QRT-PCR/Single tube: Difference between revisions
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== Basic Principle == | == Basic Principle == | ||
* RNA | * Combine RT, Taq, dNTPs, primers, probe and RNA in a single tube. | ||
* PCR | * Reverse transcribe the RNA for ~ 30' to form a template for Taq-mediated PCR. | ||
* Do 40 cycles of PCR. Measure the levels of template are quantified during each cycle with a real-time PCR machine. | |||
* Analyze the results. | |||
== Protocol == | == Protocol == | ||
Revision as of 13:15, 26 March 2007
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
- Combine RT, Taq, dNTPs, primers, probe and RNA in a single tube.
- Reverse transcribe the RNA for ~ 30' to form a template for Taq-mediated PCR.
- Do 40 cycles of PCR. Measure the levels of template are quantified during each cycle with a real-time PCR machine.
- Analyze the results.
Protocol
Analysis
Delta-Delta Ct Method. See Livak KJ, Schmittgen. Methods 25 402-408 (2001)
