by Melanie Yanek

The benefits of automating biological assays include the elimination of problems introduced through human error, increased throughput and consistency, and decreased costs. Standard PCR and quantitative PCR are applications that have been improved from utilizing automation. Indeed, industry standard reaction volumes have progressed from a 100 μl reaction volume 10 years ago, to 50, 25 and now to 10 μl. The decrease in volume has occurred due to innovations in aspiration and dispensing technology. Liquid handling systems that utilize state of the art dispensing mechanisms can accommodate PCR reactions as small as 250 nl.⁽¹⁾

Figure 1. In small volume reactions incremental changes in the component amounts, caused by pipetting errors, can significantly alter the outcome of a reaction.

The use of sophisticated liquid handling instruments in the preparation of reaction volumes offers a number of benefits to researchers: a decrease in cost per reaction; an improvement in overall reaction time; consistency of data; and the ability to process a large quantity of samples.

The most obvious benefit, immediately recognized from a reduction in reaction volume, is the cost savings. Moving from a 50 μl reaction volume to a 5 μl reaction volume will require the use of 5-fold less reagents. A further example of this comes from researchers using a Deerac Equator liquid handler (Deerac Fluidics, Dublin, Ireland), who have reduced gene expression reaction sizes from 10 μl to 2 μl and SNP genotyping assays from 5 μl to 250 nl. These researchers were able to reduce their total reaction cost by 20 fold, with the introduction of an automated liquid handling system.⁽²⁾

Another benefit to the scientist when using an automated system is the continued consistency of the data at small volumes when compared to reactions completed at higher volumes. In small volume reactions incremental changes in the component amounts, caused by pipetting errors, can significantly alter the outcome of a reaction (Figure 1). Therefore, as the volume of the reaction decreases, the kinetics of the reaction needs to remain in balance to generate the same data quality.⁽³⁾ To ensure that each component of the reaction is aliquoted in the same volume and therefore in the same concentration, automated liquid handling can offer greater accuracy than with manual pipetting.⁽⁴⁾ Indeed, when researchers utilizing the Equator system for SNP genotyping reduced their reaction size to 250nl, there was no loss in the scattergraph data integrity.⁽¹⁾ Therefore, researchers can be assured that reducing their reaction size is not going to compromise the data generated.

Figure 2. Water bath cyclers, such as the H2OBIT from ABgene, are the gold standard for ultra high throughput PCR.

As the volume of the reaction decreases, so too does the size and thickness of the reaction vessel. The result is that quicker heat transfer can occur from the PCR block through the reaction vessel and into the liquid.⁽²⁾ As the time taken for the entire reaction volume to reach its desired temperature will be shorter, it is possible to reduce the length of time required for each step in a PCR cycle. This leads to an overall reduction in the time to completion of a PCR reaction, allowing the user to increase the number of samples analyzed per day. The rate-limiting step of a PCR reaction now becomes the temperature ramping speed of the thermal cycler. Standard Peltier block ramping times are considerably slower than with a water bath cycler.⁽⁵⁾ A water bath cycler, such as the H₂OBIT (ABgene Inc., Figure 2), can keep up with the demands of ultra-high throughput PCR. The combination of reduced reaction volumes and the fast, accurate temperature changes that can be accomplished using a water bath cycler creates a reaction that takes half the time of a Peltier block reaction. When using a water bath cycler, reaction vessels can be used that offer higher throughput than standard Peltier blocks can currently support (e.g. 1536 well-plates).

Currently, the most common way to achieve increased throughput is to move from a 96-well plate to a 384-well plate. In concurrence with recent advances in liquid handling, scientists are being offered the opportunity to move their assays to a 1536-well plate format. Such high sample density and nanoliter working volumes require the use of liquid handling instruments such as those provided by Innovadyne Technologies Inc. (Santa Rosa, CA) and Deerac. Laboratory technicians using manual instruments cannot cope with the accuracy requirements of automated systems, due to the use of small reaction volumes in high quantities. Researchers should look towards liquid handling devices to maintain the consistency of their data and increase throughput.

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