Arcturus^XT™ Laser Capture Microdissection Instrument - FAQs

I am having trouble assessing the RNA quality from formalin-fixed, paraffin-embedded samples after laser capture microdissection (LCM). Can you offer some tips?

Formalin-fixed paraffin-embedded (FFPE) samples introduce unique challenges for gene expression profiling and gene expression quantitation, including chemical modification and fragmentation of RNA molecules. The initial paraffin blot preparation and storage significant affect the RNA quality. Archival FFPE samples present additional challenges due to increased RNA degradation over time. As a result, not all FFPE samples contain high quality RNA. 

In addition, a typical Bioanalyzer profile of RNA from an FFPE sample can look fragmented and degraded and as a result, that approach may not be useful in determining the transcribability of your RNA. Therefore, a tissue scrape test is recommended. You can use the entire adjacent section to isolate RNA and to access the RNA quantity and quality for intact RNA. Two sets of β-actin gene-specific primers designed to amplify short segments of DNA within the β-actin target sequence from the 5’ and 3’ of the β-actin gene can be used to estimate the amount of RNA in a given sample in relation to β-actin content using real-time PCR. The RNA quantity derived from the 3' primer set is used to quantitate the RNA in the FFPE tissue scrape. The ratio of the RNA yield obtained from both sets of PCR primers is the 3'⁄5' ratio and is used as an indication of RNA quality. If there is less product yield of the 5’ β-actin primers, it indicates that there is less full length of RNA.

I am getting poor quality of RNA from my LCM samples. How can I improve it?

The dissection and embedding steps are critical for the RNA quality for LCM samples. We suggest using the entire adjacent section or dissecting a large area of the section (>1000 cells) for RNA isolation. Before proceeding to LCM microdissection, we recommend assessing the quality of RNA isolated from the adjacent section. With good amount of RNA, one can assess the RNA quantity and quality using the Qubit RNA HS Assay Kit and the Agilent Bioanalyzer. This will help to make sure that good quality RNA is obtained from a low number of LCM cells.

What samples can be used with the Arcuturus PicoPure RNA Isolation Kit?

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.

What kit do you recommend using for DNA isolation from Arcturus Laser Capture Microdissection (LCM) samples?

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. 

What other kits have been used for RNA isolation from Arcturus Laser Capture Microdissection (LCM) samples?

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).

Which RNA isolation kit do you recommend using with Arcturus Laser Capture Microdissection (LCM) samples?

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.

How do I locate the overview image on the Arcturus XT LCM Instrument?

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.

With the ArcturusXT LCM Instrument, which laser should I use to isolate my region of interest? UV cutting laser or IR capture laser?

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.

What is Arcturus Laser Capture Microdissection (LCM)?

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.