QRT-PCR/Single tube

From OpenWetWare

< QRT-PCR
Revision as of 15:16, 26 March 2007 by Btarlow (Talk | contribs)
Jump to: navigation, search

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.
  • 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)

Personal tools