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.