Ultrasensitive RNA profiling: Counting single molecules on microarrays (2006)

The ability to analyze RNA expression of a whole genome in a single microarray experiment has had widespread impact on basic research as well as drug discovery and development (Marton et al. 1998; Brown and Botstein 1999; Bentwich et al. 2005). It also holds promise as a tool to guide treatment in the clinic (Golub et al. 1999; Perou et al. 1999; Alizadeh et al. 2000; Mattie et al. 2006; Yanaihara et al. 2006). What else lies in the future for microarray technology? Until recently, researchers have rightly limited their horizons to what the technology can do rather than what it ought to do. However, there is agreement that it ought to be able to detect RNA from small amounts of sample material, even single cells, in a way that faithfully represents RNA abundances. In addition, there would be advantages to describing abundance levels in absolute terms— numbers or molar amounts—rather than relative values, so that comparisons between genes and across many experiments can be undertaken. Furthermore, the dynamic range of microarrays should match the range of expression levels found in cells (Holland 2002). Indeed, if the sensitivity, dynamic range, and quantitative nature of measurements could be improved, the current need for cross-validation with real-time PCR would become redundant. In order to address these issues, a change in the way we look at molecules on a microarray is needed. At present, an ensemble signal is acquired from the plurality of labeled molecules that interact with probes in a microarray spot. However, if this signal were to be resolved into its constituent parts, the individual molecules, the output would be more easily quantitated because it would be digital: An individual molecule (one bit of information) can be either present or absent, the binary 1, 0. Moreover, if single molecules can be detected, then it follows that the detection is highly sensitive and the amount of sample material required can be reduced accordingly. Although the detection of individually resolvable fluorescent molecules on surfaces has been described previously (Funatsu et al. 1995; Lizardi et al. 1998; Unger et al. 1999), analysis of microarrays at the single molecule level is more challenging, requiring the high-resolution scanning of centimeter-square areas with high speed. A recent study (Hesse et al. 2006) has shown the application of a fast CCD scanning method to a conventional long oligomer microarray spotted on a nonconventional slide, at a resolution that enables individual molecules to be resolved. Although individual Cy3 and Cy5 dye molecules—which are the labels most commonly used in microarray experiments—emit enough photons to be detected by a typical microarray scanner, scanners are not set up to resolve molecules individually. This is because of a trade-off between the time it takes to perform the scan and the resolution that is achieved. Conventional pixel-bypixel scanners would take impossibly long (weeks) to scan 1 cm of a microarray at the ∼200-nm resolution required for single molecule analysis. Remarkably, the system used by Hesse et al. (2006) was able to scan a 1-cm area in under 1 h. This was done by a form of CCD operation, time-delay integration (TDI) or “scanning” mode, that is normally used in astronomy for finding trajectories of objects such as asteroids (Netten et al. 1994; Hesse et al. 2004). This technology synchronizes CCD read-off with a continuous stage movement. The imaging is done in strips, which are subsequently stitched together to reconstitute the microarray image. Simpler detection regimes could be implemented if brighter labels were used such as plasmon resonant nanoparticles (Oldenburg et al. 2002; Blab et al. 2006; also see Fig. 1A). The benefits of analyzing single molecules is clearly evident from Hesse et al.’s work. Without needing to use PCR or linear amplification, the Hesse group achieved a 100-fold decrease in the amount of sample material needed. This should open up applications where sample quantities are limiting. Also, the ability to work with small amounts of material without the need for amplification circumvents the preferential amplification of highabundance messages such as globins in blood, which is one of the more accessible tissues for microarray analysis. Hesse et al. were able to validate their single molecule results by conventional microarray hybridization done with 100-fold more material. This is impressive, as different microarray platforms often do not show high concordance. Hesse et al. (2006) also demonstrate that the dynamic range of single molecule detection is superior to conventional methods. The range of mRNA abundance levels in biological systems can approach 6 orders of magnitude, which clearly cannot be addressed by the ∼10 dynamic range of current microarray experiments. In contrast, the Hesse investigators found the dynamic range of their single molecule detection system to be 4.7 orders of magnitude. In particular, the range at the lower end, at which regulatory molecules may be expressed, can be extended with greater confidence. It is possible that by using an appropriate single molecule microarray system, the full 6 orders of magnitude of biological expression levels can be addressed in a single readout. One drawback of Hesse et al.’s implementation of single molecule detection on microarrays is that there is an upper transcript concentration limit. One of the three transcripts they studied in detail could not be analyzed at the single molecule level because its high abundance produced a density of binding events on the surface that, due to the diffraction limit of light, could not be resolved. In Hesse’s system, the concentration of each transcript in the sample needs to be <10 fM for an optically resolvable signal density to be obtained. This would not be the case if the actual probe molecules within the microarray spots were placed at low density, on average beyond a diffraction-limited separation, so that no matter what concentration the target is, the density of hybridization signal is dictated by the density of the probe molecules (Fig. 2). E-mail Kalim@well.ox.ac.uk; fax 44-1865-287533. Article published online before print. Article and publication date are at http:// www.genome.org/cgi/doi/10.1101/gr.5825506. Perspective