QRT-PCR/Single tube

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(Basic Principle)
Current revision (14:43, 9 October 2007) (view source)
m (Real-time RT-PCR moved to QRT-PCR/Single tube: This is single tube qRT-PCR.)
 
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This protocol describes one-step real time quantitivie PCR to quantify relative levels of RNA in
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This protocol describes one-step real time quantitative reverse transcription PCR to quantify relative levels of a particular mRNA sequence between two samples.  This technique is also called known commerically as Taqman, qRT-PCR, real-time PCR.  This particular protocol describes use of a flourescent labeled probe (FAM) to provide readings. 
== Starting Materials ==
== Starting Materials ==
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* Validated PCR primers & probe that are efficient over the range of RNA that you are assaying.  
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* Validated PCR primers & probe that are efficient over the range of RNA that you are assaying. Primers are typically designed with a 58 degree Tm but may vary by lab. 
* PCR primers and probe for a house keeping gene (ie B-actin, GAPDH, RPL19)
* PCR primers and probe for a house keeping gene (ie B-actin, GAPDH, RPL19)
* Taq Polymerase, MuLV RT
* Taq Polymerase, MuLV RT
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== Basic Principle ==
== Basic Principle ==
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* Combine RT, Taq, dNTPs, primers, probe and RNA in a single tube.  Add enzyme last.   
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* Combine RT, Taq, dNTPs, primers, probe and RNA in a single tube.  Add enzymes 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.   
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* Do 40 cycles of PCR.  Measure the levels of template are quantified during each cycle with a real-time PCR machine.   
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* Do 40 cycles of PCR.  Measure the levels of template after each cycle with a real-time PCR machine.   
* Analyze the results.
* Analyze the results.
== Protocol ==
== Protocol ==
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* Generate a 10x solution for each gene target that includes the appropriate forward primer, reverse primer, and labelled probe (FAM-BHQ1, FAM-TAM, etc). 
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* Combine RT, Taq, dNTPs, MgCl2 and H20 in a single tube called your master mix.  Make sure to add enzymes last. Mix by pipetting (do not vortex enzymes).
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* Add the appropriate quantity (refer to your kit) of master mix to 10x primer mix. 
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* Dilute RNA to ~ 10ng/ul.  Pipet 5 ul into each well.
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* Add PCR reaction mix for each gene target into the appropriate well. 
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* Cycles for MuLV reverse transcriptase with Amplitaq Gold from Applied Biosystems.  This program may need adjustments depending on your primer design.  It's a good starting point. 
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** 30' at 48 degrees
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** 10' at 94 degrees
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** 40 Cycles
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*** 30" 94 degrees
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*** 60" 60 degrees
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*** (optional) 60" 72 degrees
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** END
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* The first time you use a primer set it is a good idea to run the sample out to ensure that you're getting one band, thus showing that your primers are specific for your desired gene product. 
== Analysis ==
== Analysis ==
Delta-Delta Ct Method.  See Livak KJ, Schmittgen.  Methods '''25''' 402-408 (2001)
Delta-Delta Ct Method.  See Livak KJ, Schmittgen.  Methods '''25''' 402-408 (2001)
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== Comments & Tips ==
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* Use a master mix to ensure a consistent amount of enzyme in each tube that you will be comparing. 
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== Protocol Endorsements ==
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[[Category:Protocol]]
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[[Category:In vitro]]
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[[Category:RNA]]

Current revision

This protocol describes one-step real time quantitative reverse transcription PCR to quantify relative levels of a particular mRNA sequence between two samples. This technique is also called known commerically as Taqman, qRT-PCR, real-time PCR. This particular protocol describes use of a flourescent labeled probe (FAM) to provide readings.

Contents

Starting Materials

  • Validated PCR primers & probe that are efficient over the range of RNA that you are assaying. Primers are typically designed with a 58 degree Tm but may vary by lab.
  • 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 enzymes 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.

Protocol

  • Generate a 10x solution for each gene target that includes the appropriate forward primer, reverse primer, and labelled probe (FAM-BHQ1, FAM-TAM, etc).
  • Combine RT, Taq, dNTPs, MgCl2 and H20 in a single tube called your master mix. Make sure to add enzymes last. Mix by pipetting (do not vortex enzymes).
  • Add the appropriate quantity (refer to your kit) of master mix to 10x primer mix.
  • Dilute RNA to ~ 10ng/ul. Pipet 5 ul into each well.
  • Add PCR reaction mix for each gene target into the appropriate well.
  • Cycles for MuLV reverse transcriptase with Amplitaq Gold from Applied Biosystems. This program may need adjustments depending on your primer design. It's a good starting point.
    • 30' at 48 degrees
    • 10' at 94 degrees
    • 40 Cycles
      • 30" 94 degrees
      • 60" 60 degrees
      • (optional) 60" 72 degrees
    • END
  • The first time you use a primer set it is a good idea to run the sample out to ensure that you're getting one band, thus showing that your primers are specific for your desired gene product.

Analysis

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

Comments & Tips

  • Use a master mix to ensure a consistent amount of enzyme in each tube that you will be comparing.


Protocol Endorsements

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