QRT-PCR/Single tube

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

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.

Analysis

Delta-Delta Ct Method. See Livak KJ, Schmittgen. Methods 25 402-408 (2001)