Optimal RT-PCR for DENV by using Light cycler 480, Roche

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huekien2005
huekien2005's picture
Optimal RT-PCR for DENV by using Light cycler 480, Roche

I have some problems with this assay. My lab used to run RT-PCR by Biorad machine and it's good, now we change to use LC480 and Master Mix from Roche but the result doesn't same as previous. So i don't know where the problem is.
Please give me some advise! Thanks so much!
 

Ivan Delgado
Ivan Delgado's picture
There are different answers

There are different answers to your question, but one possible one is that the channels in each one of these instruments are not the same. Assuming that you are running probe-based assays (Taqman), you should check and see if the channels you specify for detection are the same. One good way to test for this is to look at the component plot of your assay. If you assay contains ROX you will be able to determine how much signal your assay is generating compared to the baseline level of ROX.
If you provide more details about your assay (chemistry, thermal profile, amplification plot, ...) we may be able to help you more.

huekien2005
huekien2005's picture
if you have a standard curve

if you have a standard curve with 8 serial dilution and you need to find the suitable primer concentation to make sure that at least  6 dilution give signal. Is it good if i only choose randomly (top concentration, sixth diluion) for optimizing?
 
 

Ivan Delgado
Ivan Delgado's picture
Running standard curves to

Running standard curves to validate your assay are definitely a very good tool to use. When choosing how to design your validation experiments I would not worry too much about how many dilutions you need to run when optimizing your primer concentrations. I would choose an average amount of template, just one dilution, and run experiments to determine the optimal concentration for your primers. Once you identify the optimal primer concentration(s) all you really need, statistically speaking, is a 5-point standard curve. I recommend 10-fold dilutions, but you can get away with 5-fold dilutions.
To answer your question more directly: it is perfectly fine to use your sixth dilution to optimize your primers, as long as once the primers are optimized you run all dilutions again and determine that your standard curve is good (slope between -3.1 and -3.6, efficiency between 90% and 110%, R2 of >0.99).
Good luck