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Assay Tutorials : Apr 1, 2013 ( )
Flexible qPCR Assay Assembly
Liquid Handler Designed to Reduce Contamination Risk and Operational Costs!--h2>
Real-time quantitative polymerase chain reaction (qPCR) is widely used throughout drug discovery to examine gene expression in response to drug stimuli. The technique is often used to confirm endogenous gene expression levels determined by a less sensitive (but more cost-effective) technique, such as a microarray or reporter gene assay. The technique has additional application in areas where high-throughput techniques might be unavailable or unnecessary, such as the detection of viral, bacterial, or fungal pathogens in food or patient samples and the detection of cancer or other disease biomarkers in patient samples.
A wide variety of master mixes, probes, and specific gene expression assay kits have been created to address varying needs of the researcher. High operating costs and advances in qPCR instrumentation to enable higher density 384- and 1,536-well microplate formats have incentivized researchers to miniaturize qPCR assays to reduce costs and increase throughput. Nonmicroplate-based formats also exist to ensure greater levels of sample density.
While precise quantitation of gene expression levels is a critical advantage of qPCR, the increased density and lower volume requirements become sensitive to variability in liquid-transfer techniques. Researchers often rely on hand-held pipettors in many instances to interface with increasingly small and complex assay formats. As a result, the value of qPCR, and the highly advanced instrumentation required to perform qPCR, is diminished by the abilities of cumbersome liquid-handling techniques. Many researchers therefore have turned to automated liquid handlers to retain the benefits of qPCR when attempting to miniaturize.
Many tip-based liquid handlers can address the throughput requirements of higher-density, lower-volume qPCR. This instrumentation usually requires tips and uses fixed-format pipetting heads, which constrains experimental usage and design.
Most generally work by employing the hand-held pipettor model to transfer reagents in a single step to 96- or 384-well formats. Researchers then require enough reagents to take into account transfer with 96 or 384 tips, plus an additional volume to account for the dead volume required to aspirate from this format.
For individual sample handling, the plastic tip presents benefits and risk. The benefit is that a pristine object can be used to transfer a unique sample, then discarded and replaced for the next sample. While the cost of an individual tip is insignificant, the cost of hundreds or thousands of tips per week presents a major operational cost hurdle. Researchers who choose to wash and reuse tips do so at the risk of introducing retained sample into other samples and experiments. This event, commonly termed “carryover”, significantly impacts the value of the technique and downstream data analysis as a result.
In this article we discuss how the Echo® 525 liquid handler from Labcyte can be utilized to reduce operational costs and eliminate the risk of carryover. The Echo platform uses low-power acoustic energy to transfer reagents to qPCR microplates. The Echo 525 platform has a 25 nL drop increment, which ensures a high degree of volume transfer flexibility.
Tipless Assay Assembly
Tipless, touchless acoustic droplet ejection with the Echo liquid handler eliminates the cost of disposable tips. This study utilized the Echo 525 liquid handler to assemble low-volume qPCR assays at five microliters per second or greater, easily keeping pace with high-throughput demands. The capability to transfer all qPCR assay components was verified by the setup of qPCR experiments for three reference genes at a 5 μL volume in a 384-well format.
The RealTime ready DNA Probes Master Mix and Universal Probe Library (Roche Applied Science) human reference genes including G6PD were used to confirm that all reagents could be successfully transferred acoustically (Figure 1). The average Cp for G6PD expression was 26.39 with a standard deviation of 0.18. Other genes tested showed similar performance levels.
The Echo 525 system uses a single acoustic transducer, capable of moving to any well of the source microplate. The destination microplate also moves to align any destination well to any location. Reagents can therefore be transferred from any well to any well.
Rather than requiring a single transfer from 384-well source wells to the 384-well qPCR plate, less than 40 source wells can be utilized for transferring all reagents into the entire 384-well qPCR microplate. This simplifies assay setup and reduces the reagent requirements typical of 384-well tip-based liquid handlers or bulk fluid dispensers.
Direct Dilution of Reagents
A key function of assay development is the ability to transfer reagents at different volumes. In high-throughput assays, volumes are generally fixed prior to starting the protocol. Gene expression assays often require an additional variation in sample amount. This could be due to a screen where the concentration of template is expected to vary significantly across a set of treatment or screening conditions.
To address this requirement with the Echo 525 liquid handler, the added volume of cDNA template was varied across multiple wells. Using a two-step process, the Echo 525 platform transferred master mix with premixed primers and probes into a qPCR microplate, followed by transfers of human reference cDNA in incremental volumes. Human reference cDNA at a concentration of 0.05 ng/μL was used to create a dilution series from 1.25 pg to 15 pg cDNA (25 to 300 nL). Reference genes G6PD, GAPDH, HPRT, and PBGD were studied.
Linear delineation in Cp can be detected for small changes in cDNA amounts (Figure 2). This was shown over a fairly broad range of Cp values.
No Cross Contamination at High Densities
Acoustic transfer requires the ejection of droplets in a controlled manner (volume and direction). Linear drop trajectory is critical to ensure no chance of cross contamination to adjacent wells. For this test, the LightCycler® 1536 was used to verify that there was no cross contamination. 450 nL of RealTime ready DNA Probes Master mix, combined with G6PD primer and probe sets, was transferred into all wells of a 1,536-well qPCR microplate with a 1.5 mm diameter well. 2.5 ng of cDNA (50 nL) was transferred into one well, surrounded by eight wells with no cDNA added. Wells were scored (Figure 3) and no cross contamination to adjacent wells was observed.
The Labcyte Echo 525 liquid handler enables exciting new capabilities for 384-well qPCR experimental setup in the 3–10 μL range. The low-volume transfer increment enables scientists to explore qPCR miniaturization without sacrificing data quality that may come with imprecise or inaccurate liquid transfer at low volumes. Positional accuracy allows accurate transfer without causing carryover in 384-well and 1,536-well formats. Superior volumetric precision ensures excellent cycle quantification even with very little target DNA in very low reaction volumes. The Echo 525 liquid handler enables scientists to fully explore the capabilities of miniaturized PCR and other genomics applications. Tip costs can be eliminated and reagent consumption reduced—without sacrificing data quality.
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