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 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.

  1. Use aersol-resistant pipette tips, and have designated pipettors and tips for pre- and post-amplification steps.
  2. Wear gloves. Change them frequently.
  3. Have designated areas for pre- and post-amplification work.
  4. 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.
  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.

 

Meet the Mighty Masked Masters of Measurement

Scientific investigation is an iterative process, for which reproducibility is key. Reproducibility, in turn, requires accuracy and precision—particularly in measurement. The unsung superheroes of accuracy and precision in the research lab are the members of your local Metrology Department. According to Promega Senior Metrologist, Keela Sniadach, it’s good when the metrology department remains unsung and behind the scenes because that means everything is working properly.

Holy Pipettes, Scientists! We have a metrology department?! Wait…what’s metrology again?

Callibration technician checks out a multipipettorMetrology (the scientific study of measurement) got its start in France, when it was proposed that an international length standard be based on a natural source. It was from this start that the International System of Units (SI), the modern metric system of measurement, was born.

Metrology even has its own day: May 20, which is the anniversary of the day that the International Bureau of Weights and Measures (BIPM) was created by the Meter Convention in Paris in 1875. The job of BIPM is to ensure worldwide standards of measurement.

For life scientists, metrology centers around making sure the equipment used everyday—from pipettes to heating blocks to centrifuges—is calibrated and measuring correctly. Continue reading “Meet the Mighty Masked Masters of Measurement”

Activating the Inflammasome: A New Tool Brings New Understanding

Innate immunity, the first line of immune defense, uses a system of host pattern recognition receptors (PRRs) to recognize signals of “danger” including invariant pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). These signals in turn recruit and assemble protein complexes called inflammasomes, resulting in the activation of caspase-1, the processing and release of the pro-inflammatory cytokines IL-1ß and IL-18, and the induction of programmed, lytic cell death known as pyroptosis.

Innate immunity and the activity of the inflammasome are critical for successful immunity against a myriad of environmental pathogens. However dysregulation of inflammasome activity is associated with many inflammatory diseases including type 2 diabetes, obesity-induced asthma, and insulin resistance. Recently, aberrant NLRP3 inflammasome activity also has been associated with age-related macular degeneration and Alzheimer disease. Understanding the players and regulators involved in inflammasome activity and regulation may provide additional therapeutic targets for these diseases.

Currently inflammasome activation is monitored using antibody-based techniques such as Western blotting or ELISA’s to detect processed caspase-1 or processed IL-1ß. These techniques are tedious and are only indirect measures of caspase activity. Further, gaining information about kinetics—relating inflammasome assembly, caspase-1 activation and pyroptosis in time—is very difficult using these methods. O’Brien et al. describe a one-step, high-throughput method that enables the direct measurement of caspase-1 activity. The assay can be multiplexed with a fluorescent viability assay, providing information about the timing of cell death and caspase-1 activity from the same sample. Continue reading “Activating the Inflammasome: A New Tool Brings New Understanding”

Celebrate World Migratory Bird Day by Reducing Your Plastic Use

One of the most noticeable phenological events of Spring in the Midwest United States is the arrival of the red winged black birds in March. These birds fly in from the South and take up residence on fence posts, power lines and tall reeds, creating a a weaving of red and yellow and black against a still brown backdrop. Shortly after the blackbirds arrive, the first robins of spring greet us and sandhill cranes fly in along with many other species.


These migratory birds that serve as heralds of spring are celebrated on World Migratory Bird Day (#WMBD #WMBD2019 #BirdDay). This day is celebrated twice a year, on the second Saturday in May and the second Saturday in October.

Continue reading “Celebrate World Migratory Bird Day by Reducing Your Plastic Use”

There’s Something About Trees: Arbor Day 2019

two girls climbing a tree
Climbing The Old Willow Tree is best with a friend

My daughter has a favorite tree, The Old Willow Tree, at Aldo Leopold Nature Center in Madison. I don’t know exactly how much time she has spent in that tree, but I suspect it is significant.

Trees have served as the source of inspiration for scientists’ careers, writers’ metaphors, and musicians’ nostaglia.

One of our science writers at Promega tells this story about an early “botanical” experiment he and his grandmother performed:

“When I was four, my grandma helped me plant a ‘helicopter’ in a butter dish. It slowly graduated to a Cool Whip container, then a family-sized takeout tub from a spaghetti joint. It’s now the largest tree on my parents’ property.” –Jordan Villanueva

When I was in college, the campus quad was lined with ancient Ginkgo trees that filled my morning walks to my fall classes with shimmering gold leaf. I remember those trees with such fondness that the first tree I planted when I moved to Wisconsin was a Ginkgo tree.

There’s something about trees…we wax poetic and become nostalgic; we include them in our literature and art as important symbols and teachers of life lessons. Continue reading “There’s Something About Trees: Arbor Day 2019”

Wetlands, Water Quality and Rapid Assays

toad

The storms of the previous day had moved eastward, leaving in their wake flooded farm fields and saturated roadside wetlands. At dusk, we loaded the Ford Escort wagon and headed south. We bumped along the maze of farm roads intent upon listening for croaks and snores in the night. At one roadside wetland, I heard my first congress of Spadefoot toads. The sound was deafening, invoking everything that a “congress of snoring toads” brings to mind. Around the corner, in a low spot of a corn field, a lone Spadefoot toad called for a mate; he was joined by a rather enthusiastic Copes Gray tree frog and several chorus frogs. The congress down the road provided a rolling bass to these more melodic anurans.

Wetlands exist in many different shapes and sizes and in many different geographies: coastal margins, mountain valleys, beaches and rocky shores, estuarine wetlands where tidal saltwater and freshwater mix, and inland wetlands. Some of them are ephemeral, some of them permanent. Wetlands serve many different functions, from providing habitat and food for plants and animals to offering protection from floods and maintaining water quality. One acre of one-foot deep wetland is estimated to hold 330,000 gallons of water. Coastal wetlands are important for reducing storm erosion by decreasing tidal surge and buffering the wind. In the US alone, this benefit has an estimated value of $23.2 billion dollars each year. Continue reading “Wetlands, Water Quality and Rapid Assays”

Control Samples: Three Terrifying Tales for Scientists

Lab science cartoon
Carl may not scare her…but did she remember the controls?

Warning: This blog contains stories about phantom serial killers, frankenfoods, mysteriously phosphorylated bands and unrequited ligations that may be disturbing to some people. Children or scientists prone to anxiety over irreproducible results should read this with their eyes shut.

I

Clouds hung low in the sky, and the late October wind howled between the buildings, rattling the window panes of the basement laboratory. The grackles cawed in desperate warning, their flocks changing the evening color palette from gray to black. I was as unsettled as the weather, watching my blot slosh back and forth. Continue reading “Control Samples: Three Terrifying Tales for Scientists”

Forensic Scientists Improve Sexual Assault Kit Turnaround Time with Y-Screening

The backlog of sexual assault kit samples in crime laboratories across the nation is a topic that hit the spotlight when a group of journalists uncovered the issue in an open records search of crime lab records in 2015. Reasons for the backlog include lack of staff, lack of funding, and simply, lack of time or a decision not to prosecute the case. Processing samples can be a labor-intensive process.

We recently interviewed Lynndsey R. Simon, Forensic Scientist II and Alternate CODIS Administrator from the Columbus Police Forensic Services Center to discuss some recent changes in sample processing in their laboratory that are helping to alleviate some of the backlog. She will be presenting a talk at the upcoming International Symposium on Human Identification (ISHI) in September.

The Columbus Police Forensic Services Center is a smaller forensic laboratory and according to Simon, one of the biggest challenges they face is strained resources. The DNA extraction and processing kits that forensic laboratories use are very expensive, and the number of DNA samples that laboratories are getting for DNA analysis are increasing. With limited resources and funding, maximizing efficiency and finding the best solutions for the laboratory becomes critical. Continue reading “Forensic Scientists Improve Sexual Assault Kit Turnaround Time with Y-Screening”

Shaping the Future by Investing in Science

“Today is certainly a great day for Promega R&D, but it is also a great day for science.”

Picture of people gathered
Community members and Promega employees gather for the ground breaking celebration for a new R&D facility.

Gary Tarpley, director of Research and Development at Promega closed his remarks for the ground breaking of the new Promega Research and Development Center in Fitchburg, WI, with those words.

With the ground breaking on this new R&D facility, Promega makes a $190 million, long-term investment in science.

But why invest in science?
Continue reading “Shaping the Future by Investing in Science”

Questions of Genome Privacy and Protection

In April 2018, law enforcement officials announced the arrest of a suspect in the Golden State Killer case (New York Times ). Shortly after the announcement, those same law enforcement officers explained that detectives had used a public forensic genealogy web site to help identify the killer.

What does it mean when a law enforcement agency accesses a public genetic genealogy database to search for a suspect in a crime? Continue reading “Questions of Genome Privacy and Protection”