QRT-PCR/Single tube

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== Starting Materials ==
== Starting Materials ==
* Validated PCR primers that are efficient over the range of RNA that you are assaying.   
* 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 ==
== Basic Principle ==

Revision as of 15:13, 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 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

  • RNA is reverse transcribed in a single well.
  • PCR reaction procedes. The levels of template are quantified during each cycle.

Protocol

Analysis

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

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