mirVana™ qRT-PCR miRNA Detection Kit - FAQs

I used the mirVana qRT-PCR miRNA Detection Kit and got no products in the no-template control (NTC) reaction. What could have happened?

Here are possible causes and solutions:

- Product from amplification of PCR primer-dimer: we recommend routinely running a no-template control reaction for each mirVana qRT-PCR Primer Set in the experiment. In successful miRNA amplification reactions, a single ~90 bp product is synthesized, which results in detection of a single product by melt curve analysis on a real-time thermal cycler. Smaller products (end-point) or products with lower Tm (real-time) may be observed in some no-template control reactions; these result from primer-dimer formation. They are typically only detectable late in the cycling protocol (>30 cycles). (Product from primer-dimer can also be amplified in no-RT control reactions.)
- Reagent contamination with PCR product: if a product of ~90 bp is observed in the no-template control reaction, there is a good chance that one or more of the PCR reagents, pipettors, or benchtops has been contaminated with PCR products. Unfortunately the only way to remedy contaminated reagent(s) is to replace them.
To avoid PCR contamination, clean the lab bench and the pipettors routinely with a DNA decontamination reagent such as DNAZap solution (Cat. No. AM9890). Always use barrier tips to pipette PCR reagents, and store completed PCRs in a different location than the PCR reagents.