PCR 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
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 Controls and Standards
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