QRT-PCR/Single tube - Revision history

Reshma P. Shetty: Real-time RT-PCR moved to QRT-PCR/Single tube: This is single tube qRT-PCR.

2007-10-09T19:43:28Z

Real-time RT-PCR moved to QRT-PCR/Single tube: This is single tube qRT-PCR.

←Older revision

Revision as of 19:43, 9 October 2007

Reshma P. Shetty at 18:08, 21 May 2007

2007-05-21T18:08:12Z

←Older revision		Revision as of 18:08, 21 May 2007
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	== Protocol Endorsements ==		== Protocol Endorsements ==
		+
		+	[[Category:Protocol]]
		+	[[Category:In vitro]]
		+	[[Category:RNA]]

Btarlow at 00:45, 27 March 2007

2007-03-27T00:45:35Z

←Older revision		Revision as of 00:45, 27 March 2007
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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).	+	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 ==
-	* Validated PCR primers & probe that are efficient over the range of RNA that you are assaying.	+	* 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 ==
-	* Combine RT, Taq, dNTPs, primers, probe and RNA in a single tube. Add ~~enzyme~~ last.	+	* 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.
	* Do 40 cycles of PCR. Measure the levels of template after each cycle with a real-time PCR machine.		* Do 40 cycles of PCR. Measure the levels of template after each cycle with a real-time PCR machine.
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	== Protocol ==		== 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 ==		== 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)
		+
		+	== Comments & Tips ==
		+	* Use a master mix to ensure a consistent amount of enzyme in each tube that you will be comparing.
		+
		+
		+	== Protocol Endorsements ==

Btarlow at 20:18, 26 March 2007

2007-03-26T20:18:01Z

←Older revision		Revision as of 20:18, 26 March 2007
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-	This protocol describes one-step real time quantitivie PCR to quantify relative levels of ~~RNA in~~	+	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 ==		== Starting Materials ==

Btarlow: /* Basic Principle */

2007-03-26T20:16:53Z

Basic Principle

←Older revision		Revision as of 20:16, 26 March 2007
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	* Combine RT, Taq, dNTPs, primers, probe and RNA in a single tube. Add enzyme last.		* 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.		* 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.	+	* 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.

Btarlow: /* Basic Principle */

2007-03-26T20:16:31Z

Basic Principle

←Older revision		Revision as of 20:16, 26 March 2007
Line 10:		Line 10:

	== Basic Principle ==		== Basic Principle ==
-	* Combine RT, Taq, dNTPs, primers, probe and RNA in a single tube.	+	* 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.		* 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.		* Do 40 cycles of PCR. Measure the levels of template are quantified during each cycle with a real-time PCR machine.

Btarlow: /* Starting Materials */

2007-03-26T20:16:08Z

Starting Materials

←Older revision		Revision as of 20:16, 26 March 2007
Line 2:		Line 2:

	== Starting Materials ==		== Starting Materials ==
-	* Validated PCR primers that are efficient over the range of RNA that you are assaying.	+	* 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		* Taq Polymerase, MuLV RT
	* dNTPs, MgCl2, nuclease free water		* dNTPs, MgCl2, nuclease free water

Btarlow: /* Basic Principle */

2007-03-26T20:15:25Z

Basic Principle

←Older revision		Revision as of 20:15, 26 March 2007
Line 9:		Line 9:

	== Basic Principle ==		== Basic Principle ==
-	* RNA ~~is reverse transcribed~~ in a single ~~well~~.	+	* Combine RT, Taq, dNTPs, primers, probe and RNA in a single tube.
-	* PCR ~~reaction procedes~~. ~~The~~ levels of template are quantified during each cycle.	+	* 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 ==		== Protocol ==

Btarlow: /* Starting Materials */

2007-03-26T20:13:39Z

Starting Materials

←Older revision		Revision as of 20:13, 26 March 2007
Line 3:		Line 3:
	== 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 ==

Btarlow at 20:10, 26 March 2007

2007-03-26T20:10:48Z

←Older revision		Revision as of 20:10, 26 March 2007
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-	~~#REDIRECT [[Quantitative~~ reverse ~~transcriptase~~ PCR]]	+	This protocol describes one-step real time quantitivie PCR to quantify relative levels of RNA in
		+
		+	== Starting Materials ==
		+	* Validated PCR primers that are efficient over the range of RNA that you are assaying.
		+	*
		+
		+	== 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)