This protocol describes one-step real time quantitivie PCR to quantify relative levels of RNA in
- Validated PCR primers that are efficient over the range of RNA that you are assaying.
- Taq Polymerase, MuLV RT
- dNTPs, MgCl2, nuclease free water
- PCR strip caps or 96-well plates with transpearant caps
- total RNA (~50ng per reaction, diluted to 10ng/ul)
- RNA is reverse transcribed in a single well.
- PCR reaction procedes. The levels of template are quantified during each cycle.
Delta-Delta Ct Method. See Livak KJ, Schmittgen. Methods 25 402-408 (2001)