Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.
Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.
Bova
G Steven
GS
Department of Pathology, Institute of Genetic Medicine, The Johns Hopkins Hospital, PELICAN Laboratory, Carnegie 628, Baltimore, MD 21287, USA. gbov@jhmi.edu
Eltoum
Isam A
IA
Kiernan
John A
JA
Siegal
Gene P
GP
Frost
Andra R
AR
Best
Carolyn J M
CJ
Gillespie
John W
JW
Su
Gloria H
GH
Emmert-Buck
Michael R
MR
D015415:Biomarkers / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D003239:Connective Tissue Cells / Q000145:classification / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D042282:Microdissection / Q000379:methods*; D008967:Molecular Biology / Q000379:methods; D020543:Proteome / Q000378:metabolism*; D013048:Specimen Handling / Q000379:methods*; D014021:Tissue Preservation / Q000379:methods*
D015415:Biomarkers; D020543:Proteome