Combining laser capture microdissection with quantitative real-time PCR: Effects of tissue manipulation on RNA quality and gene expression

Ilan A. Kerman, Bradley J. Buck, Simon J. Evans, Huda Akil, Stanley J. Watson

Molecular and Behavioral Neuroscience Institute, Department of Psychiatry, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI 48109, USA, Journal of Neuroscience Methods 153 (2006) 71–85

Laser capture microdissection (LCM) is a powerful tool that is used to obtain specific cell types from tissue sections that can be used for gene expression profiling; however, the gene expression of cells captured by LCM can be difficult due to small sample amounts. In addition, various tissue manipulations during tissue preparation for LCM may result in RNA degradation due to reactivation of RNAases. Based on this, the researchers in this study examined the effect of different experimental procedures on the quality and quantity of RNA extracted from samples collected by LCM and the impact of this on qRT-PCR results.

In this study, 8 adults Sprague-Dawley rats were used and microdissected specific subregions of the rat cerebellum both in normal tissue and following ibotenic acid injection were examined.  After sectioning at 10 μm, the tissue sections were mounted differently either one section per slide, 2 per slide, or 4 per slide onto two different slide types either on poly-lysine coated slides or onto charged slides. Some of the sections were stained with either cresyl violet stain or neutral red stain while some sections remained non-stained. The RNA integrity for samples that were obtained from different approach was measured using three different measures.

There was no difference in RNA quality between sections mounted on different types of slides; in contrast, the RNA quality decreased with increased the number of sections per slide to four, and it was significantly reduced with staining as compared to non-stained sections. Interestingly, the RNA yields increased with the staining, and it was significantly greater with neutral red staining than in either cresyl violet or the non-stained sections since the staining improves the tissue pick up during microdissection.  From the PCR results, they found that the expression of one gene was sensitive to RNA yield but not quality, while the other gene was influenced by both which indicates that the RNA yield is an important determinant of gene expression levels. In conclusion, it is necessary to decrease the amount of time for tissue preparation before LCM to decrease the possibility of mRNA degradation, and to use less complex protocols that improve the RNA quality.      

An overview of laser microdissection technologies

Graeme I. Murray
Acta histochemica 109 (2007) 171-176

In this review, Murray compared and contrasted two methods of laser microdissection, giving pros and cons for laser capture microdissection and laser cutting microdissection.  Both have the advantage over traditional microdissection techniques (requiring a scalpel or a fine stainless steel needle), because the older methods of microdissection are "slow, cumbersome, and require considerable dexterity."  In addition, using a scalpel or needle makes the tissue sample both hard to acquire and easy to contaminate.

Laser capture microdissection (as we do in our lab) requires a membrane attached to a cap, which is placed over the tissue section and melted with a laser.  The melted plastic attaches to the sample, cools within milliseconds, and allows the user to pick up only the small spot of the sample that they have chosen.  While this is convenient for collecting samples for use with assays that allow for amplification, like DNA and RNA, it is not very useful for protein assays, as the sample will be quite small.  The laser and the temperature changes do not seem to harm the sample, and the settings can be adjusted for use with tissues fixed in various ways.

Laser cutting microdissection was developed more recently and involves using a laser to "draw around" the desired cells, allowing them to detach from the slide.  The sample is then dropped or catapulted into a tube for collection.  This method does not require costly caps, heating and cooling of a membrane, or contact of any kind with the sample (thus cutting down on opportunities for contamination).  It allows for greater precision, as well, because laser capture microdissection has a larger laser diameter with a fixed shape (a circle) for sample collection, whereas laser cutting microdissection allows the user to outline their cells exactly (minimizing the collection of non-target cells that are adjacent to the target cell).  However, it works best when only a limited number of cells is needed, as laser capture microdissection allows for a more rapid collection of a large number of cells.

As to the matter of tissue preparation, Murray recommends that the tissue be exposed for as little time as possible to stains or rapid IHC techniques.  The biggest takeaway for tissue preparation is to avoid doing anything make the sample less ideal for the assays that one intends to do.  For us, because we wish to isolate the RNA, that means using fresh frozen tissue and avoiding letting the sections stay at room temperature for very long, so that the RNA has little opportunity to degrade.  Different types of molecular analysis will require different treatment of the tissue, though.