Getting Intact RNA from LCM Samples

Obtaining specific populations of cells from heterogeneous samples is a major challenge in studying gene expression profiles of specific cell types. Laser capture microdissection (LCM), developed in a collaborative effort between the NIH and Arcturus, Inc., specifically to isolate cancer cells from normal tissue, can be used to separate individual cell types from within complex tissues and organs. RNA can then be extracted from these microsections for gene expression analysis.

LCM can select and capture cells or discrete morphological structures from thin tissue sections. Frozen, O.C.T.-embedded tissue sections are prepared for the LCM process by fixation, staining, and dehydration. The section is then visualized through a thermoplastic film attached to the bottom of a microcentrifuge tube cap. A laser pulse is directed through the film onto the target cells. The plastic film melts onto the targeted area, then cools and bonds with the underlying cells. Then the film, along with the adhered target cells, is collected. RNA can be isolated from these captured cells for real-time RT-PCR and mRNA expression profiling.

Ambion has optimized the processing and staining of tissues for LCM, and here we offer some suggestions for avoiding pitfalls and obtaining high quality RNA from LCM samples.

Freezing and embedding your tissue:

• It is important to freeze samples quickly to preserve RNA. We typically use a tissue-freezing aerosol such as Cytocool II (Richard Allen Scientific) to flash freeze samples, but liquid nitrogen can also be used.
• Although nonembedded frozen tissues can be used for cryosectioning, we recommend embedding the samples in a cryoembedding matrix such as TissueTek’s O.C.T. to help preserve morphology during sectioning.
• Keep samples cold while embedding tissues; and use a disposable vinyl specimen cryomold to embed the frozen tissue on dry ice.

Cryosectioning your tissue:

• Clean the specimen stage with RNaseZap (Ambion) and apply a layer of O.C.T., then place the tissue on the stage. Settle the stage into dry ice to allow the O.C.T. to freeze before proceeding. This extra layer of embedding medium helps prevent the tissue from being knocked off the stage by the knife during cryosectioning.
• Be sure to equilibrate embedded tissue to your cryostation temperature before cryosectioning. Optimal cutting temperatures range from –10 to –60°C and are dependent on tissue fat content.
• Keep the tissue cold during sectioning to avoid RNA degradation. If tissues warm during any point in the process, use tissue-freezing aerosol spray to quickly cool tissues or instruments.
• Keep a microscope slide box on dry ice and immediately place the sectioned slides into the prechilled box. If slides will be stained soon after sectioning, it may be more convenient to simply keep them in the cryostat until you are ready to begin staining.
• Slides with cryosections can be safely frozen for later use. Add a small desiccant pack (sorbent) to the slide box, seal with laboratory film, and store at –80ºC until ready to stain.

Fixing and staining your slides:

• To avoid carrying residual ethanol from one step to another, gently tap the slide on an absorbent surface to remove excess ethanol.
• Use molecular sieves (e.g. EMD Chemicals Molecular Sieve, Type 4A, 8–12 Mesh Beads) in your stock 100% ethanol to reduce accumulation of water.
• For superior staining of frozen sections, while preserving RNA quality for downstream applications, see Ambion's LCM Staining Kit.

Ensuring thorough dehydration of cryosectioned sample:

It is important to completely dehydrate your samples before beginning LCM. Samples with residual moisture will be subject to hydrostatic forces that make it difficult to separate the tissue from the slide and from the surrounding tissue after LCM. Poorly dehydrated samples will generally appear more translucent and will have a shinier surface than thoroughly dehydrated samples.

• Change ethanol and xylene fixing solutions often.
• Extend time in ethanol/water solutions to 30 seconds per container, and extend time in 100% ethanols to 1 minute if you experience inadequate dehydration at shorter times.
• Add an additional 10 second xylene step before beginning the initial 5 minute xylene dip to remove ethanol after dehydration.
• High humidity can severely compromise LCM experiments. If humidity is a problem in your setting, use a hygrometer to monitor room humidity levels and a dehumidifier to keep the ambient humidity in the range of 25–45%.

Performing LCM and beginning the RNA extraction procedure:

• Carry out LCM as soon as possible after the slides have been fixed and stained to minimize RNA degradation.
• Use an adhesive strip (such as Prep Strip™ from Arcturus) to remove loose tissue from the slide before performing LCM.
• Remove extraneous tissue from the thermoplastic film using an adhesive strip (such as Capsure Cleanup Pads from Arcturus).
• To maximize RNA recovery from microdissected tissue, peel off the thermoplastic film from the Capsure LCM Cap (Arcturus) using sharp forceps and drop it into the lysis solution.

Extracting RNA from microdissected tissue:

Ambion’s RNAqueous-Micro kit is recommended for recovery of RNA from tissue microdissected by LCM. This kit is based on solid-phase extraction of RNA onto a silica matrix. The construction of the filter allows recovery of the RNA in a small volume (~20 µl). The kit contains also contains reagents for removal of contaminating genomic DNA.