Real-Time PCR Instrumentation
Real-Time PCR Instrumentation: An Instrument Selection Guide
from Sandrine Javorski-Miller and Ivan Delgado Orlic writing in PCR Troubleshooting and Optimization: The Essential Guide
A paper from 2008 mentions that quantitative PCR is 25 years old but routine use of this technology has only taken off during the past 12 years. The first commercial Real-Time PCR instrument, the ABI Prism 7700, was introduced to researchers in 1996 by Applied Biosystems. Since then over 40 additional Real-Time PCR instruments have been developed by more than a dozen vendors. Because there are so many Real-Time PCR instruments available utilizing a wide range of technologies, scientists face a daunting selection task. The space includes everything from entry level (single color detection, a small number of samples, low cost) to more complex (over 5 channel colors and multiplex detection, thousands of samples processed in each run, and expensive system price). Key features differentiate Real-Time PCR instruments, and various criteria should be considered when selecting the instrument that best fits a specific scientist's research needs.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from Sandrine Javorski-Miller and Ivan Delgado Orlic writing in PCR Troubleshooting and Optimization: The Essential Guide
A paper from 2008 mentions that quantitative PCR is 25 years old but routine use of this technology has only taken off during the past 12 years. The first commercial Real-Time PCR instrument, the ABI Prism 7700, was introduced to researchers in 1996 by Applied Biosystems. Since then over 40 additional Real-Time PCR instruments have been developed by more than a dozen vendors. Because there are so many Real-Time PCR instruments available utilizing a wide range of technologies, scientists face a daunting selection task. The space includes everything from entry level (single color detection, a small number of samples, low cost) to more complex (over 5 channel colors and multiplex detection, thousands of samples processed in each run, and expensive system price). Key features differentiate Real-Time PCR instruments, and various criteria should be considered when selecting the instrument that best fits a specific scientist's research needs.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
RT-PCR Optimization
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
PCR Sensitivity and Specificity
Category: Optimization
Obtaining Maximum PCR Sensitivity and Specificity
from Cameron N. Gundry and Matthew D. Poulson writing in PCR Troubleshooting and Optimization: The Essential Guide:
PCR is a highly sensitive and specific technique used in molecular biology laboratories everywhere. It is able to provide near 100% sensitivity and specificity with appropriately designed assays in controlled situations. However, results do not always match this potential. The most common problems in PCR arise from overlooking basic principles in assay design and optimization. Maximum PCR performance depends on key factors which include: 1) choosing an appropriate detection system, 2) using available software for the best primer and probe design, 3) assessing sample quality and controlling inhibitors, 4) avoiding amplicon and environmental contamination, 5) optimizing for reagent quality and concentration, and 6) modifying the thermal cycling protocol for optimal sensitivity and specificity. Addressing all of these factors will aid the investigator in designing high quality PCR assays.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from Cameron N. Gundry and Matthew D. Poulson writing in PCR Troubleshooting and Optimization: The Essential Guide:
PCR is a highly sensitive and specific technique used in molecular biology laboratories everywhere. It is able to provide near 100% sensitivity and specificity with appropriately designed assays in controlled situations. However, results do not always match this potential. The most common problems in PCR arise from overlooking basic principles in assay design and optimization. Maximum PCR performance depends on key factors which include: 1) choosing an appropriate detection system, 2) using available software for the best primer and probe design, 3) assessing sample quality and controlling inhibitors, 4) avoiding amplicon and environmental contamination, 5) optimizing for reagent quality and concentration, and 6) modifying the thermal cycling protocol for optimal sensitivity and specificity. Addressing all of these factors will aid the investigator in designing high quality PCR assays.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
Controls and Standard Curves in PCR
Category: Technology | Troubleshooting
Significance of Controls and Standard Curves in PCR
from Ian Kavanagh, Gerwyn Jones and Saima Naveed Nayab writing in PCR Troubleshooting and Optimization: The Essential Guide:
Whilst qPCR is a powerful technique, the results achieved using this method is valid only if the appropriate controls have been included in the experiment. Careful selection of controls and proper Optimisation of qPCR conditions promise generation of highly specific, repeatable, reproducible and sensitive data. There are strategies for preparing both negative and positive controls for PCR, when they should be employed and how to interpret the information they provide. Standard curves are vital for determining the initial starting amount of the target template and for assessing assay efficiency, precision, sensitivity, and dynamic range. It is important to know how to prepare standards, interpret standard curve and troubleshoot inefficient qPCR reactions.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from Ian Kavanagh, Gerwyn Jones and Saima Naveed Nayab writing in PCR Troubleshooting and Optimization: The Essential Guide:
Whilst qPCR is a powerful technique, the results achieved using this method is valid only if the appropriate controls have been included in the experiment. Careful selection of controls and proper Optimisation of qPCR conditions promise generation of highly specific, repeatable, reproducible and sensitive data. There are strategies for preparing both negative and positive controls for PCR, when they should be employed and how to interpret the information they provide. Standard curves are vital for determining the initial starting amount of the target template and for assessing assay efficiency, precision, sensitivity, and dynamic range. It is important to know how to prepare standards, interpret standard curve and troubleshoot inefficient qPCR reactions.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
Difficult Templates and Inhibitors of PCR
Category: Troubleshooting
Difficult Templates and Inhibitors of PCR
from Jack M. Gallup writing in PCR Troubleshooting and Optimization: The Essential Guide:
One of the least-acknowledged problems with PCR, RT-PCR and qPCR is reaction inhibition. Addressing or eliminating inhibition is central to allowing qPCR to be modeled by the least complex mathematics, and enables more effective troubleshooting of amplifications from difficult templates such as AT- or GC-rich sequences, repetitive sequences, and templates with prohibitive secondary structures. In the absence of inhibition, additives aimed at improving PCR, RT-PCR and qPCR performance can be assessed more directly, allowing investigators to identify and utilize better primer/probe designs, enzymes and master mixes, and formulate better reverse transcription reactions. In addition to inhibition, RNA integrity is another major concern which must be addressed both by using appropriate optical assessments and the 3':5' assay.
To address inhibition, commercial kits for removing inhibitory substances have been developed in addition to the SPUD assay and the P-Q assay-development/project-management software tool. Although reagent choice alone plays a large part in determining the success or failure of reverse transcription, PCR, RT-PCR or qPCR, there are strategies for detecting, avoiding and/or eliminating inhibition during reverse transcription, PCR, RT-PCR and qPCR. Also there are strategies to amplify difficult templates and optimize reverse transcription reactions.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from Jack M. Gallup writing in PCR Troubleshooting and Optimization: The Essential Guide:
One of the least-acknowledged problems with PCR, RT-PCR and qPCR is reaction inhibition. Addressing or eliminating inhibition is central to allowing qPCR to be modeled by the least complex mathematics, and enables more effective troubleshooting of amplifications from difficult templates such as AT- or GC-rich sequences, repetitive sequences, and templates with prohibitive secondary structures. In the absence of inhibition, additives aimed at improving PCR, RT-PCR and qPCR performance can be assessed more directly, allowing investigators to identify and utilize better primer/probe designs, enzymes and master mixes, and formulate better reverse transcription reactions. In addition to inhibition, RNA integrity is another major concern which must be addressed both by using appropriate optical assessments and the 3':5' assay.
To address inhibition, commercial kits for removing inhibitory substances have been developed in addition to the SPUD assay and the P-Q assay-development/project-management software tool. Although reagent choice alone plays a large part in determining the success or failure of reverse transcription, PCR, RT-PCR or qPCR, there are strategies for detecting, avoiding and/or eliminating inhibition during reverse transcription, PCR, RT-PCR and qPCR. Also there are strategies to amplify difficult templates and optimize reverse transcription reactions.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
A Brief History of PCR
Category: History | Technology
A Brief History of PCR
from Carl T. Wittwer and Jared S. Farrar writing in PCR Troubleshooting and Optimization: The Essential Guide:
The polymerase chain reaction (PCR) has become a fundamental tool in molecular research and clinical testing. A recent review by Wittwer and Farrar discusses the origins of PCR, its early evolution including adaptation to RNA, thermostable polymerases, automation, improvements in specificity and rapid temperature cycling. Perhaps the most significant advance is real-time PCR, combining both amplification and detection into one instrument as a superior solution for nucleic acid quantification. Real-time PCR is enabled by monitoring the reaction with double stranded DNA dyes or specific probes, including hydrolysis, hybridization, and conformation-sensitive probes. PCR product and probe melting analysis continues to improve in resolution, allowing greater sequence detail for genotyping and variant scanning. Microfluidic platforms and digital PCR are destined to find more applications in the future.
Read more: PCR Troubleshooting and Optimization: The Essential Guide
from Carl T. Wittwer and Jared S. Farrar writing in PCR Troubleshooting and Optimization: The Essential Guide:
The polymerase chain reaction (PCR) has become a fundamental tool in molecular research and clinical testing. A recent review by Wittwer and Farrar discusses the origins of PCR, its early evolution including adaptation to RNA, thermostable polymerases, automation, improvements in specificity and rapid temperature cycling. Perhaps the most significant advance is real-time PCR, combining both amplification and detection into one instrument as a superior solution for nucleic acid quantification. Real-time PCR is enabled by monitoring the reaction with double stranded DNA dyes or specific probes, including hydrolysis, hybridization, and conformation-sensitive probes. PCR product and probe melting analysis continues to improve in resolution, allowing greater sequence detail for genotyping and variant scanning. Microfluidic platforms and digital PCR are destined to find more applications in the future.
Read more: PCR Troubleshooting and Optimization: The Essential Guide
MIQE Guidelines Uncloaked
Category: Technology | Seminar
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