title: Single primer amplification (SPA) of cDNA for microarray expression analysis.
abstract: The potential of expression analysis using cDNA microarrays to address complex problems in a wide variety of biological contexts is now being realised. A limiting factor in such analyses is often the amount of RNA required, usually tens of micrograms. To address this problem researchers have turned to methods of improving detection sensitivity, either through increasing fluorescent signal output per mRNA molecule or increasing the amount of target available for labelling by use of an amplification procedure. We present a novel DNA-based method in which an oligonucleotide is incorporated into the 3' end of cDNA during second-strand cDNA synthesis. This sequence provides an annealing site for a single complementary heel primer that directs Taq DNA polymerase amplification of cDNA following multiple cycles of denaturation, annealing and extension. The utility of this technique for transcriptome-wide screening of relative expression levels was compared to two alternative methodologies for production of labelled cDNA target, namely incorporation of fluorescent nucleotides by reverse transcriptase or the Klenow fragment. Labelled targets from two distinct mouse tissues, adult liver and kidney, were compared by hybridisation to a set of cDNA microarrays containing 6500 mouse cDNA probes. Here we demonstrate, through a dilution series of cDNA derived from 10 micro g of total RNA, that it is possible to produce datasets comparable to those produced with unamplified targets with the equivalent of 30 ng of total RNA. The utility of this technique for microarray analysis in cases where sample is limited is discussed.
authors.affiliation: MRC Mammalian Genetics Unit, Harwell, Didcot, Oxfordshire OX11 0RD, UK. l.smith@har.mrc.ac.uk
authors.forename: Lee
authors.initials: L
authors.lastname: Smith
authors.forename: Peter
authors.initials: P
authors.lastname: Underhill
authors.forename: Clare
authors.initials: C
authors.lastname: Pritchard
authors.forename: Zuzanna
authors.initials: Z
authors.lastname: Tymowska-Lalanne
authors.forename: Saba
authors.initials: S
authors.lastname: Abdul-Hussein
authors.forename: Helen
authors.initials: H
authors.lastname: Hilton
authors.forename: Laura
authors.initials: L
authors.lastname: Winchester
authors.forename: Deborah
authors.initials: D
authors.lastname: Williams
authors.forename: Tom
authors.initials: T
authors.lastname: Freeman
authors.forename: Sarah
authors.initials: S
authors.lastname: Webb
authors.forename: Andy
authors.initials: A
authors.lastname: Greenfield
mesh_terms: D000818:Animals; D004256:DNA Polymerase I / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary / Q000138:chemical synthesis*; D005260:Female; D020869:Gene Expression Profiling; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D015203:Reproducibility of Results; D019914:Taq Polymerase / Q000378:metabolism
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D019914:Taq Polymerase; D012194:RNA-Directed DNA Polymerase; D004256:DNA Polymerase I