RT-qPCR and qPCR Assays—Detecting Viruses and Beyond

We have all been hearing a lot about RT-PCR, rRT-PCR and RT-qPCR lately, and for good reason. Real-Time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) is the technique used in by the Center for Disease Control (CDC) to test for COVID-19. Real-time RT-PCR, or quantitative RT-PCR (RT-qPCR)*, is a specialized PCR technique that visualizes amplification of the target sequesnce as it happens (in real time) and allows you to measure the amount of starting target material in your reaction. You can read more about the basics of this technique, and watch a webinar here. For more about RT-PCR for COVID-19 testing, read this blog.

Both qPCR and RT-qPCR are powerful tools for scientist to have at their disposal. These fundamental techniques are used to study biological process in a wide range of areas. Over the decades, Promega has supported researchers with RT-qPCR and qPCR reagents and systems to study everything from from diseases like COVID-19 and cancer to viruses in elephants and the circadian rhythm of krill.  

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Researching the Researcher: Abbeah Navasca, 2019 Real-Time PCR Grant Winner

The three winners of the 2019 Real-Time PCR Grants have been hard at work in the six months since receiving their grants. Each winner was eligible to receive up to $10,000 in free PCR reagents as well as the opportunity to collaborate with our knowledgeable technical service and training teams.

Abbeah Navasca is a plant pathology researcher with the Tagum Agricultural Development Company, Inc. (TADECO*, Philippines). She is developing treatments for viral infections that affect one of Philippines’ largest and most valuable agricultural exports: bananas. As a result of the qPCR grant, she and two of her colleagues were able to participate in sample preparation and analysis workshops with Promega Technical Services experts in Singapore. During her visit, the team worked through strategies for plant sample preparation and amplified those samples with the GoTaq® 1-Step RT-qPCR System. We had a chance to ask her more before she headed back to her lab.

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Researching the Researchers: Alberto Biscontin

2019 Real-Time PCR Grant

The three 2019 Real-Time PCR Grant Winners have been hard at work in the six months since winning their grants. Each winner was eligible to receive up to $10,000 in free PCR reagents as well as the opportunity to collaborate with our knowledgeable technical service and training teams.

One of the 2019 winners, Alberto Biscontin (University of Padova, Italy), performs research in the fields of Neurogenetics and Chronobiology. He is looking to shed greater light on the circadian rhythms of the Antarctic krill. Alberto published his most recent analysis in Nature and GoTaq® qPCR Master Mix helped him validate expression of genes for his study.

His qPCR data showed support for internal mechanisms that not only support daily living but also clarified the overwintering process of the krill. Now that Alberto has sized up some zooplankton, we asked him to share a little more about himself and his research:

Q: How long have you been a researcher?
A: I have been a researcher since 2012.

Q: How did you decide to research Antarctic krill?
A: In 2013, I had the opportunity to join the international Antarctic research program PolarTime. [It] brought together eight research groups with different scientific expertise to study seasonal and daily rhythms in the Antarctic krill Euphausia superba.

Q: When you are not busy at the bench, what do you like to do?
A: Traveling. I love strolling through open-air markets.

Q: Are there any tips or tricks you have learned that make your job easier?
A: You can easily switch from a classic RT-PCR protocol to a cheaper and faster One-step protocol using the same primers and temperatures.

Q: What comes next?
A: I would like to characterize the clock machinery of other polar organisms to understand whether high latitude clocks have developed similar strategies to cope with [the] polar environment. Moreover, a better understanding of marine circadian clocks could help to shed light on the evolution of the animal circadian machinery.

You can find Alberto’s most recent publication in Nature Scientific Reports. The 2020 Real-Time PCR Grant will be coming soon. For more information on the 2019 winners and information on the 2020 Grant, visit the Real-Time Grant web page. Be sure to follow us on social media for the most up-to-date information regarding the 2020 Grant, including application deadlines and winner notifications!

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qPCR: The Very Basics

Real-Time (or quantitative, qPCR) monitors PCR amplification as it happens and allows you to measure starting material in your reaction.
Real-Time (or quantitative, qPCR) monitors PCR amplification as it happens and allows you to measure starting material in your reaction. Data are presented graphically rather than as bands on a gel.

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.

End-Point PCR 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 Measures 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 can quantitate gene expression, measure DNA damage, and quantitate 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.

  1. Use aerosol-resistant pipette tips, and have designated pipettors and tips for pre- and post-amplification steps.
  2. Wear gloves and change them frequently.
  3. Have designated areas for pre- and post-amplification work.
  4. Use reaction “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 the reaction master mix, and all of your reactions are getting their components from the same master mix, you are reducing variability from reaction to reaction.
  5. 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.

Top 10 Tips to Improve Your qPCR or RT-qPCR Assays

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Scientists have had a love-hate relationship with PCR amplification for decades. Real-time or quantitative PCR (qPCR) can be an amazingly powerful tool, but just like traditional PCR, it can be quite frustrating. There are several parameters that can influence the success of your PCR assay. We’ve highlighted ten things to consider when trying to improve your qPCR results.

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Do you want to build a snowman? Developing and optimizing a qPCR assay to detect ice-nucleating activity

Snowflakes---MA-400x600Over 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”

Methods for Quantitating Your Nucleic Acid Sample

Nucleic acid quanitation webinarFor 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”

How to Choose a Good Reference Gene?

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?”