RNA expression profiling at the single molecule level

Abstract

We developed a microarray platform for PCR amplification-independent expression profiling of minute samples. A novel scanning system combined with specialized biochips enables detection down to individual fluorescent oligonucleotide molecules specifically hybridized to their complementary sequence over the entire biochip surface of cm2 size. A detection limit of 1.3 fM target oligonucleotide concentration—corresponding to only 39,000 molecules in the sample solution—and a dynamic range of 4.7 orders of magnitude have been achieved. The applicability of the system to PCR amplification-independent gene-expression profiling of minute samples was demonstrated by complex hybridization of cDNA derived from the equivalent of only 104 cells, which matches results obtained in ensemble studies on large samples. By counting each hybridized molecule on the microarray, the method is insusceptible to gene-specific variations of the labeling, thereby representing a principle advance to conventional ensemble-based microarray analysis.

Ultrasensitive RNA profiling: Counting single molecules on microarrays

“…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 3 dynamic range of current microarray experiments.…”

“…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.…”

“…Conventional pixel-bypixel scanners would take impossibly long (weeks) to scan 1 cm 2 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 2 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.…”

Single-molecule fluorescence imaging by total internal reflection fluorescence microscopy (IUPAC Technical Report)

“…Finally, a single-molecule approach to DNA microarrays has been developed [40], as have high-throughput [41] and single-molecule [42][43][44] DNA sequencing methods. These examples are only a few of the many ways in which TIRF has been applied, and new uses for this simple yet versatile technique continue to emerge.…”

Functionalization of polymerized 3D microstructures for biological applications

“…The determination of protein density on the SU-8 surface was carried out in a custom built single molecule scanner based on a conventional inverted epi-fluorescence microscope (Axiovert 200 M, Zeiss, Germany) (Hesch et al 2009;Hesse et al 2006;Hesse et al 2004). The DyLight650…”