The PCR Blog: The Polymerase Chain Reaction

A comprehensive list of qPCR Web Resources including qPCR machine manufacturers' websites, PCR web resources, and PCR news groups.

from Julie Logan, Kirstin Edwards and Nick Saunders in Real-Time PCR: Current Technology and Applications

Further reading: qPCR Web Resources

Labels: qPCR, real-time pcr

In weighing up the pros and cons of the different real-time PCR platforms or qPCR machines for your laboratory, factors to consider include: supported chemistries; multiplex capability for that chemistry; throughput; flexibility; format; easy-of-use and robust software package; reproducibility; speed; size; technical support; customer support and not least the cost, not only of the initial equipment outlay and servicing but also the associated cost of consumables and reagents.

The following qPCR machines are compared for various features to help you decide which instrument is most suitable for your needs.

• Applied Biosystems: ABI 7300, ABI 7500, ABI 7500 Fast, ABI 7900 Fast HT with automation accessory, ABI StepOne
• Roche: LightCycler 480, LightCycler 1.5, LightCycler 2.0
• Stratagene: Mx4000, Mx3000P, Mx3005P
• Cepheid: SmartCycler
• Corbett: Rotor-Gene 6000
• Eppendorf: Mastercycler ep realplex
• BioRad: MiniOpticon, MyiQ, Opticon2, Chromo4, iQ5

read more ...

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: qPCR, qPCR instruments, qPCR machines, real time PCR machines

In weighing up the pros and cons of the different platforms for your laboratory, factors to consider include: supported chemistries; multiplex capability for that chemistry; throughput; flexibility; format; easy-of-use and robust software package; reproducibility; speed; size; technical support; customer support and not least the cost, not only of the initial equipment outlay and servicing but also the associated cost of consumables and reagents. It is also possible to 'try before you buy', most companies will provide a loan machine. It is wise to test a few of these once you have narrowed down your choice. User experiences should not be overlooked and there are now a number of useful websites and news groups where you can address you questions and queries.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: ease-of-use, flexibility, format, multiplex, pros and cons, reproducibility, size, software, speed, supported chemistries, technical support, throughput, user experiences

Newly introduced qPCR machines include the Cepheid GeneXpert and Enigma Diagnostics Enigma ML instruments, which combine automated sample preparation and real-time PCR into a single integrated compact bench top instrument, which opens new avenues for real-time PCR in field-based and point-of-care environments. At the other end of the scale is the introduction of nanoliter high throughput quantitative PCR. The Biotrove OpenArray system allows 3072 33nl reactions on a microscope slide array format, which equates to a single operator performing 27,000 qPCR reactions in a working day. Another interesting development on the horizon is ultra fast real-time PCR chips capable of performing 40 cycles in under six minutes.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: Biotrove OpenArray, Cepheid GeneXpert, Enigma Diagnostics, nanoliter high throughput qPCR, PCR chips

The majority of instrument manufacturers supply optimised real-time PCR mastermixes, these reagents benefit from being quality controlled, are easy to use, and usually offer reproducible and reliable results. However, for certain laboratories the additional cost can be prohibitive, however with an array of third party companies (e.g. Qiagen, Eurogentec, Invitrogen) now supplying real-time reagents, competition in this market should lead to reduced costs. Target specific kits for a range of applications are available from some manufacturers and other companies (e.g. Roche, Applied Biosystems, Qiagen). Additionally, Applied Biosystems offer Assays-on-Demand and Assays-by-Design services for SNP genotyping by real-time PCR. A number of companies now offer real-time PCR services from primer design right through to assay design and validation, quality assurance and other custom services.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: Applied Biosystems, Eurogentec, Invitrogen, PCR mastermixes, Qiagen

Ideally, the analysis software supplied with the platform should be as user-friendly as possible but it is also important to check that the software can fully analyse results of the chosen probe chemistry. Some platforms and analysis software suites are biased towards certain chemistries. Some real-time instruments also have specific primer and probe design software that is either supplied with the hardware or available at extra cost. Such software can help simplify and speed up the assay design process and is optimised for that system and reagents. The LightCyclers also have specific relative quantification software that is designed to determine the exact relative nucleic acid concentration normalized to a calibrator sample. This software speeds up and greatly simplifies relative quantification.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: qPCR, qPCR software, qPCR thermal cycler, qRT-PCR, RT-PCR software, software

Some platforms offer a low to medium throughput but are more flexible. Although the format may allow only 32 or even 16 samples, thermocycling times are faster and multiple runs can be performed thereby increasing the potential throughput. Performing multiple runs rapidly may be an advantage when several applications are employed which require different cycling parameters. The SmartCycler offers a different concept to real-time PCR of random access, which means independent programming of cycling parameters for 16 different assays. Each reaction vessel has its own element so that runs in available slots may be started any time, whilst other reaction sites are already in use. It is also modular allowing up to six units to be operated by a single computer. Others, such as the Chromo4 offers a modular design for maximum flexibility.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Several of the platforms employ a standard 96-well block format or interchangeable 384-well block and offer a medium to high throughput. An advantage of employing a block format is that standard PCR plates and tubes can be used and these tend to be cheaper than instrument-specific plastics (SmartCycler, Rotor-Gene) or glass capillaries (LightCyclers 1.5 & 2.0). Also, the LightCyclers 1.5 & 2.0 and SmartCycler require centrifugation to move sample into the reaction vessel and these alternative designs may not be suitable for all applications. For the highest throughput, the ABI 7900HT combines a 384-well plate format with an automation accessory, which allows for up to 84 plates to be loaded and unloaded, providing a throughput of >5000 wells per 8-hour day or 30,000 wells for end-point analysis only. The LightCycler 480 also offers a similar level of automation and high-throughput workflow capability. These high throughput instruments are ideal for dedicated laboratories where large batches of samples are run, with few different cycling parameters. An additional specification of a few platforms is the ability to perform gradient thermocycling, which can be very useful at the assay optimisation stage.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: ABI 7900HT, LightCycler, Rotor-Gene, SmartCycler

Second, third and fourth generation real-time PCR platforms have been developed with improvements in multiplexing and increased throughput capabilities. The optical characteristics of a given platform clearly have an impact on the ability to multiplex and also determine which probe systems are compatible. In addition, the analysis software may also predetermine the appropriate chemistries.

Iit is important to point out that the platform and choice of fluorescent chemistry are strongly linked. Indeed some platforms are biased towards a particular probe system and whilst the optics permit different probe chemistries to be excited and detected, often the analysis software does not and the user is required to export the data to a spreadsheet program for detailed user analysis. For example, the Applied Biosystems platforms do not officially support any chemistries other than hydrolysis probes and SYBR Green and only support duplexing (Logan and Edwards, 2009). Therefore, the reporting chemistry required for an application should be strongly considered before a choice of platform is made.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: Applied Biosystems, chemistries, chemistry, duplexing, fluorescent chemistry, hydrolysis probes, multiplexing, SYBR Green