title: Effect of immunohistochemistry on molecular analysis of tissue samples: implications for microdissection technologies.
abstract: Laser-based tissue microdissection is an important tool for the molecular evaluation of histological sections. The technology has continued to advance since its initial commercialization in the 1990s, with improvements in many aspects of the process. More recent developments are tailored toward an automated, operator-independent mode that relies on antibodies as targeting probes, such as immuno-laser capture microdissection or expression microdissection (xMD). Central to the utility of expression-based dissection techniques is the effect of the staining process on the biomolecules in histological sections. To investigate this issue, the authors analyzed DNA, RNA, and protein in immunostained, microdissected samples. DNA was the most robust molecule, exhibiting no significant change in quality after immunostaining but a variable 50% to 75% decrease in the total yield. In contrast, RNA in frozen and ethanol-fixed, paraffin-embedded samples was susceptible to hydrolysis and digestion by endogenous RNases during the initial steps of staining. Proteins from immunostained tissues were successfully analyzed by one-dimensional electrophoresis and mass spectrometry but were less amenable to solution phase assays. Overall, the results suggest investigators can use immunoguided microdissection methods for important analytic techniques; however, continued improvements in staining protocols and molecular extraction methods are key to further advancing the capability of these methods.
authors: Pathogenetics Unit, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health (NIH), Bethesda, Maryland 20892, USA.
authors: Michael A
authors: MA
authors: Tangrea
authors: Sumana
authors: S
authors: Mukherjee
authors: Bing
authors: B
authors: Gao
authors: Sanford P
authors: SP
authors: Markey
authors: Qiang
authors: Q
authors: Du
authors: Michael
authors: M
authors: Armani
authors: Matthew S
authors: MS
authors: Kreitman
authors: Alex M
authors: AM
authors: Rosenberg
authors: Benjamin S
authors: BS
authors: Wallis
authors: Franziska C
authors: FC
authors: Eberle
authors: Francesca C
authors: FC
authors: Duncan
authors: Jeffrey C
authors: JC
authors: Hanson
authors: Rodrigo F
authors: RF
authors: Chuaqui
authors: Jaime
authors: J
authors: Rodriguez-Canales
authors: Michael R
authors: MR
authors: Emmert-Buck
mesh_terms: D000818:Animals; D004247:DNA / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D016612:Paraffin Embedding; D011506:Proteins / Q000032:analysis*; D012313:RNA / Q000032:analysis*; D051381:Rats
chemical_list: D011506:Proteins; D012313:RNA; D004247:DNA