Hi I'm faced a problem on performing Real time PCR on RNA. Amplification happened in negative samples as showed in the picture but it is not primer/dimer phenomenon.(by primer/probe dilution nothing changed)(I do not experience this problem in RNA which is extracted from positive and negative commercial cell lines) I have used TaqPath™ 1-Step Multiplex Master Mix which contain UNG. Does UNG cause this problem? or impurity of extracted RNA can be the reason? (I use MagMAX FFPE DNA/RNA Ultra Kit with bead extraction method which shows good purity on thermofisher website).
I have used a master mix with purple mustang reference passive dye and one with no passive dye but I got the same result.
Did you face the same problem and how you resolved that?
The simple fix is to not use more than 40 cycles. Any signal that late is an artifact. You can use a melt-curve or gel electrophoresis to take a look at the products.
Katie A S Burnette
Thank you for your answer.
I use a primer/probe sequence which is tested on some literatures before and all gel electrophoresis and sanger sequencing tests are performed.
I have used another primer/probe which is published on another literature too but I have the same problem.
The Ct I catch is about 32 to 37.
I experience no problem with positive cell lines but I see this in patient's prostate samples only.
I have changed master mix from a mustang purple passive dye to one without passive dye but I have the same problem yet.
Which scenario is probable?
If you are getting amplification in your negative controls, run it out on a gel. My bet is you have contamination in the other primer/probe set.
Throw away all your reagents, clean your work area, and try again.
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