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Sophisticated sample isolation and extraction systems are continually being designed to work with smaller amounts of material, such as laser-capture microdissection and ultra-low volume extraction protocols. These systems allow researchers to precisely isolate specific cells of interest and extract the required biomolecules for analysis. It is essential that quality control steps are put in place such as sample quantitation that can analyse the small quantities of material being produced.
New sampling technology provides such quality control by allowing microvolume quantitation of only 1µl of sample, while virtually eliminating the need to dilute concentrated samples. A novel cuvetteless micro-quantitation method enables researchers to enhance and often integrate the use of several existing technologies such as laser capture microdissection, real-time PCR, microarrays and protein arrays (Fig.1).
Investigators can now monitor critical steps involved with several limited-volume assays by accurately and quickly determining sample concentrations with a consistency and reliability required for success. The versatility of this technology offers investigators the opportunity to explore possibilities previously prohibited using cuvette-based spectrophotometry.
How it works
By combining modern fibre optic technology and the inherent surface tension properties of small volumes, this Thermo Fisher Scientific-developed technology has made previously unattainable small volume quantitative measurements routine.
A single microlitre of sample is pipetted directly onto the lower optical (measurement) surface. As the upper optical fibre automatically engages the sample on the lower optical surface, a liquid column of a mechanically controlled path length (1mm) is formed and held in place by the sample's surface tension. Absorbance measurements are made through the hourglass-shaped column of liquid. The resulting data is sent to a standard PC where the full-spectrum sample profile and concentration are displayed and archived.
Each sample is measured using two different path lengths - 1mm and 0.2mm - providing an extensive dynamic range, for example 2ng/µl - 3700ng/µldsDNA. The short path length allows researchers to measure samples up to 50-fold higher in concentration than can be measured on classical 1cm cuvette-based systems, virtually eliminating the need for dilutions and the associated errors, time and cost. The simple cleanup method using a laboratory wipe has proven highly effective for eliminating carryover while quickly readying the instrument for the next measurement.
The cuvetteless system is complemented by software that includes modules designed to display an absorbance spectrum of the sample along with relevant information. The modules include nucleic acids, microarray, protein A280, proteins and labels, protein BCA, protein Bradford, protein Lowry, cell cultures and general uv-vis measurement.
As an example, the microarray module displays the full uv/vis spectrum of the sample as well as the calculated concentrations of both the nucleic acid and dye components of a labelled probe. In this way, researchers can check the efficiency of the labelling process and monitor the quality of hybridisation probes or labelled proteins while saving precious sample volume.
Practical applications
In the drive to create instruments that can manage even the smallest samples, spectrophotometry finally has a technology that keeps pace. New technology allows full spectrum measurement of 1 - 2 µl samples without the need for cuvettes or capillaries. The novel technology can also enable analysis of highly concentrated samples, virtually eliminating the need to perform dilutions that can introduce errors and waste valuable time. Cuvetteless technology is already used in applications including real-time PCR and microarrays.
Accurate and efficient quantitation of RNA from patient specimens is critical to generating reliable gene expression data, particularly in laboratories receiving a large volume of samples. RNA isolates from such specimens are often so limited that quantitation using cuvette-based spectrophotometers is not feasible. In these cases, cuvetteless technology uses only 1µl of the clinical sample to obtain reliable RNA quantification, eliminating the wasted RNA preparation that can occur with traditional methods.
After isolation from both normal and cancer specimens, the RNA must be quantified prior to reverse transcription, and when preparing samples for real-time PCR, it is important to add the same amount of RNA to each reverse transcription to ensure that the reaction's efficiencies are similar. Cuvetteless technology provides the reproducible and accurate RNA concentration measurements required in this process, eliminating the errors that can occur with the traditional cuvette-requiring alternative.
In array studies, analyses are routinely performed using RNA extracted from whole blood hematocrit samples (~30µl whole blood).
Quantitation of the RNA obtained from these samples can be problematic using traditional methods; however cuvetteless technology can accurately determine the full absorbance spectrum with only 1µl of such dilute RNA samples. The cuvetteless method can also form part of quality control measures, for example assessing each step in the generation of our amplified probes for array analysis.
Conclusion
Cuvetteless technology now enables investigators to use one microlitre to accurately and easily quantitate undiluted samples. The attributes of this new technology enable scientists in various fields to perform a variety of limited-sample assays with a higher degree of confidence. The NanoDrop1000 is being integrated into many diverse workflows, ranging from real-time expression studies to tumour genotyping for cancer research. The versatility of applications allows researchers to use the instrument not only for limited volumes but also for everyday quantification.
At a hearing at the European Patent Office on November 22, 2007, the EPO allowed broad method and apparatus patent claims to Thermo Fisher Scientific pertaining to spectrophotometric measurement of small liquid samples held in place by surface tension. The European patent is expected to be granted shortly.
- Philippe Desjardins is Scientific Marketing Manager at NanoDrop products, now part of Thermo Fisher Scientific. www.nanodrop.com