30 chromatography/spectroscopy
➠
perform sample dilutions. The system automatically finds many quality control steps including the initial RNA
the optimal path in less than five seconds. extraction as well as checking the fluorescence labelling
Greatly reducing the volume necessary to perform efficiency of the final probe were simply not performed –
measurement has several advantages. Conserving limited, often resulting in failed arrays. Providing a fast, reliable
often highly valuable samples is paramount in situations way to check samples at various stages of microarray
that involve extraction of biomolecules such as DNA, probe development, from extraction through labeling,
RNA, and proteins, from a limited cell mass. There are a greatly improves the chance for a successful array.
wide variety of circumstances in which the sample mass NanoDrop instruments have been widely accepted
is very limited and require microsample quantitation in common research environments, but are increasingly
post extraction. These include samples derived from being used for clinical applications. The differentiating
laser-capture microdissection, needle biopsies, tumor factor between molecular biology techniques performed
subtyping, surgical resections, forensic specimens, in basic research environments and the same techniques
extremophiles and many more. Virtually any situation performed in medical settings is simply that the results
in which the sample is derived from limited cell mass is are used for clinical purposes. The techniques themselves
applicable for NanoDrop technology quantitation. Even remain the same. Due to the often limited amounts
though microsample pedestal measurements are ideal of material acquired from clinical samples, reducing
for limited cell mass scenarios, the speed of the system the amount of volume required for quality control
also makes it an attractive alternative to traditional steps is important. This is the main reason NanoDrop
quantitation methods for situations in which the quantity microsample quantitation is being adopted in several
of sample is plentiful. areas of molecular diagnostics.
The microsample quantitation capability has greatly One example is sequence-based genotyping in
increased the efficiency of many molecular workflows which microsample technology is being used to
throughout the life sciences. These workflows range quantify critical biomolecules at several steps during
from expression studies including microarrays and the diagnostic workflow. After a clinical specimen is
quantitative real-time PCR, to clinical workflows such acquired, DNA extraction is performed. Using a minute
as HLA (human lymphocyte antigen) typing for organ amount of elution, the NanoDrop 2000c determines the
transplantation. Scientists would often forgo taking concentration and purity of the extracted sample. This
measurements at various time points throughout a information is critical for optimising the next step in the
workflow due to limited sample, limited time, or both. process – DNA amplification by PCR (polymerase chain
Reducing the amount of sample required for measurement reaction). The microsample quality control measurement
as well as increasing the speed of the measurement itself, not only conserves the maximum amount of the original
allows for more quality control steps to be performed. By genetic material, it allows the clinician to determine
allowing measurements to be performed at crucial steps the smallest amount of template DNA that can be
throughout a given workflow, microsample quantitation used for a successful PCR reaction. Post amplification,
technology greatly increases the chances for success. the instrument can also be used to measure the final
For example, expression studies often require concentration of PCR product. This measurement is used
extracted biomolecules such as RNA to be treated at to optimise the sequencing reaction, which requires a
various steps in preparation for a downstream assay such specific ratio of DNA to primer concentration. By using
as microarray analysis. Prior to microsample quantitation, 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
into the clinical setting, microvolume quality control
Fig. 3. The distance between the optical pedestals changes in real time steps that are successful in the research environment
to optimise the path length during measurement. are applicable to molecular diagnostics. By consuming
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