QRT-PCR/Single tube

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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).

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 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)