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{{Redirect|RT-PCR|real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (qPCR) or kinetic polymerase chain reaction|real-time polymerase chain reaction}}
{{Redirect|RT-PCR|real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (qPCR) or kinetic polymerase chain reaction|real-time polymerase chain reaction}}


==Introduction==
'''Reverse transcription polymerase chain reaction''' ('''RT-PCR''') is a variant of [[polymerase chain reaction]] (PCR). It is a [[laboratory technique]] commonly used in [[molecular biology]] where a RNA strand is [[Reverse transcription|reverse transcribed]] into its [[DNA]] complement (''[[complementary DNA]]'', or ''cDNA'') using the enzyme [[reverse transcriptase]], and the resulting cDNA is amplified using PCR. Reverse transcription PCR is not to be confused with [[real-time polymerase chain reaction]] (Q-PCR/qRT-PCR), which is also sometimes abbreviated as RT-PCR.

'''Reverse transcription polymerase chain reaction''' ('''RT-PCR''') is one of many variants of [[polymerase chain reaction | polymerase chain reaction (PCR)]], in which the technique is commonly used in molecular biology to detect RNA expression levels.<ref name="pmid9894600">{{cite journal |author=Freeman WM, Walker SJ, Vrana KE |title=Quantitative RT-PCR: pitfalls and potential |journal=[[BioTechniques]] |volume=26 |issue=1 |pages=112–22, 124–5 |year=1999 |month=January |pmid=9894600 |doi= |url= |accessdate=2012-11-06}}</ref> RT-PCR is often interchanged with [[real-time polymerase chain reaction | real-time polymerase chain reaction (qPCR)]] by students and scientists alike.<ref>{{cite book |author=Mackay, Ian |title=Real-time PCR in Microbiology: From Diagnosis to Characterization |publisher=Caister Academic Press |location=Norfolk, England |year=2007 |pages=440 |isbn=1-904455-18-2 |oclc= |doi= |accessdate=}}</ref> However, they are separate and distinct techniques. While RT-PCR is used to qualitatively detect gene expression through creation of [[complementary DNA]] transcripts from RNA, qPCR is used to quantitatively measure the amplification of DNA using fluorescent probes. qPCR is also referred to as quantitative PCR,<ref>{{cite book |author=Mackay, Ian |title=Real-time PCR in Microbiology: From Diagnosis to Characterization |publisher=Caister Academic Press |location=Norfolk, England |year=2007 |pages=440 |isbn=1-904455-18-2 |oclc= |doi= |accessdate=}}</ref> quantitative real-time PCR,<ref name="pmid14706621">{{cite journal |author=Radonić A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A |title=Guideline to reference gene selection for quantitative real-time PCR |journal=[[Biochemical and Biophysical Research Communications]] |volume=313 |issue=4 |pages=856–62 |year=2004 |month=January |pmid=14706621 |doi= |url=http://linkinghub.elsevier.com/retrieve/pii/S0006291X03025646 |accessdate=2012-11-06}}</ref> and real-time quantitative PCR<ref name="pmid11846609">{{cite journal |author=Livak KJ, Schmittgen TD |title=Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method |journal=[[Methods (San Diego, Calif.)]] |volume=25 |issue=4 |pages=402–8 |year=2001 |month=December |pmid=11846609 |doi=10.1006/meth.2001.1262 |url=http://linkinghub.elsevier.com/retrieve/pii/S1046-2023(01)91262-9 |accessdate=2012-11-06}}</ref>.

Although RT-PCR and the traditional PCR both produce multiple copies of particular DNA isolates through amplification, the applications of the two techniques are fundamentally different. The traditional PCR is simply used to make billions of copies of given DNA sequences. RT-PCR is used to clone expressed genes by [[reverse transcription | reverse transcribing]] the RNA of interest into its DNA complement through the use of [[reverse transcriptase]]. Subsequently, the newly synthesized cDNA is amplified using traditional PCR.

In addition to qualitative study of gene expression, RT-PCR can be utilized for quantification of RNA, in both relative and absolute terms,<ref name="urlgroups.molbiosci.northwestern.edu">{{cite web |url=http://groups.molbiosci.northwestern.edu/morimoto/research/Protocols/IV.%20DNA/G.%20Amplification/2.%20quant.%20RT-PCR.pdf |title=groups.molbiosci.northwestern.edu |format= |work= |accessdate=2012-11-06}}</ref> by incorporating qPCR into the technique. The combined technique, described as quantitative RT-PCR<ref>{{cite doi | 10.1385/1-59259-283-X:083}}</ref> or real-time RT-PCR<ref name="pmid20515509">{{cite journal |author=Kang XP, Jiang T, Li YQ, ''et al.'' |title=A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus |journal=Virol. J. |volume=7 |issue= |pages=113 |year=2010 |pmid=20515509 |pmc=2892456 |doi=10.1186/1743-422X-7-113 |url=http://www.virologyj.com/content/7//113 |accessdate=2012-11-06}}</ref> (sometimes even quantitative real-time RT-PCR<ref name="pmid15956331">{{cite journal |author=Bustin SA, Benes V, Nolan T, Pfaffl MW |title=Quantitative real-time RT-PCR--a perspective |journal=J. Mol. Endocrinol. |volume=34 |issue=3 |pages=597–601 |year=2005 |month=June |pmid=15956331 |doi=10.1677/jme.1.01755 |url=http://jme.endocrinology-journals.org/cgi/pmidlookup?view=long&pmid=15956331 |accessdate=2012-11-06}}</ref>), is often abbreviated as qRT-PCR<ref name="pmid20204872">{{cite journal |author=Varkonyi-Gasic E, Hellens RP |title=qRT-PCR of Small RNAs |journal=Methods Mol. Biol. |volume=631 |issue= |pages=109–22 |year=2010 |pmid=20204872 |doi=10.1007/978-1-60761-646-7_10 |url=http://dx.doi.org/10.1007/978-1-60761-646-7_10 |accessdate=2012-11-06}}</ref>, RT-qPCR<ref name="pmid20215014">{{cite journal |author=Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M |title=A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines |journal=Methods |volume=50 |issue=4 |pages=S1–5 |year=2010 |month=April |pmid=20215014 |doi=10.1016/j.ymeth.2010.01.005 |url=http://linkinghub.elsevier.com/retrieve/pii/S1046-2023(10)00020-4 |accessdate=2012-11-06}}</ref>, or RRT-PCR<ref name="pmid12202562">{{cite journal |author=Spackman E, Senne DA, Myers TJ, ''et al.'' |title=Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes |journal=J. Clin. Microbiol. |volume=40 |issue=9 |pages=3256–60 |year=2002 |month=September |pmid=12202562 |pmc=130722 |doi= |url=http://jcm.asm.org/cgi/pmidlookup?view=long&pmid=12202562 |accessdate=2012-11-06}}</ref>. Compared to other RNA quantification methods, such as northern blot, qRT-PCR is considered to be the most powerful, sensitive, and quantitative assay for the detection of RNA levels. It is frequently used in the expression analysis of single or multiple genes, and expression patterns for identifying infections and diseases.<ref name="urlgroups.molbiosci.northwestern.edu">{{cite web |url=http://groups.molbiosci.northwestern.edu/morimoto/research/Protocols/IV.%20DNA/G.%20Amplification/2.%20quant.%20RT-PCR.pdf |title=groups.molbiosci.northwestern.edu |format= |work= |accessdate=2012-11-06}}</ref>

In order to avoid confusion, the following abbreviations will be used consistently throughout this article:

{| class="wikitable"
|-
! Technique !! Abbrebiation
|-
| Polymerase chain reaction || PCR
|-
| Reverse transcription polymerase chain reaction || RT-PCR
|-
| Real-time polymerase chain reaction|| qPCR
|-
| RT-PCR / qPCR combined technique || qRT-PCR

|}

==History==

Since the introduction of [[Northern blot]] in 1977, the technique has been used extensively for RNA quantification despite its shortcomings of: (a) being time-consuming, (b) requiring a large quantity of RNA for detection, and (c) being quantitatively inaccurate in the low abundance of RNA content.<ref name="pmid414220">{{cite journal |author=Alwine JC, Kemp DJ, Stark GR |title=Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes |journal=Proc. Natl. Acad. Sci. U.S.A. |volume=74 |issue=12 |pages=5350–4 |year=1977 |month=December |pmid=414220 |pmc=431715 |doi= |url= |accessdate=2012-11-06}}</ref><ref name="pmid19131955">{{cite journal |author=Streit S, Michalski CW, Erkan M, Kleeff J, Friess H |title=Northern blot analysis for detection and quantification of RNA in pancreatic cancer cells and tissues |journal=Nat Protoc |volume=4 |issue=1 |pages=37–43 |year=2009 |pmid=19131955 |doi=10.1038/nprot.2008.216 |url=http://dx.doi.org/10.1038/nprot.2008.216 |accessdate=2012-11-06}}</ref> With the discovery of [[reverse transcriptase]] during the study of viral replication of genetic material, the development of reverse transcription polymerase chain reaction (RT-PCR) was possible, and since then, it has become the method of choice for RNA detection and quantification as an alternative to [[Northern blot]]<ref name="pmid11013345">{{cite journal |author=Bustin SA |title=Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays |journal=J. Mol. Endocrinol. |volume=25 |issue=2 |pages=169–93 |year=2000 |month=October |pmid=11013345 |doi= |url=http://jme.endocrinology-journals.org/cgi/pmidlookup?view=long&pmid=11013345 |accessdate=2012-11-06}}</ref>. However, the more sensitive and reproducible RT-PCR is not without a major flaw of its own. The exponential growth of the reverse transcribed [[complementary DNA]] (cDNA) during the multiple cycles of PCR produces inaccurate end point quantification due to the difficulty in maintaining linearity<ref name="pmid14664723">{{cite journal |author=Shiao YH |title=A new reverse transcription-polymerase chain reaction method for accurate quantification |journal=BMC Biotechnol. |volume=3 |issue= |pages=22 |year=2003 |month=December |pmid=14664723 |pmc=317330 |doi=10.1186/1472-6750-3-22 |url=http://www.biomedcentral.com/1472-6750/3/22 |accessdate=2012-11-06}}</ref>. For accurate detection and quantification of RNA content in a sample, real-time RT-PCR was developed using fluorescence-based modification to monitor the amplification products during each cycle of PCR.
RT-PCR has risen to become the benchmark technology for the detection and/or comparison of RNA levels for several reasons: (a) it does not require post PCR processing ;(b) a wide range (>10^7 fold) of RNA abundance can be measured and ;(c) it provides insight into both qualitative and quantitative data.<ref name="pmid15956331">{{cite journal |author=Bustin SA, Benes V, Nolan T, Pfaffl MW |title=Quantitative real-time RT-PCR--a perspective |journal=J. Mol. Endocrinol. |volume=34 |issue=3 |pages=597–601 |year=2005 |month=June |pmid=15956331 |doi=10.1677/jme.1.01755 |url=http://jme.endocrinology-journals.org/cgi/pmidlookup?view=long&pmid=15956331 |accessdate=2012-11-06}}</ref> Due to its simplicity, specificity and sensitivity, RT-PCR is used in a wide range of applications from experiments as simple as quantification of yeast cells in wine to more complex uses as diagnostic tools for detecting infectious agents such as the avian flu virus.<ref name="pmid17088381">{{cite journal |author=Hierro N, Esteve-Zarzoso B, González A, Mas A, Guillamón JM |title=Real-time quantitative PCR (QPCR) and reverse transcription-QPCR for detection and enumeration of total yeasts in wine |journal=Appl. Environ. Microbiol. |volume=72 |issue=11 |pages=7148–55 |year=2006 |month=November |pmid=17088381 |pmc=1636171 |doi=10.1128/AEM.00388-06 |url=http://aem.asm.org/cgi/pmidlookup?view=long&pmid=17088381 |accessdate=2012-11-06}}</ref><ref name="pmid19627372">{{cite journal |author=Slomka MJ, Pavlidis T, Coward VJ, ''et al.'' |title=Validated RealTime reverse transcriptase PCR methods for the diagnosis and pathotyping of Eurasian H7 avian influenza viruses |journal=Influenza Other Respi Viruses |volume=3 |issue=4 |pages=151–64 |year=2009 |month=July |pmid=19627372 |doi=10.1111/j.1750-2659.2009.00083.x |url= |accessdate=2012-11-06}}</ref>


==Uses==
==Uses==
Line 13: Line 40:
* [http://www.realtimeprimers.org Database of validated PCR primer sets] ([http://www.genengnews.com/bestofweb/list.aspx?iid=93 website critique])
* [http://www.realtimeprimers.org Database of validated PCR primer sets] ([http://www.genengnews.com/bestofweb/list.aspx?iid=93 website critique])
* [http://www.bio.davidson.edu/Courses/immunology/Flash/RT_PCR.html Animation to illustrate RT-PCR procedure, from Cold Spring Harbor Laboratory]
* [http://www.bio.davidson.edu/Courses/immunology/Flash/RT_PCR.html Animation to illustrate RT-PCR procedure, from Cold Spring Harbor Laboratory]

==References==
{{reflist}}



{{PCR}}
{{PCR}}

Revision as of 14:51, 12 November 2012

"RT-PCR" redirects here. For real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (qPCR) or kinetic polymerase chain reaction, see real-time polymerase chain reaction.

Introduction

Reverse transcription polymerase chain reaction (RT-PCR) is one of many variants of polymerase chain reaction (PCR), in which the technique is commonly used in molecular biology to detect RNA expression levels.[1] RT-PCR is often interchanged with real-time polymerase chain reaction (qPCR) by students and scientists alike.[2] However, they are separate and distinct techniques. While RT-PCR is used to qualitatively detect gene expression through creation of complementary DNA transcripts from RNA, qPCR is used to quantitatively measure the amplification of DNA using fluorescent probes. qPCR is also referred to as quantitative PCR,[3] quantitative real-time PCR,[4] and real-time quantitative PCR[5].

Although RT-PCR and the traditional PCR both produce multiple copies of particular DNA isolates through amplification, the applications of the two techniques are fundamentally different. The traditional PCR is simply used to make billions of copies of given DNA sequences. RT-PCR is used to clone expressed genes by reverse transcribing the RNA of interest into its DNA complement through the use of reverse transcriptase. Subsequently, the newly synthesized cDNA is amplified using traditional PCR.

In addition to qualitative study of gene expression, RT-PCR can be utilized for quantification of RNA, in both relative and absolute terms,[6] by incorporating qPCR into the technique. The combined technique, described as quantitative RT-PCR[7] or real-time RT-PCR[8] (sometimes even quantitative real-time RT-PCR[9]), is often abbreviated as qRT-PCR[10], RT-qPCR[11], or RRT-PCR[12]. Compared to other RNA quantification methods, such as northern blot, qRT-PCR is considered to be the most powerful, sensitive, and quantitative assay for the detection of RNA levels. It is frequently used in the expression analysis of single or multiple genes, and expression patterns for identifying infections and diseases.[6]

In order to avoid confusion, the following abbreviations will be used consistently throughout this article:

Technique Abbrebiation
Polymerase chain reaction PCR
Reverse transcription polymerase chain reaction RT-PCR
Real-time polymerase chain reaction qPCR
RT-PCR / qPCR combined technique qRT-PCR

History

Since the introduction of Northern blot in 1977, the technique has been used extensively for RNA quantification despite its shortcomings of: (a) being time-consuming, (b) requiring a large quantity of RNA for detection, and (c) being quantitatively inaccurate in the low abundance of RNA content.[13][14] With the discovery of reverse transcriptase during the study of viral replication of genetic material, the development of reverse transcription polymerase chain reaction (RT-PCR) was possible, and since then, it has become the method of choice for RNA detection and quantification as an alternative to Northern blot[15]. However, the more sensitive and reproducible RT-PCR is not without a major flaw of its own. The exponential growth of the reverse transcribed complementary DNA (cDNA) during the multiple cycles of PCR produces inaccurate end point quantification due to the difficulty in maintaining linearity[16]. For accurate detection and quantification of RNA content in a sample, real-time RT-PCR was developed using fluorescence-based modification to monitor the amplification products during each cycle of PCR. RT-PCR has risen to become the benchmark technology for the detection and/or comparison of RNA levels for several reasons: (a) it does not require post PCR processing ;(b) a wide range (>10^7 fold) of RNA abundance can be measured and ;(c) it provides insight into both qualitative and quantitative data.[9] Due to its simplicity, specificity and sensitivity, RT-PCR is used in a wide range of applications from experiments as simple as quantification of yeast cells in wine to more complex uses as diagnostic tools for detecting infectious agents such as the avian flu virus.[17][18]

Uses

The exponential amplification via reverse transcription polymerase chain reaction provides for a highly sensitive technique in which a very low copy number of RNA molecules can be detected. RT-PCR is widely used in the diagnosis of genetic diseases and, semiquantitatively, in the determination of the abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression. Northern blot analysis is used to study the RNA's gene expression further. RT-PCR can also be very useful in the insertion of eukaryotic genes into prokaryotes. Because most eukaryotic genes contain introns which are present in the genome but not in the mature mRNA, the cDNA generated from a RT-PCR reaction is the exact (without regard to the error prone nature of reverse transcriptases) DNA sequence which would be directly translated into protein after transcription. When these genes are expressed in prokaryotic cells for the sake of protein production or purification, the RNA produced directly from transcription need not undergo splicing as the transcript contains only exons. (Prokaryotes, such as E. coli, lack the mRNA splicing mechanism of eukaryotes).

RT-PCR is commonly used in studying the genomes of viruses whose genomes are composed of RNA, such as Influenzavirus A and retroviruses like HIV.

External links

References

  1. ^ Freeman WM, Walker SJ, Vrana KE (1999). "Quantitative RT-PCR: pitfalls and potential". BioTechniques. 26 (1): 112–22, 124–5. PMID 9894600. {{cite journal}}: |access-date= requires |url= (help); Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  2. ^ Mackay, Ian (2007). Real-time PCR in Microbiology: From Diagnosis to Characterization. Norfolk, England: Caister Academic Press. p. 440. ISBN 1-904455-18-2.
  3. ^ Mackay, Ian (2007). Real-time PCR in Microbiology: From Diagnosis to Characterization. Norfolk, England: Caister Academic Press. p. 440. ISBN 1-904455-18-2.
  4. ^ Radonić A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A (2004). "Guideline to reference gene selection for quantitative real-time PCR". Biochemical and Biophysical Research Communications. 313 (4): 856–62. PMID 14706621. Retrieved 2012-11-06. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  5. ^ Livak KJ, Schmittgen TD (2001). "Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method". Methods (San Diego, Calif.). 25 (4): 402–8. doi:10.1006/meth.2001.1262. PMID 11846609. Retrieved 2012-11-06. {{cite journal}}: Unknown parameter |month= ignored (help)
  6. ^ a b "groups.molbiosci.northwestern.edu" (PDF). Retrieved 2012-11-06.
  7. ^ Attention: This template ({{cite doi}}) is deprecated. To cite the publication identified by doi: 10.1385/1-59259-283-X:083, please use {{cite journal}} (if it was published in a bona fide academic journal, otherwise {{cite report}} with |doi= 10.1385/1-59259-283-X:083 instead.
  8. ^ Kang XP, Jiang T, Li YQ; et al. (2010). "A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus". Virol. J. 7: 113. doi:10.1186/1743-422X-7-113. PMC 2892456. PMID 20515509. Retrieved 2012-11-06. {{cite journal}}: Explicit use of et al. in: |author= (help)CS1 maint: multiple names: authors list (link) CS1 maint: unflagged free DOI (link)
  9. ^ a b Bustin SA, Benes V, Nolan T, Pfaffl MW (2005). "Quantitative real-time RT-PCR--a perspective". J. Mol. Endocrinol. 34 (3): 597–601. doi:10.1677/jme.1.01755. PMID 15956331. Retrieved 2012-11-06. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  10. ^ Varkonyi-Gasic E, Hellens RP (2010). "qRT-PCR of Small RNAs". Methods Mol. Biol. 631: 109–22. doi:10.1007/978-1-60761-646-7_10. PMID 20204872. Retrieved 2012-11-06.
  11. ^ Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M (2010). "A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines". Methods. 50 (4): S1–5. doi:10.1016/j.ymeth.2010.01.005. PMID 20215014. Retrieved 2012-11-06. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  12. ^ Spackman E, Senne DA, Myers TJ; et al. (2002). "Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes". J. Clin. Microbiol. 40 (9): 3256–60. PMC 130722. PMID 12202562. Retrieved 2012-11-06. {{cite journal}}: Explicit use of et al. in: |author= (help); Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  13. ^ Alwine JC, Kemp DJ, Stark GR (1977). "Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes". Proc. Natl. Acad. Sci. U.S.A. 74 (12): 5350–4. PMC 431715. PMID 414220. {{cite journal}}: |access-date= requires |url= (help); Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  14. ^ Streit S, Michalski CW, Erkan M, Kleeff J, Friess H (2009). "Northern blot analysis for detection and quantification of RNA in pancreatic cancer cells and tissues". Nat Protoc. 4 (1): 37–43. doi:10.1038/nprot.2008.216. PMID 19131955. Retrieved 2012-11-06.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  15. ^ Bustin SA (2000). "Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays". J. Mol. Endocrinol. 25 (2): 169–93. PMID 11013345. Retrieved 2012-11-06. {{cite journal}}: Unknown parameter |month= ignored (help)
  16. ^ Shiao YH (2003). "A new reverse transcription-polymerase chain reaction method for accurate quantification". BMC Biotechnol. 3: 22. doi:10.1186/1472-6750-3-22. PMC 317330. PMID 14664723. Retrieved 2012-11-06. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: unflagged free DOI (link)
  17. ^ Hierro N, Esteve-Zarzoso B, González A, Mas A, Guillamón JM (2006). "Real-time quantitative PCR (QPCR) and reverse transcription-QPCR for detection and enumeration of total yeasts in wine". Appl. Environ. Microbiol. 72 (11): 7148–55. doi:10.1128/AEM.00388-06. PMC 1636171. PMID 17088381. Retrieved 2012-11-06. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  18. ^ Slomka MJ, Pavlidis T, Coward VJ; et al. (2009). "Validated RealTime reverse transcriptase PCR methods for the diagnosis and pathotyping of Eurasian H7 avian influenza viruses". Influenza Other Respi Viruses. 3 (4): 151–64. doi:10.1111/j.1750-2659.2009.00083.x. PMID 19627372. {{cite journal}}: |access-date= requires |url= (help); Explicit use of et al. in: |author= (help); Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)


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