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Title
A double-hybridization approach for the transcription- and amplification-free detection of specific mRNA on a microarray
AuthorHaider, Michaela ; Haselgrübler, Thomas ; Sonnleitner, Alois ; Aberger, Fritz ; Hesse, Jan
Published in
Microarrays, Basel, 2016, Vol. 5, Issue 1, page 1-10
PublishedMDPI, 2016
LanguageEnglish
Document typeJournal Article
Keywords (EN)microarray / mRNA detection / enzyme-free / gene expression / fluorescence microscopy
ISSN2076-3905
URNurn:nbn:at:at-ubs:3-5608 Persistent Identifier (URN)
DOI10.3390/microarrays5010005 
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 The work is publicly available
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A double-hybridization approach for the transcription- and amplification-free detection of specific mRNA on a microarray [1.42 mb]
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Abstract (English)

A double-hybridization approach was developed for the enzyme-free detection of specific mRNA of a housekeeping gene. Targeted mRNA was immobilized by hybridization to complementary DNA capture probes spotted onto a microarray. A second hybridization step of Cy5-conjugated label DNA to another section of the mRNA enabled specific labeling of the target. Thus, enzymatic artifacts could be avoided by omitting transcription and amplification steps. This manuscript describes the development of capture probe molecules used in the transcription- and amplification-free analysis of RPLP0 mRNA in isolated total RNA. An increase in specific signal was found with increasing length of the target-specific section of capture probes. Unspecific signal comprising spot autofluorescence and unspecific label binding did not correlate with the capture length. An additional spacer between the specific part of the capture probe and the substrate attachment site increased the signal significantly only on a short capture probe of approximately 30 nt length.

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CC-BY-License (4.0)Creative Commons Attribution 4.0 International License