Real Time PCR

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Pratiksha
Pratiksha's picture
Real Time PCR

When I run kit standards on RotorGene 3000, I get amplification with 100 copies std and not with 10 copies std. How can I  make my assay more sensitive?

Ivan Delgado
Ivan Delgado's picture
Hi Pratiksha,

Hi Pratiksha,

My recommendation is to design at least three different assays, at different locations in your gene, and see which one is more sensitive. In my experience that is the fastest, and most cost effective, way to find assays that not only work well but are the most sensitive. 

Good luck.

Pratiksha
Pratiksha's picture
I am using a commercial kit

I am using a commercial kit and so cannot design different sets of primers.

Ivan Delgado
Ivan Delgado's picture
Hi Pratiksha,

Hi Pratiksha,

If you are using a commercial kit, then likely the kit has DNA controls. If those controls are not working as they should, giving you signal at 10 copies, then you should contact the vendor and request/buy new controls. 

Pratiksha
Pratiksha's picture
Dear Ivan,

Dear Ivan,

A lyophilized vial of quantitated control was provided. I had to reconstitute, make serial 10 fold dilutions and use. High copy standard gives an expected Ct. I have done 2 assays now, once there was no ampln with 10 copy std, next time it amplified. I cannot question kit primer probe mix and the control that has been provided. I want to achieve consistent amplification with the lowest 10 copy standard. Do you think changes in GAIN settings or cycling conditions would help?

Ivan Delgado
Ivan Delgado's picture
Dear Pratiksha,

Dear Pratiksha,

If you got the 10 copy standard to work one time and the other time it failed, then likely you are trying to analyze a range in the PCR that is too weak for analysis. If you look at the amplification curves of your qPCRs you will see that they are sigmoidal curves. Likely your 100 copy standard shows a sigmoidal curve while your 10 copy standard never amplifies enough to plateau. My recommendation is look at your amplification curves and see if you are getting enough amplification or not. One rule of thumb, not a rule by any means, is that unless the Ct you are getting for your 10 copy standard is 37 or lower (i.e. the inflection point of your amplification curve is at PCR cycle 37 or earlier) you cannot use this standard for analysis. If that is the case one trick, although I personally do not recommend it, is to simply increase the number of PCR cycles from the standard 40 to 45. 

Pratiksha
Pratiksha's picture
Its a 50 cycle PCR. 10 copy

Its a 50 cycle PCR. 10 copy standard gave ct-35.5, sigmoidal curve with plateau. This time I am planning to run clinical samples along with the standards and want to see reproducibility with 10 copy standard. Thanks a lot for the inputs.

Ivan Delgado
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Anytime.  Good luck!

Anytime. 

Good luck!