Digitally guided microdissection aids somatic mutation detection in difficult to dissect tumors.

Molecular genetic testing on formalin fixed, paraffin embedded (FFPE) tumors frequently requires dissection of tumor from tissue sections mounted on glass slides. In a process referred to as "manual macrodissection," the pathologist reviews an H&E stained slide at the light microscope and marks areas for dissection, and then the laboratory performs manual dissection from adjacent sections without the aid of a microscope, using the marked reference H&E slide as a guide. Manual macrodissection may be inadequate for tissue sections with low tumor content. We compared manual macrodissection to a new method, digitally guided microdissection, on a series of 32 FFPE pancreatic cancer samples. KRAS hotspot mutation profiling was performed using the Sequenom MassARRAY system (Agena Bioscience). Digitally guided microdissection was performed on multiple smaller areas of high tumor content selected from within the larger areas marked for manual macrodissection. The KRAS mutant allele fraction and estimated neoplastic cellularity were significantly higher in samples obtained by digitally guided microdissection (p < 0.01), and 7 of the 32 samples (22%) showed a detectable mutation only with digitally guided microdissection. DNA quality and yield per cubic millimeter of dissected tissue were similar for both dissection methods. These results indicate a significant improvement in tumor content achievable with digitally guided microdissection.

Application of tissue mesodissection to molecular cancer diagnostics.

AIMS: To demonstrate clinical application of a mesodissection platform that was developed to combine advantages of laser-based instrumentation with the speed/ease of manual dissection for automated dissection of tissue off standard glass slides. METHODS: Genomic analysis for KRAS gene mutation was performed on formalin fixed paraffin embedded (FFPE) cancer patient tissue that was dissected using the mesodissection platform. Selected reaction monitoring proteomic analysis for quantitative Her2 protein expression was performed on FFPE patient tumour tissue dissected by a laser-based instrument and the MilliSect instrument. RESULTS: Genomic analysis demonstrates highly confident detection of KRAS mutation specifically in lung cancer cells and not the surrounding benign, non-tumour tissue. Proteomic analysis demonstrates Her2 quantitative protein expression in breast cancer cells dissected manually, by laser-based instrumentation and by MilliSect instrumentation (mesodissection). CONCLUSIONS: Slide-mounted tissue dissection is commonly performed using laser-based instruments or manually scraping tissue by scalpel. Here we demonstrate that the mesodissection platform as performed by the MilliSect instrument for tissue dissection is cost-effective; it functions comparably to laser-based dissection and which can be adopted into a clinical diagnostic workflow.

A gel electrophoresis loading system to prevent laboratory contamination by amplification products.

Pipet tip loading of polymerase chain reaction (PCR) and other amplification products into an electrophoresis gel represents a potential source of laboratory contamination. We have developed a prototype of the gel contamination control system (GelCCS) that enables gel loading by bottom puncture of PCR tubes. Puncture occurs within a sealed gel casing, preventing contamination of the surrounding environment. The system was designed for inexpensive manufacture so that after the results are visualized, the gel casing and PCR tubes are discarded intact with the amplification products sealed inside. We demonstrate that gel loading is reliable and that the resulting bands are equivalent in appearance to manually loaded gels.

A mill based instrument and software system for dissecting slide-mounted tissue that provides digital guidance and documentation.

BACKGROUND: Dissection of specific Areas Of Interest (AOIs) of slide-mounted tumor samples is often used to enrich for cancer cells in order to generate better signal to noise ratios in subsequent biochemical characterization. Most clinical laboratories utilize manual dissection for practical reasons and to avoid the expense and difficulties of laser microdissection systems. Unfortunately, manual methods often lack resolution and process documentation. The goal of this project was to design a dissection system for slide-mounted tissue with better precision than manual methods that also provides digital image guidance and electronic process documentation. METHODS: An instrument that is essentially a micro tissue mill was developed. It employs a specialized disposable mill bit that simultaneously dispenses liquid, cuts tissue from the slide surface, and aspirates the liquid along with the displaced tissue fragments. A software package was also developed that is capable of transferring digitally annotated AOIs between images of serially cut tissue sections to guide dissection and generate an electronic record of the process. RESULTS: The performance of this "meso" dissection system was tested using post dissection visual examination for resolution and accuracy, fluorescence based DNA quantitation for recovery efficiency, and dissection of closely situated mouse-human tissue sections followed by PCR amplification for purity determination. The minimum resolution is a dissected circle smaller than 200 microns in diameter, edge dissection accuracy is tighter than 100 microns, recovery efficiency appears greater than 95%, and recovery purity is greater than 99% relative to a different tissue located 100 microns from the dissection boundary. The system can dissect from both paraffinized and deparaffinized FFPE tissue sections that are mounted on plain glass slides, and it is compatible with DNA, RNA, and protein isolation. CONCLUSIONS: The mesodissection system is an effective alternative to manual dissection methods and is applicable for biomarker analysis of anatomical pathology samples, where enrichment of AOIs from the tissue section is helpful, but pure cell populations are not required.

A draft sequence of the rice genome (Oryza sativa L. ssp. japonica).

The genome of the japonica subspecies of rice, an important cereal and model monocot, was sequenced and assembled by whole-genome shotgun sequencing. The assembled sequence covers 93% of the 420-megabase genome. Gene predictions on the assembled sequence suggest that the genome contains 32,000 to 50,000 genes. Homologs of 98% of the known maize, wheat, and barley proteins are found in rice. Synteny and gene homology between rice and the other cereal genomes are extensive, whereas synteny with Arabidopsis is limited. Assignment of candidate rice orthologs to Arabidopsis genes is possible in many cases. The rice genome sequence provides a foundation for the improvement of cereals, our most important crops.

Gains in sensitivity with a device that mixes microarray hybridization solution in a 25-microm-thick chamber.

A microarray hybridization system that allows mixing in volumes comparable to those used by glass coverslips is presented. This system is composed of a disposable flexible lid that binds to 1 in. x 3 in. glass slides via an adhesive gasket, forming a uniform 25-microm-thick hybridization chamber. This chamber rests on a base unit for temperature control. The lid contains two air-driven bladders that continuously mix the hybridization fluid. Mixing enhances sensitivity from a typical microarray experiment 2-3-fold. Mixing is particularly effective at high spotted probe and low labeled target concentrations and overcoming local target depletion that occurs when homologous probes are spotted in close proximity. Mixing appears to be compatible with most hybridization conditions; however, mix versus no-mix control experiments should be performed. Also covered are a number of microfluidic issues related to manufacturing, filling, mixing, and packaging.