Good diluents for RNA - (Jul/19/2016 )
I need to some serial dilutions of RNA targets for RT-PCR following in vitro transcription. At the moment the RNA is about 2 ug/ul, or 10^12 copies /ul, and I need to get down to <50 copies/ul. I have tried ultra pure water, but the RNA appears to be sticking together, as the dilutions cease being linear after the 1/100 dilution (I am doing a 10-fold dilution series). I have also tried 1 mM citrate, which seems better, but still isn't great.
Does anyone know a good diluent that will reduce/remove intermolecular interactions, so I can get an accurate dilution series? Or are there any good tips out there for diluting RNA quantitatively?
I have never done this.
Is it possible to heat your sample up at each dilution to reduce annealing?
Maybe formamide or betaine or some other PCR additive would help during the dilutions?
"Formamide is a widely used organic PCR additives. Formamide is thought to work by binding in the major and minor grooves of DNA, destabilizing the template double-helix and lower melting temperature. Formamide is usually used at 1-5%."
- perhaps it would do the same with RNA.