PCR troubleshooting book available
The new book PCR Troubleshooting and Optimization: The Essential Guide edited by Suzanne Kennedy and Nick Oswald has been delivered to our distributors and is available for immediate dispatch read more ...
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 | Edited by: Suzanne Kennedy and Nick Oswald ISBN: 978-1-904455-72-1 Publisher: Caister Academic Press Publication Date: January 2011 Cover: hardback |
PCR Optimization Strategies
RT-PCR Optimization Strategies
from Martina Reiter and Michael W. Pfaffl writing in PCR Troubleshooting and Optimization: The Essential Guide
PCR technology is based on a simple principle; an enzymatic reaction that increases the amount of nucleic acids initially present in a sample but this powerful method makes it possible to detect specific mRNA transcripts in any biological sample by the application of RT-PCR. The RT-PCR quantitative analysis workflow has several steps, each of which is crucial to the success of the experiment. It starts with a sampling step, followed by nucleic acid extraction and stabilization, cDNA synthesis and finally the qPCR where the mRNA quantification takes place. PCR itself is quite a stable reaction with reproducibility between 2-8% but the number and nature of the pre-PCR steps mean that there are many sources of experimental variance in the workflow. Reliable data can only be produced when the experimental variance is minimized, so the sources of variation must be identified and optimized for each step of each experiment. Typically, however, the pre-PCR steps are neglected and optimization is done for PCR reaction only. Optimization of the whole RT-PCR workflow is important and recommendations to reduce experimental variance and produce more reproducible and reliable results should be followed.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from Martina Reiter and Michael W. Pfaffl writing in PCR Troubleshooting and Optimization: The Essential Guide
PCR technology is based on a simple principle; an enzymatic reaction that increases the amount of nucleic acids initially present in a sample but this powerful method makes it possible to detect specific mRNA transcripts in any biological sample by the application of RT-PCR. The RT-PCR quantitative analysis workflow has several steps, each of which is crucial to the success of the experiment. It starts with a sampling step, followed by nucleic acid extraction and stabilization, cDNA synthesis and finally the qPCR where the mRNA quantification takes place. PCR itself is quite a stable reaction with reproducibility between 2-8% but the number and nature of the pre-PCR steps mean that there are many sources of experimental variance in the workflow. Reliable data can only be produced when the experimental variance is minimized, so the sources of variation must be identified and optimized for each step of each experiment. Typically, however, the pre-PCR steps are neglected and optimization is done for PCR reaction only. Optimization of the whole RT-PCR workflow is important and recommendations to reduce experimental variance and produce more reproducible and reliable results should be followed.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
MIQE Guidelines Uncloaked
No matter how good you are at PCR, you can always learn something from the speakers we have lined up for our Getting the most out of PCR live online seminar series. These guy eat, sleep and drink PCR.
Next up we have MIQE Guidelines Uncloaked, in which Greg Shipley will give you the inside track on the requirements you need to satisfy to make sure your PCR results are suitable for publication. You'd be mad to miss it.
This event goes out live tomorrow (Tue 8th June) at 9am Pacific / 12pm Eastern / 5pm BST (UK) / 6pm CET. Click here to secure one of the remaining places on this live event.
You can also click here to take a look at our archive for this series, which now contains:
Magic in Solution: An Introduction and Brief History of PCR
Speaker: Carl Wittwer
Obtaining Maximum PCR Sensitivity and Specificity
Speaker: Cameron N. Gundry Attendence: 125
Significance of Controls and Standard Curves in PCR
Speaker: Ian Kavanagh
Next up we have MIQE Guidelines Uncloaked, in which Greg Shipley will give you the inside track on the requirements you need to satisfy to make sure your PCR results are suitable for publication. You'd be mad to miss it.
This event goes out live tomorrow (Tue 8th June) at 9am Pacific / 12pm Eastern / 5pm BST (UK) / 6pm CET. Click here to secure one of the remaining places on this live event.
You can also click here to take a look at our archive for this series, which now contains:
Magic in Solution: An Introduction and Brief History of PCR
Speaker: Carl Wittwer
Obtaining Maximum PCR Sensitivity and Specificity
Speaker: Cameron N. Gundry Attendence: 125
Significance of Controls and Standard Curves in PCR
Speaker: Ian Kavanagh
Getting The Most Out of PCR
We think you will be interested in an online seminar series entitled "Getting The Most Out of PCR", which is being broadcast by the popular life science blog, Bitesize Bio. Bitesize Bio is headed by Nick Oswald and Suzanne Kennedy, who co-edited our recent title "PCR Troubleshooting and Optimization".
The series lineup includes many of the authors from this book and kicks off on 18 May with a talk from LightCycler co-inventor, Carl Wittwer, entitled "Magic in Solution: An Introduction and Brief History of PCR". This will be a great learning experience with an opportunity to ask questions and learn from experts and pioneers in the PCR field. The full program is shown below.
Click here to book your place on these excellent events.
Recommended reading: PCR publications
The series lineup includes many of the authors from this book and kicks off on 18 May with a talk from LightCycler co-inventor, Carl Wittwer, entitled "Magic in Solution: An Introduction and Brief History of PCR". This will be a great learning experience with an opportunity to ask questions and learn from experts and pioneers in the PCR field. The full program is shown below.
Click here to book your place on these excellent events.
- Magic in Solution: An Introduction and Brief History of PCR
Speaker: Carl Wittwer
18 May 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET - Obtaining Maximum PCR Sensitivity and Specificity
Speaker: Cameron N. Gundry
25 May 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET - Significance of Controls and Standard Curves in PCR
Speaker: Ian Kavanagh
01 June 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET - The MBD2-based Enrichment Approach for Analyzing DNA methylation
Speaker: Chris Adams
08 June 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET - The MIQE Guidelines Uncloaked
Speaker: Greg Shipley
15 June 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET - High Resolution Melting Analysis - Beyond the SNP
Speaker: John Mackay
22 June 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET
Recommended reading: PCR publications
PCR book
New PCR book announced:
The book describes and discusses strategies for preparing effective controls and standards for PCR, when they should be employed and how to interpret the information they provide. The significance of optimization for efficiency, precision and sensitivity of PCR methodology and essential guidance on how to troubleshoot inefficient reactions. Design and optimization techniques, the use of appropriate controls, the significance of standard curves and the principles and strategies required for effective troubleshooting. The importance of sample preparation and quality, primer design, controlling inhibitors, avoiding amplicon and environmental contamination, optimizing reagent quality and concentration, and modifying the thermal cycling protocol for optimal sensitivity and specificity read more.
The book describes and discusses strategies for preparing effective controls and standards for PCR, when they should be employed and how to interpret the information they provide. The significance of optimization for efficiency, precision and sensitivity of PCR methodology and essential guidance on how to troubleshoot inefficient reactions. Design and optimization techniques, the use of appropriate controls, the significance of standard curves and the principles and strategies required for effective troubleshooting. The importance of sample preparation and quality, primer design, controlling inhibitors, avoiding amplicon and environmental contamination, optimizing reagent quality and concentration, and modifying the thermal cycling protocol for optimal sensitivity and specificity read more.
| Edited by: Suzanne Kennedy and Nick Oswald ISBN: 978-1-904455-72-1 Publisher: Caister Academic Press Publication Date: January 2011 Cover: Hardback read more ... |