Validation of a novel ultra-short immunolabeling method for high-quality mRNA preservation in laser microdissection and real-time reverse transcriptase-polymerase chain reaction.

The Journal of Molecular Diagnostics : JMD
Dorthe von SmolinskiAndreas Gebert

Abstract

Laser microdissection allows isolation of tiny samples from tissue sections for analysis of gene expression by real-time quantitative polymerase chain reaction (PCR). Although immunohistochemical labeling is often required to identify target structures, it drastically degrades mRNA so that shortened protocols are needed. Here, we present a novel method that allows fluorescence double labeling to be performed in only one incubation of 5 minutes. Fab fragments directly coupled to fluorochromes are linked to primary antibodies before these complexes are applied to sections. We quantified the influences of fixatives, labeling solutions, and incubation time on the mRNA yield and compared our method with previously proposed protocols. While tissue components, ie, vimentin and Ki67 antigen, were sufficiently stained after only 5 minutes of incubation, the new method produced a minute loss of mRNA that did not significantly differ from that of untreated sections. In contrast, incubation times of 15 and 30 minutes reduced the mRNA yield by 99.8 to 99.9%. Furthermore, incubation periods longer than 5 minutes critically affected the ratio between the target and housekeeping genes tested by factors of up to 10.6. In conclusion, the novel m...Continue Reading

References

Jan 1, 1995·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·I M Grumbach, R W Veh
Mar 1, 1995·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·J L WhitelandT J Hill
Sep 1, 1995·Journal of Photochemistry and Photobiology. B, Biology·A de With, K O Greulich
Aug 14, 1998·Nature Biotechnology·K Schütze, G Lahr
Nov 11, 1998·Nature Medicine·L FinkR M Bohle
Jun 12, 1999·Molecular Carcinogenesis·S M GoldsworthyR R Maronpot
Aug 17, 1999·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·N Panchuk-VoloshinaR P Haugland
Nov 14, 2000·The American Journal of Pathology·L FinkR M Bohle
Mar 30, 2001·Pathobiology : Journal of Immunopathology, Molecular and Cellular Biology·F FendL Quintanilla-Martinez
Aug 18, 2004·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·Hidetoshi Ino
Aug 18, 2004·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·Jeremy K BrownHugh R P Miller
Jul 29, 2005·Histochemistry and Cell Biology·Dorthe von SmolinskiAchim D Gruber

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Citations

Mar 18, 2011·Nature Protocols·Jeffrey C HansonMichael R Emmert-Buck
Oct 31, 2006·Photomedicine and Laser Surgery
Mar 25, 2011·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·Michael A TangreaMichael R Emmert-Buck
Feb 23, 2008·Experimental and Molecular Pathology·Melissa L CoxRobert W Dunstan
Jun 28, 2011·Growth Hormone & IGF Research : Official Journal of the Growth Hormone Research Society and the International IGF Research Society·Cheng-Yuan Feng, Christopher S von Bartheld
May 18, 2011·Cellular and Molecular Life Sciences : CMLS·Immo Prinz

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