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

Key words: PCR Technology, RT-PCR, RT-PCR quantitative analysis workflow, qPCR, mRNA quantification, PCR reproducibility, Pre-PCR steps, RT-PCR workflow

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

Key words: PCR Optimization, PCR Sensitivity, PCR Specificity, PCR detection system, PCR Software, PCR Primer design, PCR probe design, PCR inhibitors, Thermal cycling protocol

A new book on PCR Troubleshooting and Optimization has been announced by Caister Academic Press. Included in the book is: 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.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

Key words: Standards for PCR, Controls for PCR, Troubleshooting PCR, Optimization of PCR