Quantification controls

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phani9999
phani9999's picture
Quantification controls

Dear All,

I am working on RT-PCR for quantifying HIV,HBV,HCV viral copies by Artus kit,My problem here is  the controls that we are using not giving repeatable results/copies it is crossing the general QC limits here we are using previous lot standards as controls i would like to know is it fine or need to use commercial controls?

or is there any other method for preparing controls?

If yes Let me know about The preparation of LOW positive, MED positive and High Positive controls for Viral Quantifying assays,

Regards,

Phani kumar.

Ivan Delgado
Ivan Delgado's picture
Hi Phani,

Hi Phani,

It is not uncommon to have RNA controls degrade and lead to unreliable results. My recommendation is to obtain commercial controls and re-test. You can always make your own controls, but in my experience that will likely cause you more problems and delay your ability to run the assays. 

Once you obtain commercial controls, and they are running well, you can always prepare your own controls and run them side by side until you are confident your own controls are working well. Then you can stop using commercial controls and start using your own. 

Good luck

phani9999
phani9999's picture
Dear Ivan,

Dear Ivan,

Thank you for your response,Can you please explain the best method to prepare pooled samples / controles for HIV,HBV,HCV quantification.

Regards,

Phani

Ivan Delgado
Ivan Delgado's picture
Dear Phani,

Dear Phani,

Unfortunately I do not know how to prepare pooled samples of the various viruses. What I would recommend is determining the size of each virus and simply mix them in equal molarities. 

As for controls, if at all possible I would purchase them from a commercial source. Again I do not know if this is available. 

phani9999
phani9999's picture
Oh....ivan..

Oh....ivan..

Sorry to know this..I will go for commercial controls!

....

Phani