qPCR robotics  -  qPCR applications using pipetting robots

The Trend to qPCR Automation

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 posses 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. Let’s 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.

Choosing a Robot
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.

Here 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).

Workspace – 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.

Channels – 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 some robots.

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.

Calibration – 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.

Level Sensing – 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.

The 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.

Real 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 operator 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 and 2.7×103 copy 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".

qPCR applications using pipetting robots

High-Throughput Real-Time Quantitative Reverse Transcription PCR
Angie L. Bookout, Carolyn L. Cummins, Martha F. Kramer, Jean M. Pesola, and David J. Mangelsdorf
Current 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 DNA melting curve analysis.
Paul T. Monis, Steven Giglio, Christopher P. Saint
Analytical Biochemistry 340 (2005) 24–34

Analysis of One-Step and Two-Step Real-Time RT-PCR Using SuperScript III
Michael J. Wacker and Michael P. Godard

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. Whitman, and
Michael P. Godard
Journal of Biomolecular Techniques 17: 157–162

Staphylococcus aureus Genotyping Using Novel Real-Time PCR Formats.
Flavia Huygens, John Inman-Bamber, Graeme R. Nimmo, Wendy Munckhof, Jacqueline Schooneveldt, Bruce Harrison, Jennifer A. McMahon, and Philip M. Giffard

Preparation and Characterization of Candidate Reference Materials for Telomerase Assays.
John P. Jakupciak, Peter E. Barker, Wendy Wang, Sudhir Srivastava, and Donald H. Atha
Clinical Chemistry (2005) 51:8 143-1450

Evaluation 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)

Automated 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

Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels.
Elisabeth Fredlund, Ann Gidlund, Monica Olsen, Thomas Börjesson, Niels Henrik Hytte Spliid, Magnus Simonsson
Journal of Microbiological Methods (2008)

Robotic 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

High-throughput HBV DNA and HCV RNA detection system using a nucleic acid purification robot and real-time detection PCR: its application to analysis of posttransfusion hepatitis.
Shigeki Mitsunaga, Kayoko Fujimura, Chieko Matsumoto, Rieko Shiozawa, Shinichi Hirakawa, Kazunori Nakajima, Kenji Tadokoro, and Takeo Juji
Transfusion 42 (2002): 100-106

Automation 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

CAS-1200 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.

Experimental 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  info@dornier-ltf.com  or visit our web page:  www.dornier-ltf.com

Real quantitative PCR needs automated pipetting

Automated epMotion pipetting can show a significant reproducibility advantage, when compared to manual set-ups. With a pipetting range down to just 1µl, its unmatched accuracy, reproducibility and easy to use philosophy mean the epMotion is the unique pipetting system for qPCR. epMotion automated qPCR set-up guarantee highly consistent and reproducible results within and across experiments. Reactions can be performed in lower volumes and with fewer replicates, thereby reducing reagent cost significantly.
Real time PCR needs real speed
The Mastercycler ep realplex is the perfect partner for automated real time PCR. Powered by the highly accurate and uniform Mastercycler ep thermal engine, the realplex defines a new dimension of speed for automated real time PCR. Utilising the high speed silver block and impulse PCR technology, amplification can be performed in less than 30 minutes. 
Packaged in a compact and robust design matched with a peace of mind real time-PCR licence ensure the Mastercycler ep realplex is a real marvel in space and time.

epMotion – affordable automation:

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