DATA IN HARMONY:

MODERN
APPROACHES
TO QPCR

Custom Publishing From

Sponsored By

Page 3 Page 4 Page 5 Page 6

Optimal Thermal Creating Temperature Orchestrating Data Marching to the Beat of a
Performance Control in Unison Connectivity Different Drum: Alternative
qPCR Applications
 Evolutionary qPCR.
Transformative
connectivity and utility.

Introducing the new CFX Opus Real-Time PCR Systems

At Bio-Rad, we believe that if you can make something better, you should.

So we’re introducing the CFX Opus Systems. They have the reliable features you know
and expect from Bio-Rad, including differentiated Bio-Rad optics and CFX Maestro
Software, plus improvements in design, functionality, and connectivity. CFX Opus
Systems are also fully integrated with the new BR.io cloud platform for experimental
setup and data management. It’s the perfect combination of everything you expect and
everything you want for the modern lab.

Are the CFX Opus Systems right for you?

Visit bio-rad.com/CFXOpus to schedule a demo.

#ScienceForward
 OPTIMAL THERMAL
PERFORMANCE

S
cientists use quantitative poly- the sample should also be rapid for high effi- spread use of qPCR to diagnose SARS-CoV-2
merase chain reaction (qPCR) ciency generation of target DNA. highlights the practicality of the technique for
for a variety of sensitive appli- Peltier elements control heating and cool- infectious disease testing. qPCR delivers test
cations. From multiplexed gene ing throughout the reaction cycles in many results quickly compared to previously-used
expression profiling to infectious instruments. These devices tend to be cooler methods. Clinicians apply the technique to
disease diagnostics, researchers require reli- around their edges than in the middle, which easily determine if someone is infected with
able qPCR instruments. may create temperature variation throughout a specific disease, while researchers can use
Some thermal cycler blocks may pro- the block, particularly at the edges and sur- qPCR to quantify viral loads and monitor
duce suboptimal results, as issues with ther- face of the block.2 Insufficient heating around therapy responses.
mal uniformity affect the rate and efficiency the block edges (the edge effect) and hot and Issues of speed and uniformity are espe-
of a reaction. Because small amounts of cold spots throughout create experimental cially important for clinical sample test-
genetic material are exponentially ampli- variability. ing because quick and reliable results allow
fied, small perturbations can lead to large physicians to diagnose, treat, and stop the
problems. Instruments that precisely control Impact on Data spread of communicable diseases. Viral
reaction conditions are essential for highly The edge effect can lead to insufficient tem- diagnostic experiments are especially sen-
sensitive assays where a specific annealing plate DNA melting, especially when the tem- sitive to changes in temperature. Different
temperature is required. Reactions may fail plate has a high GC content. This decreases virus strains can be very similar, so incorrect
or the accuracy of the quantified data may nucleic acid amplification, leading research- primer binding due to temperature irregu-
be brought into question if the temperature ers to underestimate the amount of target in larities causes inaccurate diagnoses. Scien-
deviates from the ideal. their samples. Additionally, undershooting tists in this field should seek out high-qual-
the temperature may cause amplification of ity machines that monitor and tightly control
Around the Block unwanted DNA.3 In this situation, primers temperature across the block, ensuring repro-
An important consideration for qPCR may bind their target insufficiently or they ducible and accurate results.
machine performance is the time it takes to can anneal to sequences with weak sequence
heat or cool the block with each temperature homologies. Hot spots in the reaction block References
change. How quickly an instrument changes may inactivate the polymerase, which gener- 1 ) K.-R. Rogers-Broadway, E. Karteris,
temperature is an important determinant of ates false negative results. “Amplification efficiency and thermal
the duration of an experiment. Instruments Temperature variability also affects the stability of qPCR instrumentation: Current
landscape and future perspectives,” Exp
must balance the time it takes to change tem- melt curve. Identical qPCR amplicons in Ther Med, 10:1261-64, 2015.
peratures with uniformity. The temperature separate wells may appear to melt at differ- 2 ) M. Span et al., “Measurement uncertainty in
in each well should stabilize quickly across ent temperatures. Researchers then incor- calibration and compliancy testing of PCR
the block so that each sample experiences rectly interpret these results to mean that and qPCR thermal cyclers,” International
the same reaction conditions. This task is their primers are amplifying more than Congress of Metrology, 05003, 2017.
3 ) Y. Ho Kim et al., “Performance evaluation of
more complicated than it seems, as speed one product. They waste precious time and thermal cyclers for PCR in a rapid cycling
often negatively impacts thermal uniformity.1 resources optimizing primer sequences and condition,” BioTechniques,
Blocks with reduced mass heat and cool more annealing temperatures to correct a problem 44:495-505, 2008.
quickly, shortening the cycle time. However, that is not real.
blocks with higher mass maintain tempera-
ture uniformity better than those with min- Virus diagnostics
imal mass. Heat transfer from the block to Increasingly, scientists perform qPCR for
molecular diagnostics. In particular, wide-

DATA IN HARMONY: MODERN APPROACHES TO QPCR The Scientist 2020 | the-scientist.com 3

Creating Temperature
Control in Unison
Thermal uniformity is an essential characteristic
of successful qPCR assays. Uneven temperatures
in thermal cycler reaction blocks negatively affect
data accuracy and experimental reproducibility.
New qPCR instruments avoid these issues with
effective temperature monitoring and better
thermal uniformity control.

Speed Vs. Uniformity

qPCR machines with reaction blocks of minimal
mass quickly change temperature throughout
a run, but these machines may lack thermal
uniformity because of the thin metal. High mass
blocks maintain consistent temperatures at the
expense of speed. Instruments
that balance speed with uniformity
produce the most accurate results.

Too Hot to Handle

Out in the Cold Hot spots denature and inactivate the
Cold spots and edge effects undershoot polymerase, causing false negative results.
reaction temperatures, leading to
decreased primer annealing and under-
estimated target quantities. Cold
temperatures also cause off-target primer
binding to sequences with lower homology. 4
 ORCHESTRATING
DATA CONNECTIVITY

I
n modern society, countless individu- without transferring files via unsecure ing secure and redundant data centers.
als are connected electronically; cell USB drives. Within a lab’s cloud platform, scientists
phones are mini computers and arti- can generate multiple user profiles with
ficial technology controls basic func- Data Management individual usernames and passwords to
tions in many homes. But when it Cloud-based computing and data stor- further organize data and keep results
comes to laboratory technology, many age help researchers move away from secure.
scientists find themselves sent back in the confusion caused by handwritten lab
time. Out-of-date computers hooked up notebooks and poorly labeled spread- Solution for Big and Small Data
to modern lab equipment inefficiently sheets and graphs. Many cloud storage Modern molecular instruments pro-
store bits and pieces of data that await platforms for scientific data provide tools duce mountains of data thanks to mul-
analysis. Researchers should leave old for data analysis. Researchers are begin- tiplex capabilities and single cell and
technology behind and ready themselves ning to transition their data storage, whole genome analyses. Also, as the cost
for the future by storing and analyzing organization, analysis, and figure devel- of sequencing continues to decrease,
data on a cloud-based platform. opment to these web-based platforms. the amount of data generated will only
Another major benefit of cloud data increase. Researchers need cloud-based
Convenience in the Lab storage is that it simplifies collabora- strategies to keep track of this informa-
Instruments that are connected to the tion. As research teams spread across tion. Whether the dataset is small, such
cloud grant researchers freedom from the the globe, scientists easily share their as that from a qPCR experiment ana-
bench. Some machines even link to the uploaded data with lab members and lyzing the expression of a handful of
cloud directly, without the need for a ded- external collaborators. Keeping data in genes, or large like those from single cell
icated computer that is connected to the the cloud also enhances reproducibil- sequencing projects, researchers bene-
internet. By accessing the cloud with any ity because researchers can share vir- fit from working with their data in the
web browser, researchers plan and man- tual lab notebooks with collaborators cloud.
age experiments anywhere and at any and directly connect instruments to the In particular, cloud-based software
simplifies biomedical research. Research-
ers in labs across the world can remotely
view large next-generation sequencing
“Researchers should leave old technology behind and datasets that are uploaded to the cloud
ready themselves for the future by storing and analyzing and analyze them for single nucleotide
data on a cloud-based platform.” polymorphisms (SNPs), mutations, and
new gene variants. Scientists can also
integrate results from other technologies,
such as mass spectrometry and imaging,
time. They check the status of an exper- cloud to automatically record experimen- to better understand biological mecha-
iment as it runs from any computer, and tal conditions. nisms for diagnostics research.
results often upload automatically when Cloud services safeguard data
an experiment is complete. Scientists can from accidental loss or from unautho-
also analyze their data from anywhere rized users making changes by provid-

DATA IN HARMONY: MODERN APPROACHES TO QPCR The Scientist 2020 | the-scientist.com 5

 MARCHING TO THE BEAT
OF A DIFFERENT DRUM:
ALTERNATIVE QPCR
APPLICATIONS

Q
PCR is no longer employed tic acid bacteria.1 The scientists employed and distinguishing between heterozy-
only by molecular biolo- the data to enhance the viability of these gotes and homozygotes based on gene
gists in research laborato- desirable microbes in wine. dosage. Easy genotyping is also import-
ries. Scientists in diverse Scientists also use qPCR to target ant to track adventitious presence—when
fields use the technique microbial genes in environmental sam- trace amounts of an unwanted agricul-
for a variety of applications. Therefore, ples with kits that easily extract DNA or tural biotech product ends up in a field
qPCR reagents and instruments that take RNA in the field. Researchers working to population or food supply.
up minimal space and are easy to use are reduce water scarcity analyzed the safety
desirable in these industries.

Managing Microbes “qPCR is no longer employed only by molecular biologists

Microorganisms are everywhere, yet they in research laboratories. Because of their various
are invisible to the naked eye. Scientists in
backgrounds, scientists prioritize qPCR technology that is
many fields track microbes to either elimi-
nate them or support their growth. Tradi- simple to operate.”
tionally, scientists analyzed microbe pop-
ulations by counting colonies on a plate
or growing cells in liquid culture. This of roof-harvested rainwater with qPCR qPCR for All
approach lacks species specificity, only to identify pathogens in their samples.2 Because of their various backgrounds,
estimates the number of cells present, Microbial contamination is also a con- scientists prioritize qPCR technology
and takes days to obtain results, whereas cern in oil fields. Water injections that that is simple to operate. Drag and
qPCR methods detect different species stimulate oil extraction can also intro- drop tools, intuitive interfaces, kits that
and strains in a single multiplexed exper- duce hydrogen sulfide-producing micro- extract nucleic acids in the field, stand-
iment, accurately quantify the amount of organisms that generate toxic gas, lower alone instruments that use the cloud for
cells in a sample, and take mere hours to oil quality, and corrode pipelines. Scien- data storage, analysis, and sharing all
complete. tists monitor oil field conditions by pro- promote qPCR use across disciplines.
qPCR is now the method of choice for cessing samples in the field and run-
microbial monitoring in the food safety ning experiments with small-scale qPCR References
and processing fields. Scientists use spe- instruments.3 1 ) C. Beltramo et al., “Real-time PCR for
cies- and strain-specific primers to quan- characterizing the stress response of
tify and identify food-borne pathogens, Supporting Agriculture Oenococcus oeni in a wine-like medium,”
Res Microbiol, 157:267-74, 2006.
including small populations that would Agriculture researchers face the enor- 2 ) A hmed et al., “Real-time PCR detection
be hidden by more dominant ones in mous challenge of feeding the grow- of pathogenic microorganisms in
tube or plate cultures. Additionally, sci- ing world population. These scien- roof-harvested rainwater in Southeast
entists perform quantitative reverse tists improve the nutrition and output Queensland, Australia,” Appl Environ
transcription PCR (RT-qPCR) to track of crops and livestock through genetic Microbiol, 74:5490-96, 2008.
3 ) X. Lin et al., “Development and
microbial gene expression. In one exam- means. They use qPCR to analyze animal validation of in-field qPCR methods for
ple, researchers working to improve wine and plant gene expression. Additionally, water microbial analysis at oil and gas
production measured gene expression by qPCR simplifies genotyping by showing facilities,” NACE International, May 12,
qPCR of acid stress response genes in lac- the absence or presence of transgenes 2015.

DATA IN HARMONY: MODERN APPROACHES TO QPCR The Scientist 2020 | the-scientist.com 6

 Multiplex
CELL SORTING MADE
RT-qPCR SIMPLE.
beyond
Now you
your can focus onwith
expectations results,
ournot procedures.
one-step supermix.

Introducing Reliance — the right choice for multiplex RT-qPCR

Go directly from sample to data at the speed and throughput you need. Our new
Reliance One-Step Multiplex Supermix pushes the limits to get you more data, more
quickly and reliably — without compromising the quality of your results.
■ Amplify up to 5 targets per sample with maximum sensitivity and efficiency
■ Accelerate data throughput while reducing costs and reaction time
■ Achieve consistent, reproducible data run after run
■ Streamline automated workflows by bypassing plate refrigeration

Visit bio-rad.com/RelianceSelect

#ScienceForward
LOREM IPSUM DOLOR

Bulletin 7413 Ver A