Important papers and references for real-time PCR

Use this category for questions related to various quantitative PCR methods, including real time PCR, reverse transcription (RT)/PCR, qPCR, etc. For general PCR methods, continue to use the DNA/General PCR forum

Moderators: r.rosati, Suzanne

Re: Academic & Industrial Information Platform for qPCR

Postby gene.quantification » Feb 02 2007 4:25 pm

Image
http://infoportal.gene-quantification.info/


Academic & Industrial Information Platform for qPCR
The page describes and summarises all technical aspects involved in quantitative gene expression analysis using real-time qPCR & qRT-PCR. It presents a lot of cyclers, kits, dyes, chemistries, and services involved. Companies and institutions can present their qPCR technologies and services right here.

VERY HOT PAPER:
The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments.
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT.
Clin Chem. 2009 Feb 26.
http://MIQE.gene-quantification.info - latest MIQE press review on - http://MIQE-press.Gene-Quantification.info


A lot of interesting topics are grouped in qPCR themes:

http://qPCR.gene-quantification.info real-time PCR summary / overview of interesting qPCR papers
http://NEWS.gene-quantification.info NEWS on http://www.Gene-Quantification.Info pages
http://qPCRnews.gene-quantification.info MONTHLY NEWSLETTER of the Gene Quantification pages
http://strategy.gene-quantification.info Quantification strategies in qRT-PCR:
- http://absolute.gene-quantification.info Absolute Quantification in real-time PCR
- http://relative.gene-quantification.info Relative Quantification in real-time PCR

UPDATED REST software version (Dec 2009) => http://REST-2009.gene-quantification.info/

http://normalisation.gene-quantification.info Normalisation strategies & Housekeeping Genes & multiple HKGs
http://optimisation.gene-quantification.info Optimisation of reaction setup and qPCR procedure - updated
http://bioinformatics.gene-quantification.info software, REST, DATAN, qBase, algorithm, primers, statistics
http://events.gene-quantification.info Meetings, Symposia, Seminars, workshops: qPCR 2010 in Vienna, qPCR 2009, qPCR 2007, qPCR 2005, qPCR 2004 inFreising, Germany
http://cyclers.gene-quantification.info real-time PCR machines updated
http://RT.gene-quantification.info reverse transcription mRNA transcript analysis
http://dyes.gene-quantification.info detection dyes and chemistries in real-time PCR
http://efficiency.gene-quantification.info determination of real-time qPCR efficiency

----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Image

Call for TALK & POSTER Abstracts -- deadline 31. Jan 2017
qPCR dPCR & NGS 2017
8th international Gene Quantification Event
Symposium & Industrial Exhibition & Application Workshops
#GQ2017

Main topics: Liquid Biopsy, Integrative Big Data Analysis, Biomarker Signature … and beyond
3 - 7 April 2017, in Freising-Weihenstephan,
School of Life Sciences, Technical University of Munich, Weihenstephan, Germany
http://call.qPCR-dPCR-NGS-2017.net

Keynote Speakers & Event Announcement => http://Announcement.qPCR-dPCR-NGS-2017.net
Confirmed Speakers & Talk Titels => http://Talks.qPCR-dPCR-NGS-2017.net
Workshop topics & Workshop short agendas => http://Workshops.qPCR-dPCR-NGS-2017.net

Scientific Symposium Sessions:
• Liquid Biopsy & Circulating Nucleic Acids
• Advanced Molecular Diagnostics
• Integrative Big Data Analysis
• Biomarker Signatures
• Digital PCR
• Non-coding RNAs -- microRNA, isomiRs, small RNAs, long non-coding RNAs
• MicroGenomics & Single-Cells Diagnostics
• Next Generation Sequencing (NGS)
• Molecular Diagnostics in Life Science
• MIQE & QM & Standardisation Strategies in Molecular Diagnostics

Register and submit your scientific contribution (talk or poster abstracts) => http://Registration.qPCR-dPCR-NGS-2017.net
Event location on Google maps https://goo.gl/maps/6d3BNCyUkKk
Contact us [email protected]

----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Image

Join our new eConferences streaming webportal
eConferences - Amplify your knowledge!


http://www.eConferences.de - Free access to all talk streams and poster PDFs !

This portal is dedicated to scientists from the community of qPCR, digital-PCR, NGS and Molecular Diagnostics. You’ll find here all the records from around 200 presentations held at qPCR & NGS Events in the past years – qPCR 2010 in Vienna, qPCR 2011 and qPCR & NGS 2013 in Freising-Weihenstephan.
We provide the presentations via movie streaming technology in high quality - high resolution and perfect sound quality in high speed - on any internet browser or mobile device, including Android or iOS.

Specialized media channel topics: MIQE guidelines qPCR data analysis and digital PCR
- MIQE guidelines http://www.econferences.de/miqe-talks/
- qPCR data analysis http://www.econferences.de/data-analysis-all/
- digital PCR http://www.econferences.de/digital-pcr-talks/

Follow our Partner Channels => http://www.econferences.de/partner-channels/

Enjoy the presentations!
Last edited by gene.quantification on Dec 29 2016 8:57 am, edited 28 times in total.
www.GENE-QUANTIFICATION.info main focus is quantitative gene expression analysis - qPCR dPCR & NGS Events http://Events.Gene-Quantification.info - Talk Streaming Server www.eConferences.de - qPCR NEWS http://qPCRnews.Gene-Quantification.info
gene.quantification
PI of Posters
PI of Posters
 
Posts: 351
Joined: Dec 18 2002 11:43 am
Location: Germany , Bavaria, Freising

Re: Important papers and references for real-time PCR

Postby gene.quantification » Feb 02 2007 4:27 pm

Suzanne wrote: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.


****************************

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

****************************

REVIEW: RNA integrity and the effect on the real-time qRT-PCR performance.

http://rna-integrity.gene-quantification.info/

Molecular Aspects of Medicine 27 (2006) 126–139
Simone Fleige & Michael W. Pfaffl
Physiology Weihenstephan, Center of Life and Food Sciences (ZIEL), Technical University of Munich, 85350 Freising, Germany, TATAA Biocenter Germany, Freising-Weihenstephan, Germany

***************************

General Help and Quantification Strategies

http://strategy.gene-quantification.info/

Chapter 3: Quantification strategies in real-time PCR by Michael W. Pfaffl
in
A-Z of quantitative PCR ( Editor: S.A. Bustin ) International University Line (IUL), La Jolla, CA, USA,

This chapter analyzes the quantification strategies in real-time RT-PCR and all corresponding markers of a successful real-time RT-PCR. The following aspects are describes in detail: RNA extraction, reverse transcription (RT), and general quantification strategies—absolute vs. relative quantification, real-time PCR efficiency calculation, data evaluation, automation of quantification, data normalization, and statistical comparison. The discussion turns into practical considerations with focus on specificity and sensitivity.

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

****************************

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.

****************************

Housekeeping Gene Selection

http://normalisation.gene-quantification.info/

New REST software version => http://REST-2008.gene-quantification.info/

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.

*****************

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.
Last edited by gene.quantification on Mar 30 2009 8:23 am, edited 4 times in total.
www.GENE-QUANTIFICATION.info main focus is quantitative gene expression analysis - qPCR dPCR & NGS Events http://Events.Gene-Quantification.info - Talk Streaming Server www.eConferences.de - qPCR NEWS http://qPCRnews.Gene-Quantification.info
gene.quantification
PI of Posters
PI of Posters
 
Posts: 351
Joined: Dec 18 2002 11:43 am
Location: Germany , Bavaria, Freising

Postby AaJHE » Mar 23 2007 7:15 pm

You guys did a great job!!

I would like to add one more link from Current Protocols in Molecular Biology (My favorite ~~):

UNIT 15.8
High-Throughput Real-Time Quantitative Reverse Transcription PCR

:arrow: http://www.mrw.interscience.wiley.com/e ... urrent/pdf
AaJHE
technician-in-training
technician-in-training
 
Posts: 8
Joined: Mar 23 2007 2:50 pm
Location: KY

Postby wendell008 » Dec 21 2008 10:56 am

Thank you very much!


wendell
Si Chuan agricultural university
wendell008
newcomer
newcomer
 
Posts: 3
Joined: Dec 21 2008 10:47 am
Location: Ya’an,Sichuan,China

Postby JMG » Jan 22 2009 12:20 am

Recent support has been given to the following idea as well:

A current publication on IJBS at:
http://www.ijbs.org/User/ContentFullTex ... rtPage=273
Last edited by JMG on Sep 15 2011 12:15 pm, edited 1 time in total.
JMG
PI of Posters
PI of Posters
 
Posts: 620
Joined: Dec 18 2008 10:13 pm

10 tips for consistent real-time PCR

Postby Suzanne » Feb 13 2009 3:19 pm

10 tips for consistent real-time PCR

To get more details about each point, read it at www.bitesizebio.com (click the link above)

1. Always mix the reagents well before use

2. Store primers in a buffer to protect their stability

3. Aliquot the primers to avoid excessive freeze/thawing (and contamination)

4. Use pipettors accurate for low volumes when making dilutions and adding template

5. Perform a standard curve for every new primer pair to check efficiency first

7. Follow the three room rule: separate the rooms for extraction, reaction set up, and cycler location.

8. Double check the cycling conditions are correct before starting the run

9. Dilute the template (less is more)

10. Make dilutions fresh - do not store dilute solutions unless using a carrier or low retention plastic tubes
http://www.mobio.com/blog
Visit The Culture Dish!
Suzanne
ModSquad
ModSquad
 
Posts: 999
Joined: Feb 13 2003 8:59 pm
Location: Southern California

Minimum Information for Publication of QPCR Experiments

Postby Suzanne » Mar 12 2009 8:04 pm

New paper:

The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments.

Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT.

Clin Chem. 2009 Feb 26.
Suzanne
ModSquad
ModSquad
 
Posts: 999
Joined: Feb 13 2003 8:59 pm
Location: Southern California

Postby JMG » Mar 12 2009 9:08 pm

Thanks Suzanne~!

There will also be another paper coming out shortly
which ties directly into the publication above:

Look for this one too:

"SURVEY AND SUMMARY
RDML: structured language and reporting guidelines for real-time
quantitative PCR data by Steve Lefever, Jan Hellemans, Filip
Pattyn, Daniel R. Przybylski, Chris Taylor, Rene´Geurts,
Andreas Untergasser and Jo Vandesompele, on behalf
of the RDML consortium."

The galley proof looks excellent.

JMG
JMG
PI of Posters
PI of Posters
 
Posts: 620
Joined: Dec 18 2008 10:13 pm

Features of Real-time PCR Platforms and qPCR Machines

Postby Suzanne » Apr 28 2009 1:14 pm

Features of Real-time PCR Platforms and qPCR Machines (and a side by side comparison):

http://www.horizonpress.com/pcr/qPCR-machines.html
http://www.mobio.com/blog
Visit The Culture Dish!
Suzanne
ModSquad
ModSquad
 
Posts: 999
Joined: Feb 13 2003 8:59 pm
Location: Southern California

Statistical Significance of qPCR

Postby adrien » May 28 2009 1:49 am

Who is teaching you cloning ?
adrien
technophile
technophile
 
Posts: 18
Joined: May 23 2008 2:01 am

Re: Important papers and references for real-time PCR

Postby JMG » Mar 02 2010 8:11 pm

I am posting these (Suzanne's particular) choices again because they should never be
forgotten as they still impact every move we make and every breath we take when we
consider which reference ("housekeeping") genes we use for our qPCR:

From 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.

...j
Last edited by JMG on Sep 15 2011 12:15 pm, edited 1 time in total.
JMG
PI of Posters
PI of Posters
 
Posts: 620
Joined: Dec 18 2008 10:13 pm

Re: Important papers and references for real-time PCR

Postby Suzanne » Mar 12 2010 9:32 pm

Here is a new paper on a subject that comes up a lot- using plasmids for standards.

Serious Overestimation in Quantitative PCR by Circular (Supercoiled) Plasmid Standard: Microalgal pcna as the Model Gene
Yubo Hou¤a*, Huan Zhang, Lilibeth Miranda¤b, Senjie Lin
Department of Marine Sciences, University of Connecticut, Groton, Connecticut, United States of America

http://www.plosone.org/article/info%3Ad ... ne.0009545

Anyone up for reading it and then discussing the data- like a journal club? I can start a new thread for it.

Suzanne
http://www.mobio.com/blog
Visit The Culture Dish!
Suzanne
ModSquad
ModSquad
 
Posts: 999
Joined: Feb 13 2003 8:59 pm
Location: Southern California

Previous

Return to Real-Time qPCR/qRT-PCR Methods

Who is online

Users browsing this forum: No registered users and 1 guest