Formalin Fixed Paraffin Embedded samples (FFPE) have been a mainstay of the pathology lab for over 100 years. Initially FFPE blocks were sectioned, stained with simple dyes and used for studying morphology, but now a variety of biomolecules can be analyzed in these samples. Over the past 10 years we have discovered that there is a treasure trove of genomics data waiting to be unearthed in FFPE tissue. While viral RNAs and miRNA were some of the first molecules found to be present and accessible for analysis starting in the 1990s, improvements to DNA and RNA extraction methods have demonstrated that PCR, qPCR, SNP genotyping, Exome and WGS are possible. This has resulted scientific publications of DNA and RNA data generated from FFPE samples starting in 2006, and today we see immense amounts of data generated from FFPE—with nearly 2000 citations in 2018 reporting sequencing of FFPE samples.
Depending on the type of project, prospective or
retrospective, the genomics scientist has an opportunity to affect the
probability of success by better understanding the fixation process. The
challenge with FFPE is the host of variables that have the potential to
negatively affect downstream assays.
For those of us well versed in traditional, end-point PCR, wrapping our minds and methods around real-time or quantitative (qPCR) can be challenging. Here at Promega Connections, we are beginning a series of blogs designed to explain how qPCR works, things to consider when setting up and performing qPCR experiments, and what to look for in your results.
First, to get our bearings, let’s contrast traditional end-point PCR with qPCR.
Visualizes by agarose gel the amplified product AFTER it is produced (the end-point)
Visualizes amplification as it happens (in real time) via a detection instrument
Does not precisely measure the starting DNA or RNA
Allows you to measure how many copies of DNA or RNA you started with (quantitative = qPCR)
Less expensive; no special instruments required
More expensive; requires special instrumentation
Basic molecular biology technique
Requires slightly more technical prowess
Quantitative PCR (qPCR) can be used to answer the same experimental questions as traditional end-point PCR: Detecting polymorphisms in DNA, amplifying low-abundance sequences for cloning or analysis, pathogen detection and others. However, the ability to observe amplification in real time and detect the number of copies in the starting material allow quantitation of gene expression, measurement of DNA damage, and quantitation of viral load in a sample and other applications.
Anytime that you are preforming a reaction where something is copied over and over in an exponential fashion—contaminants are just as likely to be copied as the desired input. Quantitative PCR is subject to the same contamination concerns as end-point PCR, but those concerns are magnified because the technique is so sensitive. Avoiding contamination is paramount for generating qPCR results that you can trust.
Use aersol-resistant pipette tips, and have designated pipettors and tips for pre- and post-amplification steps.
Wear gloves. Change them frequently.
Have designated areas for pre- and post-amplification work.
Use “master mixes” to minimize variability. A master mix is a ready-to-use mixture of your reaction components (excluding primers and sample) that you create for multiple reactions—because you are pipetting larger volumes to make it, and all of your reactions are getting their components from the same master mix, you are reducing variability from reaction to reaction.
Dispense your primers into aliquots to minimize freeze-thaw cycles and the opportunity to introduce contaminants into a primer stock.
These are very basic tips that are common to both end-point and qPCR, but if you get these right, you are off to a good start no matter what your experimental goals are.
If you are looking for more information regarding qPCR, watch this supplementary video below.
Summer, a much-looked forward to season. We typically pack in the activities and make the most of the daylight. We work hard and we play hard. This summer will be no exception, and at the BTC Institute, we are already getting set to host as many students as we can. We will see middle and high schoolers, K-12 teachers, college students, graduate students, college and university faculty and staff, and professionals in the biotech community under our roof at some point. You may want to join us too!
Our programs for advanced learners, geared toward the graduate student or biotech professional, offer much more than just a rigorous immersion in molecular biology theory and practice. Held at the BTC Institute at Promega Headquarters, they are taught by highly knowledgeable scientists, coming from both industry and academia. These instructors offer a wealth of information and share their expertise as well as life experiences with students. Informal discussions about career trajectories and access to industry are important added benefits to attending these off-campus workshops. Continue reading “Pack a Little Science into Your Summer with Advanced Courses from BTCI”
Over the last few months we have published several blogs about qPCR—from basic pointers on avoiding contamination in these sensitive reactions to a collection of tips for successful qPCR. Today we look in depth at a paper that describes the design and and optimization of a qPCR assay, and in keeping with the season of winter in the Northern hemisphere, it is only fitting that the assay tests for the abundance and identity of ice-nucleating bacteria.
Ice-nucleating bacteria are gram-negative bacteria that occur in the environment and are able to “catalyze” the formation ice crystals at warmer temperatures because of the expression of specific, ice-nucleating proteins on their outer membrane. Ice-nucleating bacteria are found in abundance on crop plants, especially grains, and are estimated to cause one-billion dollars in crop damage from frost in the United States alone.
In addition to their abundance on crop plants, ice-nucleating bacteria are also found on natural vegetation and have been isolated from soil, snow, hail, cloud water, in the air above crops under dry conditions and during rain fall. They have even been isolated from soil, seedlings and snow in remote locations in Antarctica. For the bacteria, ice nucleation may be a method to promote dissemination through rain and snow.
Although ice-nucleating bacteria have been isolated from clouds, ice and rain, little is known about their true contribution to precipitation or other events such as glaciation. Are such bacteria the only source of warm-temperature (above temperatures at which ice crystals form without a catalyst) ice nucleation? Can they trigger precipitation directly? What are the factors that trigger their release from vegetation into the atmosphere? Can we determine their abundance and variety in the environment? Continue reading “Do you want to build a snowman? Developing and optimizing a qPCR assay to detect ice-nucleating activity”
Almost from the moment the science recognized the value of PCR amplification, it has been a bit of a love-hate relationship. One of the latest additions to the PCR portfolio, real-time or quantitative PCR (qPCR), can be an amazingly powerful tool. However, just like traditional PCR, qPCR can be frustrating. There are a number of parameters that can influence the success of your qPCR assay. Below I have highlighted ten things to consider when trying to improve your qPCR results. Continue reading “Top 10 Things that Might Improve Your qPCR or RT-qPCR Assays”
For most molecular biology applications, knowing the amount of nucleic acid present in your purified sample is important. However, one quantitation method might serve better than another, depending on your situation, or you may need to weigh the benefits of a second method to assess the information from the first. Our webinar “To NanoDrop® or Not to NanoDrop®: Choosing the Most Appropriate Method for Nucleic Acid Quantitation” given by Doug Wieczorek, one of our Applications Scientists, discussed three methods for quantitating nucleic acid and outlined their strengths and weaknesses. Continue reading “Methods for Quantitating Your Nucleic Acid Sample”
Yesterday I listened in on the Webinar “Getting the Most Out of Your DNA Analysis from Purification to Downstream Assays”, presented by Eric Vincent–a Product Manager in the Promega Genomics group.
This is the webinar for you if you have ever wondered about the relative advantages and disadvantages of the many methods available for DNA purification, quantitation and analysis, or if you are comparing options for low- to high-throughput DNA purification. Eric presents a clear analyses of each of the steps in a basic DNA workflow: Purification, Quantitation, Quality Determination, and Downstream Analysis, providing key considerations and detailing the potential limitations of the methods commonly used at each step.
The DNA purification method chosen has an affect on the quality and integrity of the DNA isolated, and can therefore affect performance in downstream assays. Accuracy of quantitation also affects success, and the various downstream assays themselves (such as end-point PCR, qPCR, and sequencing) each have different sensitivities to factors such as DNA yield, quality, and integrity, and the presence of inhibitors. Continue reading “DNA Purification, Quantitation and Analysis Explained”
A Researcher’s work is never easy but it is even harder when relative data are to be interpreted. This is especially true for Real-Time PCR. It is one of the most accurate ways to evaluate gene expression. However, despite it being such a powerful technique, it still carries many pitfalls which can lead a scientist to the wrong conclusion. Often a new user does not have thorough sample/RNA preparation, equipment or knowledge. So what are the considerations and aspects that the researcher should pay attention to? Continue reading “How to Choose a Good Reference Gene?”
Tiny particles found in clothing, cosmetics, food, electronics or furniture enter our bodies and behave in unexpected (sometimes unwanted) ways. However, in the realm of medicine another type of particle called the nanoparticle can bring untold potential. We can load them with drugs, for example, and deliver them precisely to a diseased organ or cell. Mark Davis from the California Institute of Technology has created nanoparticles that deliver siRNA specifically into melanomas. Davis and his colleagues have not shied away from making bold claims about the therapeutic potential of their work. They write:
“When taken together, the data presented here provide the first, to our knowledge, mechanistic evidence of RNAi in a human from an administered siRNA. Moreover, these data demonstrate the first example of dose-dependent accumulation of targeted nanoparticles in human tumours. ….These data demonstrate that RNAi can occur in a human from a systemically delivered siRNA, and that siRNA can be used as a gene-specific therapeutic.” Davis et al. 2010.Continue reading “Nanoparticles – Workhorses That Bring Tremendous Benefit”
Over the years we have produced several articles and technical resources on PCR. One of the most widely used is a chapter on PCR in the Protocols and Applications guide, featuring an explanation of the techniques involved, tips for reaction optimization, and example protocols for routine PCR, qPCR and RT-PCR. The protocols and applications guide is part of the Promega App, available on the promega.com web site, and as an iPhone/iPad and Android App.
Recently, we released an update to the Android app that includes customizable PCR protocols. Right now, three protocols are available in this format (Basic PCR, Hot-Start PCR and qPCR). These custom protocols allow you to input specific values for your experiment, such as concentration of stock solutions, desired number of reactions, etc. These values are used to calculate the volumes required at each step in the procedure, and a customized protocol is generated with the volumes of each reagent calculated and incorporated. Once generated, protocols can be saved for re-use, or emailed to an account for subsequent printing or incorporation into a lab notebook. Continue reading “PCR Protocols for Android”