Gene expression analyses of neurons, astrocytes, and oligodendrocytes isolated by laser capture microdissection from human brain: detrimental effects of laboratory humidity.
Laser capture microdissection (LCM) is a versatile computer-assisted dissection method that permits collection of tissue samples with a remarkable level of anatomical resolution. LCM's application to the study of human brain pathology is growing, although it is still relatively underutilized, compared with other areas of research. The present study examined factors that affect the utility of LCM, as performed with an Arcturus Veritas, in the study of gene expression in the human brain using frozen tissue sections. LCM performance was ascertained by determining cell capture efficiency and the quality of RNA extracted from human brain tissue under varying conditions. Among these, the relative humidity of the laboratory where tissue sections are stained, handled, and submitted to LCM had a profound effect on the performance of the instrument and on the quality of RNA extracted from tissue sections. Low relative humidity in the laboratory, i.e., 6-23%, was conducive to little or no degradation of RNA extracted from tissue following staining and fixation and to high capture efficiency by the LCM instrument. LCM settings were optimized as described herein to permit the selective capture of astrocytes, oligodendrocytes, and noradrenergic neurons from tissue sections containing the human locus coeruleus, as determined by the gene expression of cell-specific markers. With due regard for specific limitations, LCM can be used to evaluate the molecular pathology of individual cell types in post-mortem human brain.
Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, USA. ordway@etsu.edu
Gregory A
GA
Ordway
Attila
A
Szebeni
Michelle M
MM
Duffourc
Sophie
S
Dessus-Babus
Katalin
K
Szebeni
D000293:Adolescent; D000328:Adult; D001253:Astrocytes / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002452:Cell Count; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D006813:Humidity / Q000009:adverse effects*; D007834:Lasers; D008125:Locus Coeruleus / Q000378:metabolism; D042282:Microdissection / Q000379:methods*; D008875:Middle Aged; D009474:Neurons / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism; D009836:Oligodendroglia / Q000378:metabolism*; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D055815:Young Adult
D012313:RNA; D009638:Norepinephrine