RNA contamination in primers - (Dec/05/2008 )

I made up two sets of new primers with TE, ran an RT-PCR, and the NTC was amplified. I went back and tried various combinations of reagents and discovered the TE used to make up the primers must be contaminated with RNA (and possibly some DNA). Is it possible to remove this contamination or would it require completely new primers? Thanks.

---

If you are doing a real-time PCR, then it is expected that you will get some amplification in your NTC. However, this should not cross threshold or appear until >30 cycles (much after your amplification in your reactions containing template).

If you are just doing a RT-PCR, where you made cDNA and are using that as a template, it is possible that you get product in your reactions where you did not use RT (-RT controls -- are these what you mean by NTC?). This would mean you have genomic DNA contamination in your RNA prep and would simply need to repeat your RNA prep and perhaps include an RNase-free DNase treatment.

Finally, if you really think it is your primers which are contaminated, I would make sure the size of your amplicon is not the same as a primer dimer.

Hope that helps!

---

If you can afford it, get new primers. Getting new primers might save you time in questioning your results with the contaminated primer set and having to repeat your analysis.

---