qPCR
High Resolution Melting Analysis
High Resolution Melting Analysis
from John F. Mackay and Carl T. Wittwer writing in PCR Troubleshooting and Optimization: The Essential Guide
Real-time qPCR using SYBR Green and melting curve analysis to verify specific product amplification has become a standard laboratory technique for rapid, high throughput gene quantification. An extension of this melting curve method - High Resolution melting analysis (HRMA) is now doing the same for the analysis of sequence variation, allowing rapid cost-effective discrimination of sequences to SNP level in an automated closed-tube method. Two PCR primers are typically required as with SYBR Green quantification but HRMA differs in its requirement for the use of a saturating dye, precise reaction temperature control and software algorithms to cluster the melting curves. Originally described for SNP analysis (and still the leading application), HRMA is now being used in a wider context- HLA comparisons, microsatellite genotyping and methylation status of DNA sequences. New developments such as unlabeled probes and snapback elements on the PCR primers allow the simultaneous genotyping of a desired SNP with the scanning of the whole amplicon for other sequence variation.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from John F. Mackay and Carl T. Wittwer writing in PCR Troubleshooting and Optimization: The Essential Guide
Real-time qPCR using SYBR Green and melting curve analysis to verify specific product amplification has become a standard laboratory technique for rapid, high throughput gene quantification. An extension of this melting curve method - High Resolution melting analysis (HRMA) is now doing the same for the analysis of sequence variation, allowing rapid cost-effective discrimination of sequences to SNP level in an automated closed-tube method. Two PCR primers are typically required as with SYBR Green quantification but HRMA differs in its requirement for the use of a saturating dye, precise reaction temperature control and software algorithms to cluster the melting curves. Originally described for SNP analysis (and still the leading application), HRMA is now being used in a wider context- HLA comparisons, microsatellite genotyping and methylation status of DNA sequences. New developments such as unlabeled probes and snapback elements on the PCR primers allow the simultaneous genotyping of a desired SNP with the scanning of the whole amplicon for other sequence variation.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
MIQE Guidelines
The MIQE Guidelines Uncloaked
from Gregory L. Shipley writing in PCR Troubleshooting and Optimization: The Essential Guide
The MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines have been presented to serve as a practical guide for authors when publishing experimental data based on real-time qPCR. Each item is presented in tabular form as a checklist within the MIQE manuscript. However, this format has left little room for explanation of precisely what is expected from the items listed and no information on how one might go about assimilating the information requested. An expanded explanation of the guideline items on how those requirements might be met should be consulted prior to publication.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from Gregory L. Shipley writing in PCR Troubleshooting and Optimization: The Essential Guide
The MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines have been presented to serve as a practical guide for authors when publishing experimental data based on real-time qPCR. Each item is presented in tabular form as a checklist within the MIQE manuscript. However, this format has left little room for explanation of precisely what is expected from the items listed and no information on how one might go about assimilating the information requested. An expanded explanation of the guideline items on how those requirements might be met should be consulted prior to publication.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
qPCR Data Analysis
qPCR Data Analysis: Unlocking the Secret to Successful Results
from Jan Hellemans and Jo Vandesompele writing in PCR Troubleshooting and Optimization: The Essential Guide
Real-time quantitative PCR (qPCR) is the gold standard for fast, accurate, sensitive and cost-efficient gene expression analysis. Despite its conceptual simplicity and ease of use, the multi-step qPCR workflow contains many potential pitfalls. An intelligent experiment design and setup, high quality reagents and assays, quality controls in each step of the workflow, proper quantification models and appropriate bio-statistical analyses pave the way to successful gene expression results. Data analysis aspects include the evaluation of pilot studies and quality controls, through universally applicable quantification models and bio-statistics, to the reporting of experiment results.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from Jan Hellemans and Jo Vandesompele writing in PCR Troubleshooting and Optimization: The Essential Guide
Real-time quantitative PCR (qPCR) is the gold standard for fast, accurate, sensitive and cost-efficient gene expression analysis. Despite its conceptual simplicity and ease of use, the multi-step qPCR workflow contains many potential pitfalls. An intelligent experiment design and setup, high quality reagents and assays, quality controls in each step of the workflow, proper quantification models and appropriate bio-statistical analyses pave the way to successful gene expression results. Data analysis aspects include the evaluation of pilot studies and quality controls, through universally applicable quantification models and bio-statistics, to the reporting of experiment results.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
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
Detection of Microbes in Water
Molecular techniques based on genomics, proteomics and transcriptomics are rapidly growing as complete microbial genome sequences are becoming available and advances are made in sequencing technology, analytical biochemistry, microfluidics and data analysis. While the clinical and food industries are increasingly adapting these techniques, there appear to be major challenges in detecting health-related microbes in source and treated drinking waters. This is due in part to the low density of pathogens in water, necessitating significant processing of large volume samples. Quantitative PCR is a state-of-the-art technique available for pathogen detection and characterization from water.
Although quantitative PCR is almost 15 years old, only recently has it become a tool for diagnostic purposes in water microbiology. Conventional PCR and its variations largely give qualitative results (MPN-PCR being an exception) and are most useful when presence-absence of the target is to be noted. Since the product is measured at the end of the PCR, where the amount of amplicon (product) in a given reaction tube is likely to have been affected by saturation effects of excess amplicons or poorly optimized reactions, the yield of amplicon does not relate to the original starting concentration. Furthermore, a second step is always required for verification of the product. Because there is a quantitative relationship between amount of starting target and amount of PCR product during the exponential phase of the PCR process, if the yield of amplicons are made in the exponential or initial linear phases of amplification, which is the case in qPCR, then the data obtained can provide a quantitative relationship to the starting concentration.
In qPCR, fluorescent dyes and probes are generally used in addition to regular PCR primers, thus allowing for in situ assay of the targeted amplicon. With increasing cycles of PCR, the increase in target is directly quantified by an increase in fluorescence that is emitted by increased intercalation of fluorescent dye or hybridization of fluorescent oligonucleotide probe(s) to the target. These techniques are not "quantitative" in the strictest sense, as they measure a kinetic reaction. Often called "real time" or kinetic PCR, they do not measure the reaction as it occurs, but measure the results of the reaction in a pause between cycles. Perhaps the most correct descriptor is Kinetic PCR, but that term has not been adopted in molecular microbiology.
Further reading: Environmental Microbiology: Current Technology and Water Applications
Although quantitative PCR is almost 15 years old, only recently has it become a tool for diagnostic purposes in water microbiology. Conventional PCR and its variations largely give qualitative results (MPN-PCR being an exception) and are most useful when presence-absence of the target is to be noted. Since the product is measured at the end of the PCR, where the amount of amplicon (product) in a given reaction tube is likely to have been affected by saturation effects of excess amplicons or poorly optimized reactions, the yield of amplicon does not relate to the original starting concentration. Furthermore, a second step is always required for verification of the product. Because there is a quantitative relationship between amount of starting target and amount of PCR product during the exponential phase of the PCR process, if the yield of amplicons are made in the exponential or initial linear phases of amplification, which is the case in qPCR, then the data obtained can provide a quantitative relationship to the starting concentration.
In qPCR, fluorescent dyes and probes are generally used in addition to regular PCR primers, thus allowing for in situ assay of the targeted amplicon. With increasing cycles of PCR, the increase in target is directly quantified by an increase in fluorescence that is emitted by increased intercalation of fluorescent dye or hybridization of fluorescent oligonucleotide probe(s) to the target. These techniques are not "quantitative" in the strictest sense, as they measure a kinetic reaction. Often called "real time" or kinetic PCR, they do not measure the reaction as it occurs, but measure the results of the reaction in a pause between cycles. Perhaps the most correct descriptor is Kinetic PCR, but that term has not been adopted in molecular microbiology.
Further reading: Environmental Microbiology: Current Technology and Water Applications