QRT-PCR - Revision history

Philip Zimmermann: /* Reference genes */

Philip Zimmermann — Mon, 05 Jul 2010 06:46:58 GMT

Reference genes

←Older revision		Revision as of 06:46, 5 July 2010
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	=== Reference genes ===		=== Reference genes ===
-	Most common method. Frequently, a panel is used for normalization, e.g. [http://www.bioline.com/h_prod_detail.asp?user_prodname=Human%20Endogenous%20Control%20Gene%20Panel] not just a single reference gene and including data on suitability as reference genes. Often ''housekeeping gene''  is used here instead of reference gene but the term is poorly defined and can be misleading. It is to be noted that panels are often composed of genes that are supposed to be stable based on their function. However, more than 100 peer-reviewed articles report problems related to genes chosen from a panel, because they were not suitable for a particular context. A recent approach is to select a reference gene based on its stability across microarrays done within one's condition of interest. There is a public tool called RefGenes that searches a microarray database of more than 50,000 arrays to identify genes that are stable across subsets of conditions. It is available at the Genevestigator website [www.genevestigator.com].	+	Most common method. Frequently, a panel is used for normalization, e.g. [http://www.bioline.com/h_prod_detail.asp?user_prodname=Human%20Endogenous%20Control%20Gene%20Panel] not just a single reference gene and including data on suitability as reference genes. Often ''housekeeping gene''  is used here instead of reference gene but the term is poorly defined and can be misleading. It is to be noted that panels are often composed of genes that are supposed to be stable based on their function. However, more than 100 peer-reviewed articles report problems related to genes chosen from a panel, because they were not suitable for a particular context. A recent approach is to select a reference gene based on its stability across microarrays done within one's condition of interest. There is a public tool called RefGenes that searches a microarray database of more than 50,000 arrays to identify genes that are stable across subsets of conditions. It is available at the Genevestigator website [http://www.genevestigator.com].

	=== RNA ===		=== RNA ===

Philip Zimmermann: /* Reference genes */

Philip Zimmermann — Mon, 05 Jul 2010 06:46:35 GMT

Reference genes

←Older revision		Revision as of 06:46, 5 July 2010
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	=== Reference genes ===		=== Reference genes ===
-	Most common method. Frequently, a panel is used for normalization, e.g. [http://www.bioline.com/h_prod_detail.asp?user_prodname=Human%20Endogenous%20Control%20Gene%20Panel] not just a single reference gene and including data on suitability as reference genes. Often ''housekeeping gene''  is used here instead of reference gene but the term is poorly defined and can be misleading. It is to be noted that panels are often composed of genes that are supposed to be stable based on their function. However, more than 100 peer-reviewed articles report problems related to genes chosen from a panel, because they were not suitable for a particular context. A recent approach is to select a reference gene based on its stability across microarrays done within one's condition of interest. There is a public tool called RefGenes that searches a microarray database of more than 50,000 arrays to identify genes that are stable across subsets of conditions. It is available at the Genevestigator website.	+	Most common method. Frequently, a panel is used for normalization, e.g. [http://www.bioline.com/h_prod_detail.asp?user_prodname=Human%20Endogenous%20Control%20Gene%20Panel] not just a single reference gene and including data on suitability as reference genes. Often ''housekeeping gene''  is used here instead of reference gene but the term is poorly defined and can be misleading. It is to be noted that panels are often composed of genes that are supposed to be stable based on their function. However, more than 100 peer-reviewed articles report problems related to genes chosen from a panel, because they were not suitable for a particular context. A recent approach is to select a reference gene based on its stability across microarrays done within one's condition of interest. There is a public tool called RefGenes that searches a microarray database of more than 50,000 arrays to identify genes that are stable across subsets of conditions. It is available at the Genevestigator website [www.genevestigator.com].

	=== RNA ===		=== RNA ===

Philip Zimmermann: /* Reference genes */

Philip Zimmermann — Mon, 05 Jul 2010 06:45:33 GMT

Reference genes

←Older revision		Revision as of 06:45, 5 July 2010
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	=== Reference genes ===		=== Reference genes ===
-	Most common method. ~~Best practise is~~ a panel, e.g. [http://www.bioline.com/h_prod_detail.asp?user_prodname=Human%20Endogenous%20Control%20Gene%20Panel] not just a single reference gene and including data on suitability as reference genes. Often ''housekeeping gene''  is used here instead of reference gene but the term is poorly defined and can be misleading.	+	Most common method. Frequently, a panel is used for normalization, e.g. [http://www.bioline.com/h_prod_detail.asp?user_prodname=Human%20Endogenous%20Control%20Gene%20Panel] not just a single reference gene and including data on suitability as reference genes. Often ''housekeeping gene''  is used here instead of reference gene but the term is poorly defined and can be misleading. It is to be noted that panels are often composed of genes that are supposed to be stable based on their function. However, more than 100 peer-reviewed articles report problems related to genes chosen from a panel, because they were not suitable for a particular context. A recent approach is to select a reference gene based on its stability across microarrays done within one's condition of interest. There is a public tool called RefGenes that searches a microarray database of more than 50,000 arrays to identify genes that are stable across subsets of conditions. It is available at the Genevestigator website.

	=== RNA ===		=== RNA ===

Torsten Waldminghaus at 17:29, 23 February 2009

Torsten Waldminghaus — Mon, 23 Feb 2009 17:29:36 GMT

←Older revision		Revision as of 17:29, 23 February 2009
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		+	{{back to protocols}}
	'''Quantitative reverse transcriptase PCR''' (QRT-PCR or qRT-PCR) is a [[PCR techniques\|PCR technique]] used to determine the amount of cDNA in a sample. It is the most commonly used form of [[quantitative PCR]] (qPCR). This technique is also called ''real-time reverse transcriptase PCR''.		'''Quantitative reverse transcriptase PCR''' (QRT-PCR or qRT-PCR) is a [[PCR techniques\|PCR technique]] used to determine the amount of cDNA in a sample. It is the most commonly used form of [[quantitative PCR]] (qPCR). This technique is also called ''real-time reverse transcriptase PCR''.

Jakob Suckale: /* External links */

Jakob Suckale — Wed, 14 May 2008 17:34:51 GMT

External links

←Older revision		Revision as of 17:34, 14 May 2008
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	* [[Image:3stars.png]] excellent, detailed [http://pathmicro.med.sc.edu/pcr/realtime-home.htm qPCR tutorial] with many figures by Margaret and Richard Hunt, University of South Carolina		* [[Image:3stars.png]] excellent, detailed [http://pathmicro.med.sc.edu/pcr/realtime-home.htm qPCR tutorial] with many figures by Margaret and Richard Hunt, University of South Carolina
	* [[Image:3stars.png]] detailed [http://qcom.etsu.edu/mbcf/pcr.htm qPCR tutorial] by East Tennessee State University facility		* [[Image:3stars.png]] detailed [http://qcom.etsu.edu/mbcf/pcr.htm qPCR tutorial] by East Tennessee State University facility
		+	* [[Image:3stars.png]] excellent, up-to-date [http://www.dddmag.com/reliability-of-qPCR-data.aspx discussion on how to do qPCR experiments] with Maurice Exner, Mark Anderson, and Stephen Bustin
	* [[Image:2stars.png]] [http://www.gene-quantification.info/ gene-quantification.info - a site dedicated to qPCR] edited by M W Pfaffl, Technical University Munich (very busy design, lots of advertisement but a real trove of qPCR info)		* [[Image:2stars.png]] [http://www.gene-quantification.info/ gene-quantification.info - a site dedicated to qPCR] edited by M W Pfaffl, Technical University Munich (very busy design, lots of advertisement but a real trove of qPCR info)
	* [[Image:2stars.png]] [http://dorakmt.tripod.com/genetics/realtime.html qPCR (real-time PCR) backgrounder] without figures but with many links by Tevfik Dorak		* [[Image:2stars.png]] [http://dorakmt.tripod.com/genetics/realtime.html qPCR (real-time PCR) backgrounder] without figures but with many links by Tevfik Dorak

Jakob Suckale: /* qPCR data quality */

Jakob Suckale — Wed, 14 May 2008 17:17:47 GMT

qPCR data quality

←Older revision		Revision as of 17:17, 14 May 2008
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	both experiments above from [Bustin 2002 PMID 12200227, figure 4]		both experiments above from [Bustin 2002 PMID 12200227, figure 4]
		+
		+	=== More information ===
		+	* [http://www.dddmag.com/reliability-of-qPCR-data.aspx Partial transcript of a webcast discussing qPCR data quality]

	== Notes ==		== Notes ==

Jakob Suckale: /* Sources of variability */

Jakob Suckale — Wed, 14 May 2008 17:15:32 GMT

Sources of variability

←Older revision		Revision as of 17:15, 14 May 2008
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	Efficiency (and C<sub>t</sub> values) can also be calculated from the fluorescence data of a single PCR run or preferably replicates of the same PCR. The '''Miner''' algorithm (PMID 16241897) is an example for this type of method and can be used online at [http://www.miner.ewindup.info/miner/].		Efficiency (and C<sub>t</sub> values) can also be calculated from the fluorescence data of a single PCR run or preferably replicates of the same PCR. The '''Miner''' algorithm (PMID 16241897) is an example for this type of method and can be used online at [http://www.miner.ewindup.info/miner/].

-	== ~~Sources of variability~~ ==	+	== qPCR data quality ==
-	=== Operator ===	+
		+	=== Sources of variability: Operator ===
		+
	Due to the small amount of liquid handled and the sensitivity of the technique, operator variability is high. Bustin reports that the same qPCR experiment repeated by 3 people using the same reagents lead to very different copy number estimations [Bustin 2002 PMID 12200227, figure 3]:		Due to the small amount of liquid handled and the sensitivity of the technique, operator variability is high. Bustin reports that the same qPCR experiment repeated by 3 people using the same reagents lead to very different copy number estimations [Bustin 2002 PMID 12200227, figure 3]:
	* person A: 8·7 × 10<sup>5</sup>		* person A: 8·7 × 10<sup>5</sup>
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	* person C: 2·7 × 10<sup>3</sup> different by a factor of 300!!		* person C: 2·7 × 10<sup>3</sup> different by a factor of 300!!

-	=== Reagent lots/age ===	+	=== Sources of variability: Reagent lots/age ===
		+
	Different lots of reagents can lead to different results. Experiment repeated by same operator 5 times, same RNA sample, different kits; values are copies/μg total RNA:		Different lots of reagents can lead to different results. Experiment repeated by same operator 5 times, same RNA sample, different kits; values are copies/μg total RNA:
	* kit 1: 13±32 × 10<sup>7</sup>		* kit 1: 13±32 × 10<sup>7</sup>

Jakob Suckale: /* Linear regression on dilution curve C_t data */

Jakob Suckale — Wed, 14 May 2008 17:12:56 GMT

Linear regression on dilution curve C<sub>t</sub> data

←Older revision		Revision as of 17:12, 14 May 2008
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	:'''efficiency = 10^(-1/slope)'''		:'''efficiency = 10^(-1/slope)'''

-	Slopes between -3.3 and -4 will thus give you estimated primer efficiencies between 100% and 78% respectively. It can happen that the calculated efficiencies are above 100% [http://www.protocol-online.org/forums/index.php?showtopic=22170] [http://www.protocol-online.org/forums/index.php?showtopic=28319]. This may be due to incorrect template concentrations, too concentrated template, inhibition of the PCR reaction, unspecific PCR amplification, mistakes in the calculation [http://www.protocol-online.org/forums/index.php?showtopic=29925], etc.	+	Slopes between -3.3 and -4 will thus give you estimated primer efficiencies between 100% and 78% respectively. It can happen that the calculated efficiencies are above 100% [http://www.protocol-online.org/forums/index.php?showtopic=7611] [http://www.protocol-online.org/forums/index.php?showtopic=22170] [http://www.protocol-online.org/forums/index.php?showtopic=28319]. This may be due to incorrect template concentrations, too concentrated template, inhibition of the PCR reaction, unspecific PCR amplification, mistakes in the calculation [http://www.protocol-online.org/forums/index.php?showtopic=29925], etc.

	See a figure explaining the fitting process from the Hunts' qPCR tutorial [http://pathmicro.med.sc.edu/pcr/PCR_Standard_Curve2.htm].		See a figure explaining the fitting process from the Hunts' qPCR tutorial [http://pathmicro.med.sc.edu/pcr/PCR_Standard_Curve2.htm].

Jakob Suckale: /* References */

Jakob Suckale — Wed, 14 May 2008 16:20:48 GMT

References

←Older revision		Revision as of 16:20, 14 May 2008
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	</biblio>		</biblio>

-	[[Category:Protocol]] [[Category:In vitro]] [[Category:RNA]]	+	[[Category:Protocol]] [[Category:In vitro]] [[Category:RNA]] [[Category:DNA]]

Jakob Suckale: /* Efficiency */

Jakob Suckale — Wed, 14 May 2008 16:18:48 GMT

Efficiency

←Older revision		Revision as of 16:18, 14 May 2008
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	(Ct = cycle threshold; point when fluorescence reading surpasses a set baseline)		(Ct = cycle threshold; point when fluorescence reading surpasses a set baseline)

-	== ~~Efficiency~~ ==	+	== Primer efficiency estimation ==
		+
	A Ct difference of 1 between two samples has a different meaning depending on the efficiency of the primers used. If primers are 100% efficient, then ΔC<sub>t</sub> = 1 means one sample has twice the amount of template compared to the other. The simple ΔΔC<sub>t</sub> method, described above, often wrongly assumes perfect efficiency. It is better to experimentally verify the primer efficiency and use the Pfaffl method instead. The standard method takes the primer efficiency into account via the standard curve run with each sample. However, primer efficiencies in the standard curve dilutions and the actual samples are not necessarily the same.		A Ct difference of 1 between two samples has a different meaning depending on the efficiency of the primers used. If primers are 100% efficient, then ΔC<sub>t</sub> = 1 means one sample has twice the amount of template compared to the other. The simple ΔΔC<sub>t</sub> method, described above, often wrongly assumes perfect efficiency. It is better to experimentally verify the primer efficiency and use the Pfaffl method instead. The standard method takes the primer efficiency into account via the standard curve run with each sample. However, primer efficiencies in the standard curve dilutions and the actual samples are not necessarily the same.

QRT-PCR - Revision history

Philip Zimmermann: /* Reference genes */

Philip Zimmermann: /* Reference genes */

Philip Zimmermann: /* Reference genes */

Torsten Waldminghaus at 17:29, 23 February 2009

Jakob Suckale: /* External links */

Jakob Suckale: /* qPCR data quality */

Jakob Suckale: /* Sources of variability */

Jakob Suckale: /* Linear regression on dilution curve Ct data */

Jakob Suckale: /* References */

Jakob Suckale: /* Efficiency */

Jakob Suckale: /* Linear regression on dilution curve C_t data */