Real-time PCR - Revision history

Torsten Waldminghaus at 17:30, 23 February 2009

2009-02-23T17:30:08Z

←Older revision		Revision as of 17:30, 23 February 2009
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		+	{{back to protocols}}
	{{dablink\|[[QRT-PCR]]}}		{{dablink\|[[QRT-PCR]]}}

Jakob Suckale: primer design section moved to qRT-PCR

2008-01-14T18:12:29Z

primer design section moved to qRT-PCR

←Older revision		Revision as of 18:12, 14 January 2008
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	Generation of product is detected in one of two ways <cite>MeasuringGeneExpression</cite>. First, the amount of double stranded DNA in the tube can be measured using fluorescent dyes which intercalcate double-stranded DNA (like the DNA binding dye [[SYBR Green I]]). The intensity of fluorescence is proportional to the quantity of DNA present in the reaction. Second, the amount of PCR product can be measured by monitoring the hybridization of a set concentration of fluorescently labeled probe oligonucleotide. The oligo probe provide selectivity and only monitors the concentration of PCR product with a particular sequence. In contrast, SYBR green I will bind even nonspecific PCR products.		Generation of product is detected in one of two ways <cite>MeasuringGeneExpression</cite>. First, the amount of double stranded DNA in the tube can be measured using fluorescent dyes which intercalcate double-stranded DNA (like the DNA binding dye [[SYBR Green I]]). The intensity of fluorescence is proportional to the quantity of DNA present in the reaction. Second, the amount of PCR product can be measured by monitoring the hybridization of a set concentration of fluorescently labeled probe oligonucleotide. The oligo probe provide selectivity and only monitors the concentration of PCR product with a particular sequence. In contrast, SYBR green I will bind even nonspecific PCR products.
-
-	~~== Primer selection ==~~
-	An excellent and fast way to select primers is with the free online-tool [http://fokker.wi.mit.edu/primer3/input.htm Primer3], currently in v0.3. [http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi Primer3Plus], a variation of Primer3 has qPCR settings. Or just apply the following or similar settings to Primer3:
-	*pair towards 3' end (often more specific, some cDNAs don't contain)
-	*pair separated by an exon-exon boundary (reduces genomic background) e.g. last exon & penultimate
-	*amplified region must be no biger than 200 bp; usally 60-150 bp
-	*GC content: 50-60%
-	*min length: 18, max length 24 (best: 20 nt)
-	*melting temperature: min 60, max 63, best 60
-	*max Tm difference: 10 (shouldn't be more than 1 in final pair)
-	*max 3' self complementary: 1
-	*max poly-x: 3
-
-	Verify by blasting the primers sequences. Target gene should come out with the lowerst E value. No other gene should be close. Also check whether possible isoforms will be detected by the candidate primer pair.

	==Notes==		==Notes==

Jakob Suckale: placed disambiguation link to QRT-PCR

2008-01-14T18:09:10Z

placed disambiguation link to QRT-PCR

←Older revision		Revision as of 18:09, 14 January 2008
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		+	{{dablink\|[[QRT-PCR]]}}
		+
	'''Real-time PCR''' is a [[PCR techniques\|PCR technique]] used to quantify starting amounts of nucleic acid template by analysing the amount of DNA produced during each cycle of PCR. It is a form of quantitative PCR ([[Q-PCR]]).		'''Real-time PCR''' is a [[PCR techniques\|PCR technique]] used to quantify starting amounts of nucleic acid template by analysing the amount of DNA produced during each cycle of PCR. It is a form of quantitative PCR ([[Q-PCR]]).

Jakob Suckale: lead rewritten

2008-01-14T16:50:45Z

lead rewritten

←Older revision		Revision as of 16:50, 14 January 2008
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-	'''Real-time PCR''' is used to quantify starting amounts of nucleic acid template by analysing the amount of DNA produced during each cycle of PCR. It is a form of quantitative PCR or [[Q-PCR]].	+	'''Real-time PCR''' is a [[PCR techniques\|PCR technique]] used to quantify starting amounts of nucleic acid template by analysing the amount of DNA produced during each cycle of PCR. It is a form of quantitative PCR ([[Q-PCR]]).

-	Real-time PCR is often preceded by reverse transcription to ~~detect~~ RNA via their cDNA. In fact, mRNA quantification is one of the most frequent uses of real-time PCR. The sub-technique is sometimes called [[qRT-PCR]] for '''q'''uantitative '''r'''everse '''t'''ranscription PCR.	+	Real-time PCR is often preceded by reverse transcription to quantifiy RNA via their cDNA. In fact, mRNA quantification is one of the most frequent uses of real-time PCR. The sub-technique is sometimes called [[qRT-PCR]] for '''q'''uantitative '''r'''everse '''t'''ranscription PCR.

	== Principle ==		== Principle ==

Reshma P. Shetty: /* Notes */

2007-10-10T23:55:23Z

Notes

←Older revision		Revision as of 23:55, 10 October 2007
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	==Notes==		==Notes==
-	*When using [[SYBR Green I]] to measure DNA concentration, it is important to run the PCR product out on a gel to verify that there is only a single amplification product.	+	*When using [[SYBR Green I]] to measure DNA concentration, it is important to run the PCR product out on a gel to verify that there is only a single amplification product <cite>MeasuringGeneExpression</cite>.
-	*When using [[SYBR Green I]], the amount of fluorescence in a PCR product depends on the length and base composition of the product. So it is not possible to compare the concentrations of two different templates without having control templates of known concentration for each target DNA region.	+	*When using [[SYBR Green I]], the amount of fluorescence in a PCR product depends on the length and base composition of the product <cite>MeasuringGeneExpression</cite>. So it is not possible to compare the concentrations of two different templates without having control templates of known concentration for each target DNA region.

	== See also ==		== See also ==

Reshma P. Shetty: /* Principle */

2007-10-10T23:54:49Z

Principle

←Older revision		Revision as of 23:54, 10 October 2007
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	Real-time PCR is more precise than previously used reverse transcription PCR (RT PCR) because the generation of product is continuously monitored during the PCR run (this is where the term "real time" comes in), rather than at the end of a PCR reaction ("endpoint" PCR).		Real-time PCR is more precise than previously used reverse transcription PCR (RT PCR) because the generation of product is continuously monitored during the PCR run (this is where the term "real time" comes in), rather than at the end of a PCR reaction ("endpoint" PCR).

-	Generation of product is detected in one of two ways<cite>MeasuringGeneExpression</cite>. First, the amount of double stranded DNA in the tube can be measured using fluorescent dyes which intercalcate double-stranded DNA (like the DNA binding dye [[SYBR Green I]]). The intensity of fluorescence is proportional to the quantity of DNA present in the reaction. Second, the amount of PCR product can be measured by monitoring the hybridization of a set concentration of fluorescently labeled probe oligonucleotide. The oligo probe provide selectivity and only monitors the concentration of PCR product with a particular sequence. In contrast, SYBR green I will bind even nonspecific PCR products.	+	Generation of product is detected in one of two ways <cite>MeasuringGeneExpression</cite>. First, the amount of double stranded DNA in the tube can be measured using fluorescent dyes which intercalcate double-stranded DNA (like the DNA binding dye [[SYBR Green I]]). The intensity of fluorescence is proportional to the quantity of DNA present in the reaction. Second, the amount of PCR product can be measured by monitoring the hybridization of a set concentration of fluorescently labeled probe oligonucleotide. The oligo probe provide selectivity and only monitors the concentration of PCR product with a particular sequence. In contrast, SYBR green I will bind even nonspecific PCR products.

	== Primer selection ==		== Primer selection ==

Reshma P. Shetty: /* Principle */

2007-10-10T23:51:43Z

Principle

←Older revision		Revision as of 23:51, 10 October 2007
Line 8:		Line 8:
	Real-time PCR is more precise than previously used reverse transcription PCR (RT PCR) because the generation of product is continuously monitored during the PCR run (this is where the term "real time" comes in), rather than at the end of a PCR reaction ("endpoint" PCR).		Real-time PCR is more precise than previously used reverse transcription PCR (RT PCR) because the generation of product is continuously monitored during the PCR run (this is where the term "real time" comes in), rather than at the end of a PCR reaction ("endpoint" PCR).

-	Generation of product is detected in one of two ways. First, the amount of double stranded DNA in the tube can be measured using fluorescent dyes which intercalcate double-stranded DNA (like the DNA binding dye [[SYBR Green I]]). The intensity of fluorescence is proportional to the quantity of DNA present in the reaction. Second, the amount of PCR product can be measured by monitoring the hybridization of a set concentration of fluorescently labeled probe oligonucleotide. The oligo probe provide selectivity and only monitors the concentration of PCR product with a particular sequence.	+	Generation of product is detected in one of two ways<cite>MeasuringGeneExpression</cite>. First, the amount of double stranded DNA in the tube can be measured using fluorescent dyes which intercalcate double-stranded DNA (like the DNA binding dye [[SYBR Green I]]). The intensity of fluorescence is proportional to the quantity of DNA present in the reaction. Second, the amount of PCR product can be measured by monitoring the hybridization of a set concentration of fluorescently labeled probe oligonucleotide. The oligo probe provide selectivity and only monitors the concentration of PCR product with a particular sequence. In contrast, SYBR green I will bind even nonspecific PCR products.

	== Primer selection ==		== Primer selection ==

Reshma P. Shetty at 22:45, 9 October 2007

2007-10-09T22:45:15Z

←Older revision		Revision as of 22:45, 9 October 2007
Line 1:		Line 1:
	'''Real-time PCR''' is used to quantify starting amounts of nucleic acid template by analysing the amount of DNA produced during each cycle of PCR. It is a form of quantitative PCR or [[Q-PCR]].		'''Real-time PCR''' is used to quantify starting amounts of nucleic acid template by analysing the amount of DNA produced during each cycle of PCR. It is a form of quantitative PCR or [[Q-PCR]].

-	Real-time PCR is often preceded by reverse transcription to detect RNA via their cDNA. In fact, mRNA quantification is one of the most frequent uses of real-time PCR. The sub-technique is sometimes called [[qRT-PCR]] for '''q'''uantitative '''r'''everse ~~'''~~ '''t'''ranscription PCR.	+	Real-time PCR is often preceded by reverse transcription to detect RNA via their cDNA. In fact, mRNA quantification is one of the most frequent uses of real-time PCR. The sub-technique is sometimes called [[qRT-PCR]] for '''q'''uantitative '''r'''everse '''t'''ranscription PCR.

	== Principle ==		== Principle ==

Reshma P. Shetty at 22:44, 9 October 2007

2007-10-09T22:44:35Z

←Older revision		Revision as of 22:44, 9 October 2007
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	* Wikipedia entry on [[wikipedia:Quantitative polymerase chain reaction\|Q-PCR]]		* Wikipedia entry on [[wikipedia:Quantitative polymerase chain reaction\|Q-PCR]]
	* The venerable [http://tech.groups.yahoo.com/group/qpcrlistserver/ qpcrlistserv]. Anyone doing qPCR should be subscribed to this list.		* The venerable [http://tech.groups.yahoo.com/group/qpcrlistserver/ qpcrlistserv]. Anyone doing qPCR should be subscribed to this list.
		+
		+	==References==
		+	<biblio>
		+	#MeasuringGeneExpression isbn=0415374723
		+	</biblio>

	[[Category:Protocol]]		[[Category:Protocol]]

Reshma P. Shetty: /* Notes */

2007-10-09T22:04:52Z

Notes

←Older revision		Revision as of 22:04, 9 October 2007
Line 25:		Line 25:

	==Notes==		==Notes==
-	*When using [[SYBR ~~green~~ I]] to measure DNA concentration, it is important to run the PCR product out on a gel to verify that there is only a single amplification product.	+	*When using [[SYBR Green I]] to measure DNA concentration, it is important to run the PCR product out on a gel to verify that there is only a single amplification product.
-	*When using [[SYBR ~~green~~ I]], the amount of fluorescence in a PCR product depends on the length and base composition of the product. So it is not possible to compare the concentrations of two different templates without having control templates of known concentration for each target DNA region.	+	*When using [[SYBR Green I]], the amount of fluorescence in a PCR product depends on the length and base composition of the product. So it is not possible to compare the concentrations of two different templates without having control templates of known concentration for each target DNA region.

	== See also ==		== See also ==