Laser Capture Microdissection (LCM) Support—Getting Started

The ArcturusXT™ LCM Instrument has a proprietary combination of a gentle IR laser and a powerful UV laser that work in conjunction to efficiently isolate cells from frozen sections or paraffin embedded sections without changing morphology or integrity of the biological content. The IR laser helps to capture the cells of interest, and the UV laser microdissects the captured cells. This is done without affecting the morphology of the cells, and allows for visual inspection of the remaining specimen to help ensure the quality of the sample collected. In addition to the flexibility that the dual laser system provides, there is the flexibility of multiple stage inserts for various sample types. A wide-slide stage format is available for neurobiology researchers working with very large sections of brain tissue. The petri dish stage insert enables live-cell microdissection applications such as stem cell research and other rare-cell isolations.

• For a few cells: The gentle IR-only approach is the best recommendation for preserving nucleic acids and verifying that the desired material is collected on the cap. 
• For groups of cells or large tumor regions: Mount your tissue on a membrane slide and take advantage of the power of the IR, or IR + UV cutting laser to cut around your region of interest quickly and cleanly.

First, optimize the visualization of the image in the live video at 2X by increasing or decreasing the brightness setting, then right-click on the overview image and select "remember settings". This resets your optimized image settings, and when you select "re-acquire overview image" you will now generate a perfectly exposed overview image.

• The Arcturus™ PicoPure™ RNA Isolation Kit (Cat. Nos. KIT0204, KIT0214) is specifically designed to consistently recover high-quality total RNA from fewer than 10 cells, even from a single cell. This kit is designed to provide consistent, efficient RNA recovery without sacrificing quality. Small volume elution allows you to maximize your recovery of RNA from small numbers of cells for use with your gene expression analysis experiments. 
• For LCM cells from paraffin sections, we recommend using the Arcturus™ Paradise™ Plus RNA Extraction and Isolation Kit (Cat. No. KIT0312I) that unlocks RNA from formalin-fixed, paraffin-embedded (FFPE) tissues. As little as 5 ng of the isolated fixed RNA can then be amplified using reagents and methods that have been specially optimized to overcome the challenges of cross-linked FFPE templates. 
• We also recommend the Arcturus™ Paradise™ PLUS QC Kit (Cat. Nos. KIT0303, KIT0313). This kit offers a simple solution for evaluating the quality of RNA from formalin-fixed paraffin-embedded (FFPE) tissues before proceeding with LCM and downstream molecular analysis. The reagents provided in the kit are optimized to enable efficient extraction, isolation, and reverse transcription of total RNA from FFPE tissue scrapes. The RNA quantity and quality is then measured using quantitative real-time PCR with a housekeeping gene like beta actin. 

Some other kits have been used for RNA isolation from LCM cells. These kits include the RecoverAll™ Total Nucleic Acid Isolation Kit for Formalin-Fixed Paraffin-Embedded (FFPE) (Cat. No. AM1975), RecoverAll™ Multi-Sample RNA/DNA Isolation Workflow (Cat. No. A26135), and the RNAqueous™- Micro Total RNA Isolation Kit (Cat. No. AM19310).

The Arcturus™ PicoPure™ DNA Extraction Kit (Cat. No. KIT0103) offers an easy, streamlined genomic extraction procedure that produces PCR-ready DNA. It allows you to extract and amplify DNA in the same tube, without organic extraction or spin columns, and to recover DNA from as few as 10 cells prepared by laser capture microdissection (LCM) or other tissues. The kit provides conveniently packaged, stable Proteinase K, PCR-compatible DNA Reconstitution Buffer and a complete user guide. 

With this kit, you can amplify as little as 5 ng of RNA isolated from formalin-fixed, paraffin embedded (FFPE) tissues.

The Arcturus™ PicoPure™ RNA Isolation Kit removes total cellular RNA from pico-scale samples including frozen sections, alcohol fixed-, or fresh samples. Please note, samples fixed with formaldehyde are not recommended for use with this kit.

The dual-laser (IR) and UV nature of the ArcturusXT ™ Laser Capture Microdissection System offers ultimate flexibility for tissue preparation. A wide variety of tissues can be mounted on the system and subsequently microdissected off. We offer three types of slides that can be used for microdissection:

• Glass slides—uncharged, uncoated, standard glass slides that are useful for researchers looking to use only the IR laser to isolate their cells of interest. 
• PEN membrane glass slides—enable the combined use of IR LCM and UV Laser Cutting.
• PEN membrane frame slides—provide additional flexibility by allowing the choice of microdissection from non-dehydrated sample preparations, and thick sections. 

• The Arcturus™ HistoGene™ LCM Frozen Section Staining Kit (Cat. No. KIT0401) comes complete with all the reagents and supplies needed for preparing frozen tissue sections for laser capture microdissection (LCM). Designed as a fast-penetrating stain, this kit provides excellent contrast while preserving nucleic acid integrity and retaining low-abundance mRNAs.
• The Arcturus™ Paradise™ Plus FFPE LCM Staining Kit (Cat. No. KIT0312J) offers a simple solution to stain formalin-fixed, paraffin-embedded (FFPE) tissues before proceeding with laser capture microdissection (LCM) and downstream molecular analysis. FFPE samples introduce unique challenges for gene expression profiling, including chemical modification and fragmentation of RNA molecules. Archiving of FFPE samples adds to these challenges through increased RNA degradation over time. 
• The Arcturus™ HistoGene™ LCM Immunofluorescence Staining Kit (Cat. No. KIT0420) is designed to retrieve high-quality nucleic acids from immunofluorescently stained, frozen tissue using a quick 15-minute process. The kit provides reagents and slides for convenient and reliable immunofluorescent staining as well as protocols that are streamlined and optimized to maintain nucleic acid quality. The staining kit is used with a biotinylated monoclonal primary antibody against an antigen of choice provided by the user, and Cyanine 3–conjugated streptavidin is included in the kit.

The cresyl violet stain is a hydrophilic, basic stain that binds to negatively charged nucleic acids. It is often used to identify target cells for LCM. If the tissue morphology has been damaged, cresyl violet might not clearly show cell distribution, and therefore acridine orange stain may provide better contrast between clusters of cells. Acridine orange is a fluorescent stain that intercalates with nucleic acids and can offer an advantage when microdissecting single cells as the cells will fluoresce on the cap after microdissection allowing for easy retrieval.

For recovery of miRNA from formalin-fixed samples, we recommend using RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE. You can isolate total and miRNA using the RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE, then use that prep for enrichment of miRNA using the enrichment protocol described in the instructions for the mirVana™ kit. For unfixed LCM samples, you could use an RNAqueous™ kit.

While we have not tested this in-house, there is evidence that it is possible to use tissues preserved in RNAlater™ Stabilization Solution and then perform laser capture microdissection. Please see the reference below:
Thelen P, Burfeind P, Grzmil M et al. (2004) cDNA microarray analysis with amplified RNA after isolation of intact cellular RNA from neoplastic and non-neoplastic prostate tissue separated by laser microdissections. Int J Oncol 24(5):1085-1092.