2007 Arcturus® Laser Capture Microdissection (LCM) Users Meetings
Thanks to everyone who attended the Arcturus LCM Users Meeting in Downingtown, PA. Special thanks to our presenters, Ms. Pecherskaya from Fox Chase Cancer Center and Dr. Winrow from Merck Research for helping to make this users meeting a great success.
The presentation topics are listed below and available for viewing in PDF format. If a presentation is not available here, please contact the author directly for more information.
- Arcturus LCM and Microgenomics Product Overview
Steve Blakely, Molecular Devices
Laser Capture Microdissection (LCM) is a proven technique for isolating pure cell populations for downstream molecular analysis. Combined UV laser cutting and LCM using the IR laser, found only with Arcturus® LCM Instruments, allows for rapid and precise isolation of larger numbers of cells, while maintaining cellular and nucleic acid integrity necessary for downstream analysis.
Molecular Devices, a Division of Molecular Devices, recently introduced the ArcturusXT™ Microdissection Instrument. The open-architecture ArcturusXT system is built upon the Nikon TE2000U® inverted research grade microscope, and combines infrared (IR) laser-enabled laser capture microdissection (LCM) and ultraviolet (UV) laser cutting in a single modular instrument, offering researchers superior speed, precision and flexibility. By utilizing the Nikon TE2000U, the ArcturusXT is completely modular and fully upgradeable, allowing the system utility to expand as research requirements grow and microdissection technologies emerge.
Molecular Devices offers the full solution of Microgenomics reagent products that support and complement the LCM technology, from kits for tissue section staining and nucleic acid extraction to kits for RNA amplification and labeling for microarray analysis. The microgenomics platform benefits the researcher as it ensures high efficiency recovery of quality total RNA from as little as a single cell, obtained through LCM. High sensitivity linear amplification of mRNA is possible from as little as 100 picograms (10 LCM cells) of total RNA, generating enough aRNA for replicate microarray hybridizations. Linear amplification is followed by non-enzymatic labeling, which allows the use of unlabeled nucleotides during amplification. This enables the use of lesser amounts of starting material and results in higher aRNA yields and higher %P calls during microarray analysis. This complete microgenomics platform has been validated for use with all common microarray platforms.
- Practical Applications of LCM
Anna Pecherskaya, MS, Fox Chase Cancer Center
Laser capture microdissection (LCM) is the procedure for procuring pure cell population from the specific microscopic regions of tissue sections and can facilitate comparative genomics studies between different cell populations from the same individual. Different aspects of sample collection, tissue fixation and staining can affect the quality of RNA isolated from LCM material.
Therefore, establishment of reliable protocols and quality control steps for both frozen and formalin-fixed paraffin-embedded (FFPE) tissues is important.
Bioanalyzer profiles are a reliable means to assess RNA quality from frozen tissue. Functional RT-PCR assays using a panel of gene-specific primers provide a more reliable indication of the quality of RNA isolated from FFPE tissues in downstream analysis. Bioanalyzer profiles and agarose gels are used to control the quality of amplified material. NanoDrop analysis is used to measure dye incorporation before processing to microarray hybridization. The success of the dye incorporation as well as the strength of the signal on microarray step substantially depends on the quality of the aRNA. The usefulness of the qualitative and quantitative analysis of amplification performance and following labeling is demonstrated for the different RNA profiles.
Using these protocols we show practical approaches of the LCM technology and that it is possible to obtain reliable microarray data from FFPE samples collected from clinical resection specimens. The Arcturus Paradise Reagent System, which allows robust RNA isolation and amplification using very low RNA input, and custom-designed 22,000 oligonucleotide arrays (Agilent) were used in the present study.
An in-depth comparison of microarray data generated from FFPE human colon adenocarcinoma tissue with that obtained from frozen matched specimens was accomplished and high correlation coefficients for the gene expression profiles were obtained.
A set of practical recommendations for evaluating RNA integrity and quality control, which can guide the implementation of the developed technology in any laboratory setting was established. Because formalin-fixation paraffin embedding is the universal protocol for routine pathological diagnosis, the proposed procedure represents an invaluable tool with which to correlate the genomic characteristics of a sample with wealth of morphological and clinical information available for the same specimen.
- Applying Microdissection and Expression Profiling for CNS Studies
Chris Winrow, Ph.D., Merck Research
- Presentation not available. Contact author for more information.
- Arcturus LCM Protocols and Troubleshooting
Deren Lomago, Molecular Devices
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