qPCR
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
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
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
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