Protocol

Drosophila

Volume 420 of the series Methods in Molecular Biology pp 303-317

Linear RNA Amplification for the Production of Microarray Hybridization Probes

  • Ansgar KlebesAffiliated withInstitut fü Biologie-Genetik, Freie Universitä Berlin
  • , Thomas B. KornbergAffiliated withDepartment of Biochemistry and Biophysics, University of California

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Abstract

To understand Drosophila development and other genetically controlled processes, it is often desirable to identify differences in gene expression levels. An experimental approach to investigate these processes is to catalog the transcriptome by hybridization of mRNA to DNA microbar-rays. In these experiments mRNA-derived hybridization probes are produced and hybridized to an array of DNA spots on a solid support. The labeled cDNAs of the complex hybridization probe will bind to their complementary sequences and provide quantification of the relative concentration of the corresponding transcript in the starting material. However, such approaches are often limited by the scarcity of the experimental sample because standard methods of probe preparation require microgram quantities of mRNA template. Linear RNA amplification can alleviate such limitations to support the generation of microarray hybridization probes from a few 100 pg of mRNA. These smaller quantities can be isolated from a few 100 cells. Here, we present a linear amplification protocol designed to preserve both the relative abundance of transcripts as well as their sequence complexity.

Key Words

Drosophila melanogaster expression profiling in vitro transcription linear RNA amplification microarray nucleic acid purification reverse transcription T7 RNA polymerase