TY - JOUR
T1 - Effect of immunohistochemistry on molecular analysis of tissue samples
T2 - Implications for microdissection technologies
AU - Tangrea, Michael A.
AU - Mukherjee, Sumana
AU - Gao, Bing
AU - Markey, Sanford P.
AU - Du, Qiang
AU - Armani, Michael
AU - Kreitman, Matthew S.
AU - Rosenberg, Alex M.
AU - Wallis, Benjamin S.
AU - Eberle, Franziska C.
AU - Duncan, Francesca C.
AU - Hanson, Jeffrey C.
AU - Chuaqui, Rodrigo F.
AU - Rodriguez-Canales, Jaime
AU - Emmert-Buck, Michael R.
PY - 2011
Y1 - 2011
N2 - 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.
AB - 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.
KW - Dna
KW - Expression microdissection
KW - Immuno-lcm
KW - Immunohistochemistry
KW - Immunostaining
KW - Proteomics
KW - Rna
KW - Tissue microdissection
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U2 - 10.1369/0022155411404704
DO - 10.1369/0022155411404704
M3 - Article
C2 - 21430260
AN - SCOPUS:79960908292
SN - 0022-1554
VL - 59
SP - 591
EP - 600
JO - Journal of Histochemistry and Cytochemistry
JF - Journal of Histochemistry and Cytochemistry
IS - 6
ER -