This protocol describes one-step real time quantitivie PCR to quantify relative levels of a particular mRNA sequence between two samples. This method describes use of a flourescent labeled probe (FAM).
- Validated PCR primers & probe that are efficient over the range of RNA that you are assaying.
- PCR primers and probe for a house keeping gene (ie B-actin, GAPDH, RPL19)
- 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)
- Combine RT, Taq, dNTPs, primers, probe and RNA in a single tube. Add enzyme last.
- Reverse transcribe the RNA for ~ 30' to form a template for Taq-mediated PCR.
- Do 40 cycles of PCR. Measure the levels of template after each cycle with a real-time PCR machine.
- Analyze the results.
Delta-Delta Ct Method. See Livak KJ, Schmittgen. Methods 25 402-408 (2001)