QRT-PCR/Single tube
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This protocol describes one-step real time quantitivie PCR to quantify relative levels of RNA in
Starting Materials
- 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)
Basic Principle
- RNA is reverse transcribed in a single well.
- PCR reaction procedes. The levels of template are quantified during each cycle.
Protocol
Analysis
Delta-Delta Ct Method. See Livak KJ, Schmittgen. Methods 25 402-408 (2001)
