Gene expression analyses of neurons, astrocytes, and oligodendrocytes isolated by laser capture microdissection from human brain: Detrimental effects of laboratory humidity

Abstract

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.

Translational Research in Pediatrics IV: Solid Tissue Collection and Processing

“…67,177,[230][231][232][233][234] The parameters affecting solid tissue qualities are summarized in Table 4.…”

“…210 RNA integrity can be influenced by degradation, tissue type, BMI, and by other tissue quality markers, including pH, PMI, agonal factors, and relative humidity of the processing laboratory. [230][231][232][233] In some tissues, RNA degradation increases with longer PMIs. 177,230 Prolonged agonal factors, such as coma and hypoxia, together with a pH ≤5.9, can also affect the RNA integrity and have a major impact on gene expression.…”

“…Freezers should be equipped with an alarm system to avoid catastrophic failure and nucleotide degradation. The inner-chamber temperature 231 ACI, the average correlation index; ASR, agonal stress rating; GTP, guanosine triphosphate; IHC, immunohistochemistry; LCM, laser microdissection; MAP, microtubule-associated proteins; mRNA, messenger RNA; RT, room temperature. by guest on May 12, 2018 http://pediatrics.aappublications.org/ Downloaded from of the freezer should be monitored at least weekly and recorded in a log.…”

“…298 Commercial companies now offer a large variety of containers for temperature-sensitive shipments. 299 β-ray sterilized plastic bags and vacuum sealing of fresh excised specimens at surgical theaters, followed by time-controlled • RNA should be extracted from fresh or snap frozen material at -70°C or below 225,226 • DNA can be obtained from sections stained with hematoxylin and eosin, 225 but toluidine blue is preferred if microdissection is required 225 • RNA can be extracted from fi xed embedded tissue, but it has usually suffered more from the treatment of the specimen before fi xation than from the processing itself 225 • When DNA is isolated by LCM from frozen solid tissue, relative humidity in the laboratory must be controlled 231 • When LCM is used, and to avoid RNA degradation, it is imperative to keep tissue sections as dry as possible after fi xation 231 Tissue fi xation and preservation…”

Abordagem para análise proteômica de neurônios contendo neuromelanina na substância negra, isolados por microdissecção a laser

“…As definições são influenciadas pelo tipo de tecido, células ou regiões que serão isoladas, pela espessura do corte, pela energia do feixe do laser aplicada e pelo tampão utilizado na amostra. Além disso, a MCL pode ser interferida pela temperatura e umidade (Ordway et al, 2009). Essas condições afetam as configurações de energia necessárias para a microdissecção e este processo deve ter ajuste diário.…”