Its a question of conservation

The conservation of rare and low-yielding biological samples is becoming increasingly important as investigators routinely work with methods using progressively smaller amounts of isolated sample. Here Philippe Desjardins tells us how to be successfully thrifty with spectrophotometry samples

Novel spectroscopic technology enables absorbance and fluorescence measurements using sample volumes of just 1μL. Microsample analysis has now progressed in the areas of higher throughput spectrophotometry and limited-mass fluorometry.

The ability to concurrently measure multiple 1μL samples addresses the demand for increased throughput using absorbance spectroscopy. The development of small-volume fluorescence assays for use with microsample detection techniques has also dramatically reduced the amount of material needed for fluorescence analysis. By combining microvolumes with the sensitivity of fluorescence, detection of as little as 2pg of dsDNA is possible. Increased-throughput absorbance analysis of minute samples and reduction in total mass required per fluorescent assay represent the latest breakthroughs in microvolume quantitation technology.

In order to meet the demand for simple microvolume measurements, Thermo Fisher Scientific, developed a sample retention system. This system allows absorbance and fluorescence analysis of 1μL samples without the use of cuvettes or capillaries. This technology greatly reduces the amount of time required for spectral analysis by removing time-consuming steps such as cleaning cuvettes and preparing dilutions. However, performing spectral analysis one sample at a time is often impractical when many samples are involved.

Investigators involved with high sample turnover, such as genotyping facilities and molecular repositories, not only require conservation of sample for downstream application, but often handle hundreds or thousands of samples at any given time. Although the advent of 1μL spectrophotometry allows microvolume quantitation with maximum conservation of sample, the limited throughput is often an issue. Consequently, some investigators simply forgo desired quality-control checks or apply assumptions based on sampling. Many researchers use fluorescent plate readers that provide higher-throughput capability, but they are often displeased with the inherent variability of the results, the sample preparation time required, and the amounts of material consumed in each reaction. To address this issue and further improve lab productivity, Thermo Fisher Scientific has introduced the NanoDrop 8000 8-Sample spectrophotometer that allows for higher throughput than the single sample spectrophotometer. Using an eight-channel pipettor, 1μL samples are pipetted directly onto the sample stage that consists of an array of eight optical surfaces (Figure 1). The eight-sample UV/VIS spectrophotometer then uses the inherent surface tension to capture and hold the 1-uL samples between the upper and lower optical surfaces during the spectral readings. The entire measurement cycle for 8 samples (sample loading, spectral reading, and sample removal) takes less than 30 seconds. Using this new system, researchers can now perform quality control checks at critical points throughout workflows in ways that are either difficult or infeasible using single sample spectrophotometers or plate readers. Incorporating more quality-control measurements leads to a higher degree of confidence in the validity of data and ultimately improves productivity.

New advances in microvolume fluorometry are improving options for quantitation and downstream applications. The NanoDrop 3300 fluorospectrometer has cuvette-less sample retention technology that holds a single 1-2μL sample and measures the fluorescence emission spectrum in less than ten seconds (Figure 2). Fluorophore excitation occurs from one of three light-emitting diodes (LEDs): UV, blue, or white. The use of the broad spectrum, nonfiltered white LED is enabled by the combination of direct coupling of the sample to the optics and signal-processing technology. The broad excitation range allows for a wide range of common fluorophores to be measured (without the need for filter changes or a monochromator). This system provides versatility for experienced fluorescence users and simplicity for investigators less acquainted with fluorescence-based techniques. Microvolume fluorescent measurement capability allows widely used nucleic acid quantitation assays, such as PicoGreen (dsDNA) and RiboGreen (RNA) assays, to be scaled down to small total reaction volumes such as 10μL. This reduced reaction volume provides a substantial reduction in total sample mass required when compared to that required for plate readers and traditional 1cm cuvette fluorometers. While microvolume fluorometry may not measure ultra-low concentrations, it does lower the fluorescent detection limit of sample mass by more than an order of magnitude.

Using the PicoGreen dye, the microvolume fluorometer can detect as little as 2pg dsDNA, while a cuvette or microplate PicoGreen assay needs a minimum of 25-50pg dsDNA for detection. When working with samples of limited biomass (such as with microgenomics and microproteomics), lowering the total mass needed per measurement is more important than the ability to measure samples of low concentration. Initial field studies have shown that the ability to quantitate total RNA extracted from individual Laser Capture Microdissection (LCM) samples using the 10μL reaction volume has the potential to eliminate the practice of sample pooling, thus allowing analysis of individual samples.

Pooling samples is often necessary in order to obtain the minimum sample mass required for traditional larger volume plate readers or cuvette-based fluorometers. By reducing the amount of sample mass required for analysis, researchers save time acquiring initial material of interest, which often involves using costly time-consuming isolation and extraction techniques. By greatly reducing the total reaction volume of a fluorescence assay, less sample is required for basic quantitation, conserving the majority of the sample for downstream applications.

In addition to basic quantitation assays, such as PicoGreen and RiboGreen, the microvolume fluorometer is capable of performing more sophisticated fluorescent measurements. Excitation across a broad wavelength range enables the emission profile of several fluorophores in a single measurement. The broad spectral output lends itself to FRET analysis for homogeneous fluorescent assays. FRET assays are widely used throughout the scientific community in order to confirm the existence of a specific target, as well as revealing the interaction between molecules. Microvolume FRET analysis was conducted with FRET/Cy5 oligo nucleotide using the filtered 470nm LED in the presence and in the absence of complementary sequence. FRET oligo alone produced FITC (donor) fluorescence with no significant signal at the Cy5 (acceptor) emission wavelength (680nm). Once complimentary sequence target was introduced and hybridised to the probe, a marked increase of Cy5 signal and decrease in FITC fluorescence was observed.

Molecular beacon probes are similar to FRET oligo probes yet differ by using a nonfluorescent quencher to replace the acceptor. In the absence of a target sequence, the molecular beacon resides as a “stem loop” conformation via an intra-complimentary sequence allowing the quencher to come in close proximity to the fluorophore and suppress the fluorescent signal. The inherent single fluorophore/ quencher construction of the molecular beacon allows the user to take full advantage of all three excitation sources, the most versatile of which is the broad range white LED (465nm-650 nm). Additionally, using the white LED source for excitation enables the measurement of multiple molecular beacons within the same sample.

Researchers at the Public Health Research Institute in Newark, NJ, have successfully measured molecular beacons utilising the microvolume fluorescence technology. The quality of several HPLC purified molecular beacon probes were determined by comparing signal to background values. The probes were diluted to a concentration of 0.1μM and measured in the absence (background) and in the presence (signal) of the complimentary target sequence. Comparable results were obtained on the ND-3300 using 75-fold less sample than the larger volume reference instrument (Table 1). The overall reduction of sample required for fluorescent experimentation using a microvolume fluorometer coupled with a broad excitation range allows for more information to be generated from a single measurement while conserving a majority of the stock sample for downstream applications.

Philippe Desjardins became Scientific Marketing Manager at NanoDrop products (now part of Thermo Fisher Scientific) five years ago. He previously performed 10 years of genetic research at the University of Pennsylvania where he received a master of biotechnology degree.