qPCR robotics - qPCR
applications using pipetting robots
The Trend to qPCR
Real-time quantitative PCR (qPCR) is now a mainstay of molecular
biology. Just a few short years ago the purchase of a real-time
instrument was considered a luxury. Now most laboratories either
a real-time instrument or have easy access to one. We predict the same
will happen for robotic automation of qPCR setup; it will soon be
commonplace and probably expected.
A trend to automation is easy to understand. Firstly, the sheer numbers
of reactions that are run (all with one or more replicates) means the
workload is increasing dramatically. Secondly, and more importantly,
qPCR setup requires great skill and lots of practice because even a
very small variation when pipetting a DNA or RNA sample translates into
big differences after amplification. There is also the question of repeatability.
Identical results from different
operators and between
laboratories are expected but that can be very hard to achieve.
not forget that setting up qPCR assays is also tedious and time
consuming! A small and affordable liquid handling robot with the
precision necessary to tackle qPCR setup would address all these
issues. The good news is robots like this are now available.
Automated reaction setup offers some other practical benefits too.
Providing the robot has sufficient precision, it is possible to
routinely set up smaller reactions than you can manually. This can add
up to significant savings by stretching reagents and samples much
further (typically by 20–50%). Depending on the protocol, even plastic
consumable use can be reduced. Importantly, robots don’t tire or lose
concentration and so don’t make human errors such as mixing up sample
order, omitting a sample, or delivering two samples to the same well.
Besides the obvious expense, it can be disastrous (and also
embarrassing) when an experiment using rare sample material must be
repeated because of a simple human error.
Time savings can also be significant because it normally requires less
time to prepare reactions using a robot. If you use pre-aliquoted
standards and pre-programmed sample configurations the operator need
only prepare the mastermix and set up the deck. This equates to about a
33%-66% total time saving to set up a 96-well plate of 12–40 replicated
samples (e.g. 15–60 minutes versus 45–90 minutes). If only hands-on
time is considered, the time required by the operator to prepare
samples is further reduced to just 10-15 minutes.
There are a wide variety of liquid handling robots available but
unfortunately most are not suitable for qPCR setup. This is because
they lack the precision needed for quantitative reactions, don’t allow
fractional microlitres to be specified (e.g. 2.6 µL vs. 3.0
µL), or don’t support the various qPCR reaction tube formats
available. Some are just too big and complex to be practical. These
large robots were not designed specifically for qPCR but for
large-scale repetition of standard procedures. They are usually
difficult to program and were never intended for casual use by multiple
operators. One of the biggest discriminators for any robotic platform
is the software. A good robot has software that is easy to use and
quick to learn. So look for a robot that was designed specifically for
qPCR setup, is compatible with shared use by a number of people, and
has user-friendly software.
are a few things to consider before purchasing a robot:
Size – you
don’t want to sacrifice any more bench space than necessary.
Look out for vertical height clearance too; some robots have hoods that
lift up high over the robots work surface. Smaller robots may even fit
inside a laminar flow cabinet, which may be a requirement for some
laboratories’ standard PCR procedures. In which case, if the robot has
a hood, ask if it is removable (as it won’t be needed if it’s installed
inside a laminar flow hood).
Look for at least six separate spaces on the robot deck
(each space is typically the size of a standard 96-well plate). It is
very limiting to have less than this. For example, if there are only
four spaces on the deck and at least one must be used to hold a rack of
tips, this will leave only three spaces for everything else.
choose a single channel robot for qPCR work. A single
channel robot has one pipetting head only, allowing it to cherry-pick
from any location to any other location on the work surface for maximum
flexibility. More importantly, a single channel is more precise because
the same pipetting head mechanism is used for all operations. This
means any minor calibration error will be consistent to all tubes and
largely cancel out (this applies to manual setup too where you should
try to work with a single pipettor where possible).
Hood – it’s a
good idea to have a hood over the work surface; it cuts
down on dust contamination risk and prevents accidents (someone may
bump the robot arm while its running or worse, if someone inadvertently
leans over the robot deck, the arm may unexpectedly move and injure
them if there is no protective hood. If a hood is fitted it is also
possible to add UV sterilization and even HEPA filtration options to
Tip Discard –
discarding used tips to an external waste bag or
container is usually better than into a bin on the robot’s deck area.
Not only does an internal waste bin take up valuable space that could
be used to hold more tip racks, plates, tubes etc it also presents an
increased contamination and overfill risk.
how easy is it for the robot to be calibrated for a new
type of tube or plate? There are many variations of a 0.2 mL tube or
96-well plate for example and a robot needs to know the exact
dimensions of your particular plasticware to work properly (typically
where the centre of the well is and how deep it is). Make sure you are
not locked into specific plates or tubes if you want to use the
plasticware you are already familiar with.
– check the robot has a mechanism for sensing or tracking
liquid levels in all tubes. This is important for determining pipetting
behavior to improve precision and reliability.
Importance of Software
Experience shows that the software interface is all-important. If the
robot is difficult to learn or program users won’t be bothered.
Software designed specifically for qPCR setup is invariably better. Be
aware that there is often some initial fear or mistrust of robots
anyway and it is normal for new users to do a couple of experiments
before they feel relaxed and confident operating a robot.
Look for “smart” software, i.e. software that automatically keeps track
of liquid levels, how many tips are used, whether or not a particular
tip can be re-used or not etc, otherwise the onus is placed on the user
to know what they are doing at all times. Make sure you can define
custom plate or tube types. Otherwise you will be limited to only those
exact plates and tubes defined in the software.
Options such as “mix before aspirate” and “mix after dispense” are very
useful for qPCR setup. Check that you can specify fractional
microlitres such as 1.6 µL and not have to round-up to 2.0
µL for example. If possible, check that pipetting is actually
precise to this extent as well.
It is best if the robot is operated from a separate computer rather by
an on-board controller. On-board controllers have a very small screen
(compared to a PC) which makes programming more of a challenge.
Furthermore, it requires the user to program the robot while standing
in front of it.
Besides quantitative PCR reaction setup, check that the software is
versatile enough to tackle a range of other pipetting tasks in the
laboratory (for example replicate plating and sample pooling). A good
example of a very useful application is sample concentration
normalization, i.e. where you have a set of stock samples (such as
total RNA) that all vary in concentration. Having determined the
concentration of each sample (e.g. by spectrophotometry) it is often
desirable to dilute them all to a standard pre-determined
concentration. This is no easy task to tackle manually, since you need
to calculate the number of microlitres of each sample and diluent
needed for every separate dilution (without making math errors) and
then manually adjust the pipettor to a different setting for every
pipetting event. Not easy and sure to make your hands sore. A robot can
do this for you very easily with the right software. You simply specify
the starting concentration of each sample and the final concentration
you want and the robot will do the rest.
Finally, another application that is very useful for qPCR is setting up
a dilution series for quantification standards. Again, good software
makes this easy and will let you also let you determine several
dilution ratios (e.g. 1:2, 1:5. 1:10) in a series.
time RT-PCR expression results depend pivotal
on the quality of pipetting!
| Stephen Bustin demonstrated very nicely
in his review "Quantification of
mRNA using real-time reverse
transcription PCR (RT-PCR): trends and problems.", that the
introduce addtional variability in the mRNA quantification, when
manually pipetting real-time RT-PCR. In his experiments the
replicate Cts obtained by three individuals vaired between 8.7×105
numbers/µg total RNA. Stephen Bustin concludes these "results
provide a convincing argument for
the use of robots when
reproducibility and inter-laboratory
comparability are the main concerns".
using pipetting robots
Real-Time Quantitative Reverse Transcription PCR
L. Bookout, Carolyn L. Cummins, Martha F. Kramer, Jean M. Pesola,
and David J. Mangelsdorf
Protocols in Molecular Biology (2006) unit 15.8 suppl. 73
This unit describes
the use of real-time quantitative PCR (qPCR) for
high-throughput analysis of RNA expression. The topics covered include:
the standard curve method; production and
quantification of RNA standards (see Support Protocol 1); an
efficiency-corrected Ct (cycle time, also called cycle threshold or
crossing point) method (see Basic Protocol 2); the comparative cycle
time, or delta-delta-Ct method (see Alternate Protocol); and design and
validation of QPCR primers and probes for both SYBR Green– and
TaqMan-based assays. While the unit describes
the use of the Applied Biosystems 7900HT (high-throughput, 384-well)
instrument, the protocols may be utilized for any real-time PCR
instrument. The highthroughput design allows analysis of the levels of
transcripts from a number of genes of interest (GOIs) at one time by
using the appropriate primer set for each gene. (Within this unit, the
term GOI will refer to the actual gene of interest as well as its RNA
product or cDNA copy.) ... ...
|Comparison of SYTO9
and SYBR Green I for real-time polymerase chain reaction and
investigation of the effect of dye concentration on amplification and
melting curve analysis.
T. Monis, Steven Giglio, Christopher P. Saint
Biochemistry 340 (2005) 24–34
of One-Step and Two-Step Real-Time RT-PCR Using SuperScript III
Michael J. Wacker and Michael P. Godard
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 16, ISSUE 3, SEPTEMBER 2005
|Age-Related Changes in Relative Expression
of Real-Time PCR Housekeeping Genes in Human Skeletal Muscle.
Chad D. Touchberry, Michael J. Wacker, Scott R. Richmond, Samantha A.
Michael P. Godard
Journal of Biomolecular Techniques 17: 157–162
aureus Genotyping Using Novel Real-Time PCR Formats.
Huygens, John Inman-Bamber, Graeme R. Nimmo, Wendy Munckhof, Jacqueline
Schooneveldt, Bruce Harrison, Jennifer A. McMahon, and Philip M. Giffard
OF CLINICAL MICROBIOLOGY, Oct. 2006, p. 3712–3719
and Characterization of Candidate Reference Materials for Telomerase
John P. Jakupciak, Peter E. Barker, Wendy Wang, Sudhir Srivastava, and
Donald H. Atha
Clinical Chemistry (2005) 51:8 143-1450
and Automation of Hematopoietic Chimerism Analysis Based on Real-Time
Quantitative Polymerase Chain Reaction.
Tania N. Masmas, Hans O. Madsen, Soren L. Petersen, Lars P. Ryder, Arne
Svejgaard, Mehdi Alizadeh, Lars L. Vindelov
Biology of Blood and Marrow Transplantation 11:558-566 (2005)
high-throughput immunomagnetic separation-PCR for detection of
Mycobacterium avium subsp. paratuberculosis in bovine milk.
Christoph Metzger-Boddien, Daryush Khaschabi, Michael Schönbauer,
Sylvia Boddien, Thomas Schlederer, Johannes Kehle
International Journal of Food Microbiology 110 (2006) 201–208
evaluation of Fusarium DNA extraction from mycelia and wheat for
down-stream real-time PCR quantification and correlation to mycotoxin
Elisabeth Fredlund, Ann Gidlund, Monica Olsen, Thomas Börjesson,
Niels Henrik Hytte Spliid, Magnus Simonsson
Journal of Microbiological Methods (2008)
automation performs a nested RT-PCR analysis for HCV without
introducing sample contamination.
Theodore E. Mifflin, Christopher A. Estey, and Robin A. Felder
Clinica Chimica Acta Volume 290, Issue 2, 5 January 2000, Pages 199-211
HBV DNA and HCV RNA detection system using a nucleic acid purification
robot and real-time detection PCR: its application to analysis of
Shigeki Mitsunaga, Kayoko Fujimura, Chieko Matsumoto, Rieko Shiozawa,
Shinichi Hirakawa, Kazunori Nakajima, Kenji Tadokoro, and Takeo Juji
Transfusion 42 (2002): 100-106
of a fluorescence-based multiplex PCR for the laboratory confirmation
of common bacterial pathogens.
Karen Smith Mathew A. Diggl and Stuart C. Clarke
Journal of Medical Microbiology (2004), 53, 115–117
Automated PCR Setup robot
The standard curve
charts and calculated data below are from a recent customer
demonstration and provided courtesy
of Chris Jay, PhD (Sr.
Research Associate, Gradalis, Inc., Dallas, Texas). miRNA Profiling for Diagnosis and
Prognosis of Human Cancer. Chris repeatedly
set up standards and
96-well reactions with a CAS-1200. Tip re-use was set to 8 times (the
CAS-1200 can be set to intelligently reuse tips
a specified number of times). Further setup details can be found at the
end of this document. Each data set is
summarized from standard PCR quantification reports (base line
subtracted curve fit data [FAM] run and analyzed on
a Bio-Rad iCycler instrument and
software. Similar results can be
expected with any other 96-well real-time instrument.
notes from Chris Jay:
“We actually made a
master mix. The volumes below are for 1 well. If done manually, I
multiply the master mix times the total number of wells, and add 10%
for pipetting error. For the robot, I set up the reaction mix per well,
and the software calculated how much primer, 2x Taq mix, and water I
needed, and the robot made the master mix. Then it transferred 18.67 μL
MM per well and added 1.33 μL of appropriate DNA.” => application note
||PIRO – the ultimate
comfort in qPCR setup
The PIRO is
a newly developed
pipetting robot, based on long term knowledge and experience from users
and developers. The PIRO has been designed for the needs of qPCR
laboratories, allowing versatility, precision, reproducibility, and
safety combined with highly intuitive software for easy setup of
reactions. Even though the PIRO has a small bench top outlay, it allows
for 16 positions to be used. Setting up 384-well plates without running
out of tips, pipetting primary tubes without restrictions to its depth,
newly developed software features to facilitate the ease of use, or
interchangeable pipetting heads (in progress) are just a few features
integrated in the PIRO.
For more information, please download our
flyer contact firstname.lastname@example.org or visit our web
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