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perform sample dilutions. The system automatically finds the optimal path in less than five seconds. Greatly reducing the volume necessary to perform
measurement has several advantages. Conserving limited, often highly valuable samples is paramount in situations that involve extraction of biomolecules such as DNA, RNA, and proteins, from a limited cell mass. There are a wide variety of circumstances in which the sample mass is very limited and require microsample quantitation post extraction. These include samples derived from laser-capture microdissection, needle biopsies, tumor subtyping, surgical resections, forensic specimens, extremophiles and many more. Virtually any situation in which the sample is derived from limited cell mass is applicable for NanoDrop technology quantitation. Even though microsample pedestal measurements are ideal for limited cell mass scenarios, the speed of the system also makes it an attractive alternative to traditional quantitation methods for situations in which the quantity of sample is plentiful. The microsample quantitation capability has greatly
increased the efficiency of many molecular workflows throughout the life sciences. These workflows range from expression studies including microarrays and quantitative real-time PCR, to clinical workflows such as HLA (human lymphocyte antigen) typing for organ transplantation. Scientists would often forgo taking measurements at various time points throughout a workflow due to limited sample, limited time, or both. Reducing the amount of sample required for measurement as well as increasing the speed of the measurement itself, allows for more quality control steps to be performed. By allowing measurements to be performed at crucial steps throughout a given workflow, microsample quantitation technology greatly increases the chances for success. For example, expression studies often require
extracted biomolecules such as RNA to be treated at various steps in preparation for a downstream assay such as microarray analysis. Prior to microsample quantitation,
many quality control steps including the initial RNA extraction as well as checking the fluorescence labelling efficiency of the final probe were simply not performed – often resulting in failed arrays. Providing a fast, reliable way to check samples at various stages of microarray probe development, from extraction through labeling, greatly improves the chance for a successful array. NanoDrop instruments have been widely accepted in common research environments, but are increasingly being used for clinical applications. The differentiating factor between molecular biology techniques performed in basic research environments and the same techniques performed in medical settings is simply that the results are used for clinical purposes. The techniques themselves remain the same. Due to the often limited amounts of material acquired from clinical samples, reducing the amount of volume required for quality control steps is important. This is the main reason NanoDrop microsample quantitation is being adopted in several areas of molecular diagnostics. One example is sequence-based genotyping in which microsample technology is being used to quantify critical biomolecules at several steps during the diagnostic workflow. After a clinical specimen is acquired, DNA extraction is performed. Using a minute amount of elution, the NanoDrop 2000c determines the concentration and purity of the extracted sample. This information is critical for optimising the next step in the process – DNA amplification by PCR (polymerase chain reaction). The microsample quality control measurement not only conserves the maximum amount of the original genetic material, it allows the clinician to determine the smallest amount of template DNA that can be used for a successful PCR reaction. Post amplification, the instrument can also be used to measure the final concentration of PCR product. This measurement is used to optimise the sequencing reaction, which requires a specific ratio of DNA to primer concentration. By using microsample quantitation instrumentation, quality control steps are easy to perform throughout the process, without compromising accuracy or consuming large portions of sample. The same is true for many molecular diagnostic workflows, such as microarray-based diagnostics and tissue typing for patient-donor crossmatching. Furthermore, laboratories involved in medical
research are continually developing new clinical tests that use molecular biology techniques. For example, the development of solid tumor testing is often extremely difficult due to the small amounts of available tumor cell mass. The samples are often difficult or simply impossible to reacquire. The amount of genetic material extracted from a specific solid tumor may be so limited that the only possible method of measuring the sample is by microsample quantitation. As more molecular biology techniques are integrated
Fig. 3. The distance between the optical pedestals changes in real time to optimise the path length during measurement.
into the clinical setting, microvolume quality control steps that are successful in the research environment are applicable to molecular diagnostics. By consuming
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