QRT-PCR/Single tube

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(Starting Materials)
(Basic Principle)
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== Basic Principle ==
== Basic Principle ==
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* Combine RT, Taq, dNTPs, primers, probe and RNA in a single tube.
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* 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.   
* 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.   
* Do 40 cycles of PCR.  Measure the levels of template are quantified during each cycle with a real-time PCR machine.   

Revision as of 15:16, 26 March 2007

This protocol describes one-step real time quantitivie PCR to quantify relative levels of RNA in

Contents

Starting Materials

  • 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)

Basic Principle

  • 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 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)

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