Important papers and references for real-time PCR

[Suzanne]

Hi,
I thought it might be helpful to list here references for real-time PCR that all of us might want. These are in no particular order. The link to the article is here if it is free online. You may need to copy and paste the link into your browser to go to the paper.


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RT-PCR and Negative Control Issues, "Monte Carlo Effect"

Pitfalls of Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction
Stephen A Bustin and Tania Nolan
J. Biomol. Tech., Sep 2004; 15: 155 - 166.
http://www.ncbi.nlm.nih.gov/pubmed/15331581

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General Help and Quantification Strategies

Critical Factors for Successful Real-Time PCR
http://www1.qiagen.com/literature/brochures/pcr/QT/1037490_AG_PCR_0206_Int_lr.pdf

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SYBR Green and Melt Curves

Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis.
PT Monis, S Giglio, and CP Saint

Anal Biochem, May 2005; 340(1): 24-34.

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Housekeeping Gene Selection

Guideline to reference gene selection for quantitative real-time PCR
Aleksandar Radonic,a Stefanie Thulke, Ian M. Mackay, Olfert Landt,
Wolfgang Siegert, and Andreas Nitschea

Biochemical and Biophysical Research Communications 313 (2004) 856–862.

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Equivalence test in quantitative reverse transcription polymerase chain reaction: confirmation of reference genes suitable for normalization.
F Haller, B Kulle, S Schwager, B Gunawan, A von Heydebreck, H Sultmann, and L Fuzesi

Anal Biochem, Dec 2004; 335(1): 1-9.[/u]

[Suzanne]

This paper by Ian (Iteo) discusses the importance of carrier DNA in samples with low copy numbers:

LightCycler qPCR optimisation for low copy number target DNA.
IA Teo, JW Choi, J Morlese, G Taylor, and S Shaunak

J Immunol Methods, Dec 2002; 270(1): 119-33.

This paper can't be linked, so if you want a copy, you'll have to email Ian.

Suzanne

[Suzanne]

Fluorescent Dyes and Quencher Combinations;

http://www.bio.umass.edu/biology/genomics/files/Which_Quencher.pdf

[Suzanne]

GREAT SITE

Calculator for determining the number of copies of a template

http://www.uri.edu/research/gsc/resources/cndna.html


Suzanne

[Suzanne]

This paper uses real-time PCR to assay 2-fold gene expression changes (TaqMan-based qRT-PCR)

Real-time quantitative PCR analysis can be used as a primary screen to identify patients with CML treated with imatinib who have BCR-ABL kinase domain mutations.

Susan Branford, Zbigniew Rudzki, Ian Parkinson, Andrew Grigg, Kerry Taylor, John F. Seymour, Simon Durrant, Peter Browett, Anthony P. Schwarer, Chris Arthur, John Catalano, Michael F. Leahy, Robin Filshie, Kenneth Bradstock, Richard Herrmann, David Joske, Kevin Lynch, and Tim Hughes

Blood, Nov 2004; 104: 2926 - 2932.

[Suzanne]

Check out this great review and other significant real-time PCR articles in the July 2005 BioTechniques. You can view these free online by signing up on the website:

Real-time PCR for mRNA quantitation
Marisa L. Wong and Juan F. Medrano
BioTechniques Vol. 39, No. 1: pp 75-85 (July 2005)


Simple enzymatic means to neutralize DNA contamination in nucleic acid amplification
John Ashkenas, James W. Dennis, and Chi Yip Ho
BioTechniques Vol. 39, No. 1: pp 69-73 (July 2005)


Impact of RNA quality on reference gene expression stability
Claudina Angela Pérez-Novo, Cindy Claeys, Frank Speleman, Paul Van Cauwenberge, Claus Bachert, and Jo Vandesompele
BioTechniques Vol. 39, No. 1: pp 52-56 (July 2005)

[Suzanne]

References for using multiple housekeeping genes for qPCR normalization

1. Jo Vandesompele, Katleen De Preter, Filip Pattyn, Bruce Poppe, Nadine Van Roy, Anne De Paepe and Frank Speleman
Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.
Genome Biology 2002, 3, RESEARCH 0034.
*This is the classic; it is available at
http://genomebiology.com/2002/3/7/research/0034.1

2. J. Huggett et al., 2005, Real-time Rt-PCR normalisation; strategies and
considerations. Genes and Immunity 6, 279.

[Suzanne]

New paper: Check it out in your February issue of BioTechniques!

Short Technical Reports

Comparison of in vitro and in vivo reference genes for internal standardization of real-time PCR data

Ralf Gilsbach, Mina Kouta, Heinz Bönisch, and Michael Brüss

BioTechniques® February 2006
Volume 40, Number 2: pp 173-177


Abstract


Real-time PCR is a powerful technique for gene expression studies, which have become increasingly important in a large number of clinical and scientific fields. The significance of the obtained results strongly depends on the normalization of the data to compensate for differences between the samples. The most widely used approach is to use endogenous reference genes (housekeeping genes) as internal standards. This approach is controversially discussed in the literature because none of the reference genes is stably expressed throughout all biological samples. Therefore, candidate reference genes have to be validated for each experimental condition. In our studies, we introduced and evaluated an in vitro synthesized reference cRNA for internal standardization of relative messenger RNA (mRNA) expression patterns. This reference, consisting of the in vitro transcribed coding sequence of aequorin, a jellyfish protein, was incorporated in the extracted RNA. The experimental significance of this approach was representatively tested for the expression of the neurotrophin-3 mRNA in distinct regions of mouse brains. A comparison to three stably expressed reference genes [β-actin, glyceralde-hyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine phosphoribosyl-transferase 1 (HPRT1)] gave evidence that the spiking of template RNA with in vitro transcribed cRNA is a valuable tool for internal standardization of real-time PCR experiments.

[mateusz]

Suzanne, you've done a graet job !!!

I would like to add 2 things to your list:

1)The real-time polymerase chain reaction. Review

by: M. Kubista et al
published in: Molecular Aspects of Medicine, 2006;27:95-125
available also free at:
http://www.molbiotech.chalmers.se/research/mk/publications/publications.htm

2)http://dorakmt.tripod.com/genetics/realtime.html

a great page on real time PCR with loads of links!!!

[hyperchicken]

Thank you for posting these links, they've been a great help when my PhD supervisors mysteriously vanish!

[Suzanne]

This paper gives equations for calculating the amount of change in real-time PCR using PCR efficiency.

A new mathematical model for relative quantification in real-time PCR
Nucleic Acids Research, 2001, Vol. 29, No. 900, pages 2002-2007

http://nar.oxfordjournals.org/cgi/reprint/29/9/e45


Best,
Suzanne

[Suzanne]

This is handy:

http://www.stratagene.com/techtoolbox/calc/qpcr_slope_eff.aspx

[Suzanne]

Hi All,
This looks to be very helpful!

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Together with MultiD we are developing tutorials on real-time PCR data
processing, including selection of reference genes, relative normalization,
and expression profiling by classification. They are available on:

http://www.multid.se/demonstration.html

Best

Mikael

<http://www.tataa.com/>
Mikael Kubista
TATAA Biocenter AB
Sahlgrenska Science Park
Medicinaregatan 8A
413 46 Göteborg
Sweden
Tel: +46 31 741 18 03
Mob: +46 730 794326 (Sweden)
Mob: +34 663308117 (Spain)

[Suzanne]

An attempt to produce a standardised rt-qpcr protocol has ben
published in Nature Protocols and can be downloaded from this URL

http://www.nature.com/nprot/journal/v1/ ... 06.236.pdf

Stephen A Bustin
www.sabustin.org

[gene.quantification]

This paper gives equations for calculating the amount of change in real-time PCR using PCR efficiency.

A new mathematical model for relative quantification in real-time PCR
MW Pfaffl, Nucleic Acids Research, 2001, Vol. 29, No. 900, pages 2002-2007
http://www.gene-quantification.de/pfaffl-et-al-nar-2002.pdf

Best,
Suzanne

Hello,
Further papers about RELATIVE QUANTIFICATION and EFFICIENCY CALCULATION in real-time PCR you find here:
=> http://relative.gene-quantification.info/
=> http://efficiency.gene-quantification.info/
=> http://REST.gene-quantification.info/
Updated REST software version => http://REST-2009.gene-quantification.info/
Best regards
Michael