Laser capture microdissection protocol for gene expression analysis in the brain

Abstract

Laser capture microdissection (LCM) allows the isolation of specific cell populations from complex tissues that can be then used for gene expression studies. However, there are no reproducible protocols to study RNA in the brain and, particularly, in the substantia nigra. RNA is a very labile biomolecule that is easily degraded during manipulation. LCM studies use low amounts of material and special precautions must be taken to preserve RNA yield and integrity, which are decisive for PCR analysis. The RNA yield and/or integrity can be affected negatively by tissue manipulation, LCM process and RNA extraction. We have optimized these three critical steps using nigral tissue sections, and developed a LCM protocol to obtain high-quality RNA for gene expression analysis. The optimal LCM protocol requires the use of 20 µm-thick tissue sections mounted on glass slides and processed for rapid tyrosine hydroxylase immunofluorescence. Additionally, a total microdissected tissue area of 1 mm² and a column-based RNA extraction method were used to obtain a high RNA yield and integrity. In the rat substantia nigra, we demonstrated the expression of RNA for the angiotensin type 1 and type 2 receptors using this optimized LCM protocol. In conclusion, the LCM protocol reported here can be used to study the expression of both scarcely or abundantly expressed genes in the different brain regions of mammals under both physiological and pathological conditions.

Keywords: Brain; Gene expression; Laser capture microdissection; RNA integrity and yield; RT-PCR; Substantia nigra.