I have been using taqman microRNA assays for 3 years and have recently come across a very serious problem with one particular microRNA. RT-qPCR analysis shows the miRNA to be expressed at higher level in blank RT Product(RNAase-free water) than my tissue samples which contain 1000ng of RNA. I have always noticed primer dimerisation in samples but never to this extent where the Blank produces Ct value of 27 while Tissue samples produce 28-31 Ct with my positive control showing 24 Ct. This has occurred with several samples, fresh taqman primers, fresh reagents
- Troubleshooting so far
- RNAse free water is fine it produces no amplification with number other Taqman miRNA primers, and have utilised Other sources of RNAse free water
- Brand new Primers have been utilised with same results
- Brand new RT reagents have been utilised
- Dedicated RNAse free lab space, pipettes etc
Looking for advice from anyone who has encountered and hopefully resolved such an issue