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Striptease on glass:: Validation of an improved stripping procedure for in situ microarrays
被引:6
作者:
Hahnke, Karin
Jacobsen, Marc
Gruetzkau, Andreas
Gruen, Joachim R.
Koch, Markus
Emoto, Masashi
Meyer, Thomas F.
WaIduck, Anna
Kaufmann, Stefan H. E.
Mollenkopf, Hans-Joachim
机构:
[1] Max Planck Inst Infect Biol, Microarray Core Facil, D-10117 Berlin, Germany
[2] German Rheumatism Res Ctr, D-10117 Berlin, Germany
[3] Oligene GmbH, D-10117 Berlin, Germany
关键词:
in situ microarrays;
stripping protocol;
reuse;
data analysis;
ART;
NO;
E9;
DNA MICROARRAYS;
CDNA MICROARRAYS;
EXPRESSION;
AMPLIFICATION;
HYBRIDIZATION;
MEMBRANES;
CHIPS;
D O I:
10.1016/j.jbiotec.2006.09.003
中图分类号:
Q81 [生物工程学(生物技术)];
Q93 [微生物学];
学科分类号:
071005 ;
0836 ;
090102 ;
100705 ;
摘要:
Microarrays have rapidly become an indispensable tool for gene analysis. Microarray experiments can be cost prohibitive, however, largely due to the price of the arrays themselves. Whilst different methods for stripping filter arrays on membranes have been established, only very few protocols are published for thermal and chemical stripping of microarrays on glass. Most of these protocols for stripping microarrays on glass were developed in combination with specific surface chemistry and different coatings for covalently immobilizing presynthesized DNA in a deposition process. We have developed a method for stripping commercial in situ microarrays using a multi-step procedure. We present a method that uses mild chemical degradation complemented by enzymatic treatment. We took advantage of the differences in biochemical properties of covalently linked DNA oligonucleotides on in situ synthesized microarrays and the antisense cRNA hybridization probes. The success of stripping protocols for microarrays on glass was critically dependent on the type of arrays, the nature of sample used for hybridization, as well as hybridization and washing conditions. The protocol employs alkali hydrolysis of the cRNA, several enzymatic degradation steps using RNAses and Proteinase K, combined with appropriate washing steps. Stripped arrays were rehybridized using the same protocols as for new microarrays. The stripping method was validated with microarrays from different suppliers and rehybridization of stripped in situ arrays yielded comparable results to hybridizations done on unused, new arrays with no significant loss in precision or accuracy. We show that stripping of commercial in situ arrays is feasible and that reuse of stripped arrays gave similar results compared to unused ones. This was true even for biological samples that show only slight differences in their expression profiles. Our analyses indicate that the stripping procedure does not significantly influence data quality derived from post-primary hybridizations. The method is robust, easy to perform, inexpensive, and results after reuse are of comparable accuracy to new arrays. (c) 2006 Elsevier B.V. All rights reserved.
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页码:1 / 13
页数:13
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