Striptease on glass: validation of an improved stripping procedure for in situ microarrays.
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.
Hahnke
Karin
K
Max Planck Institute for Infection Biology, Microarray Core Facility, Schumannstr. 21/22, 10117 Berlin, Germany.
Jacobsen
Marc
M
Gruetzkau
Andreas
A
Gruen
Joachim R
JR
Koch
Markus
M
Emoto
Masashi
M
Meyer
Thomas F
TF
Walduck
Anna
A
Kaufmann
Stefan H E
SH
Mollenkopf
Hans-Joachim
HJ
D003363:Cost Control; D017743:Equipment Reuse; D020869:Gene Expression Profiling / Q000191:economics / Q000295:instrumentation* / Q000379:methods; D005898:Glass; D007202:Indicators and Reagents / Q000191:economics; D020411:Oligonucleotide Array Sequence Analysis / Q000191:economics / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results
D007202:Indicators and Reagents