Microdissection of gonadal tissues for gene expression analyses

Laser microdissection permits isolation of specific cell types from tissue sections or cell cultures. This may be beneficial when investigating the role of specific cells in a complex tissue or organ. In tissues with easily distinguishable morphology, a simple hematoxylin staining is sufficient, but in most cases a more specific staining is required to identify which cells to microdissect. We have established two staining protocols for frozen sections (1) Oil red O, which stains lipid droplet in fat cells and steroid-producing cells and (2) NBT BCIP, which stains cells expressing an alkaline phosphatase enzyme, such as fetal germ cells, testicular carcinoma in situ cells, and putatively also other early stem cell populations. We have applied these protocols for microdissection of rat Leydig cells, fetal human and zebrafish germ cells, and human testicular germ cell tumors, but the staining protocols could also be used in other species and for other cell types containing lipid droplets or expressing alkaline phosphatase. Both protocols ensure a morphology that enables microdissection of single cells with RNA quality sufficient for subsequent gene expression analysis. However, RNA yields after microdissection and purification are small, and therefore, two rounds of linear amplification are recommended prior to gene expression analysis.