB), however all of them varied between the control and treated samples. What should I do?



#2 pcrman

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Posted 26 September 2013 - 07:33 PM

Most of the housekeeping genes should not be affected by any treatment. Do they vary in a consistent way among samples? If they vary wildly, your results should not be trusted. 



#3 Renato Ivan de Ávila

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Posted 27 September 2013 - 01:44 AM

The Ct was higher than 0.5 between control (untreated) and treated. I don't know what I do. The 8 housekeeping genes that I tried are the most used in qRT-PCR, not? Do I have that to try other endogenous controls? Or is there another way?



#4 pcrman

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Posted 27 September 2013 - 01:53 AM

>>The Ct was higher than 0.5 between control (untreated) and treated.

That does not tell you that your housekeeping genes vary. They could have different Ct values between control and treated samples, resulted from unequal loading of RNA, but they should have a similar pattern. 



#5 Tabaluga

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Posted 27 September 2013 - 09:44 AM

If you want to try other ones you could also try Lamin B1 and RPLP0, they work fine for me...

Edited by Tabaluga, 27 September 2013 - 09:44 AM.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#6 Rajpoot

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Posted 29 September 2013 - 08:06 AM

A difference of 0.5 doesn't look quite significant smile.png



#7 Renato Ivan de Ávila

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Posted 01 October 2013 - 05:50 PM

Thanks all for the help!  I was not paying attention to loading of cDNA between the groups. Now my experiment works. Thanks a lot!







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