I performed RT-PCR to validate my differentially expressed microRNAs. But I didn't get any accurate results. What is the reason? I also performed standard PCR (with a gradient temperature) to select the best temperature and got bands at almost all temperatures. My reference gene (U6 SnRNA) worked well in RT-PCR, but miRNA-specific primers didn't show any results (amplification has occurred).
Please help me to find the correct reason.
Dear Sreedevi, it is quite hard to answer your question, since there are multiple reasons why qPCR might go wrong. Can you please tell what does "miRNA-specific primers didn't show any results" mean here? There were no product, or expression was inconsistent, or replicates returned different Cqs?
May be your primer specificity or PCR amplification enzyme, you have to check.
Anastasiia Hubiernatorova Ma'am, The main problem is I got CT values above 35. My reference gene's CT values are below 30. Amplifications occurred at the end of the cycles.
- Badges
- Science topic
- Similar topics
- Engineering
- Modeling