Enzymatic Synthesis of High-Density RNA Microarrays

Author(s)

Erika Schaudy, Jory Lietard, Mark M. Somoza

Abstract

Oligonucleotide microarrays are used to investigate the interactome of nucleic acids. DNA microarrays are commercially available, whereas equivalent RNA microarrays are not. This protocol describes a method to convert DNA microarrays of any density and complexity into RNA microarrays using only readily available materials and reagents. This simple conversion protocol will facilitate the accessibility of RNA microarrays to a wide range of researchers. In addition to general considerations for the design of a template DNA microarray, this procedure describes the experimental steps of hybridization of an RNA primer to the immobilized DNA, followed by its covalent attachment via psoralen-mediated photocrosslinking. The subsequent enzymatic processing steps comprise the extension of the primer with T7 RNA polymerase to generate complementary RNA, and finally the removal of the DNA template with TURBO DNase. Beyond the conversion process, we also describe approaches to detect the RNA product either by internal labeling with fluorescently labeled NTPs or via hybridization to the product strand, a step that can then be complemented by an RNase H assay to confirm the nature of the product.

Organisation(s)

Department of Inorganic Chemistry

External organisation(s)

Technische Universität München

Journal

Current protocols

Volume

3

ISSN

2691-1299

DOI

https://doi.org/10.1002/cpz1.667

Publication date

02-2023

Peer reviewed

Yes

Austrian Fields of Science 2012

106002 Biochemistry, 106023 Molecular biology, 206001 Biomedical engineering

Keywords

ASJC Scopus subject areas

General Neuroscience, General Biochemistry,Genetics and Molecular Biology, General Immunology and Microbiology, Pharmacology, Toxicology and Pharmaceutics(all), Health Informatics, Medical Laboratory Technology

Portal url

https://ucrisportal.univie.ac.at/en/publications/eea99f36-f977-42e3-9a04-1fdee5b8907a