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.