Target labelling for the detection and profiling of microRNAs expressed in CNS tissue using microarrays.
BACKGROUND
MicroRNAs (miRNA) are a novel class of small, non-coding, gene regulatory RNA molecules that have diverse roles in a variety of eukaryotic biological processes. High-throughput detection and differential expression analysis of these molecules, by microarray technology, may contribute to a greater understanding of the many biological events regulated by these molecules. In this investigation we compared two different methodologies for the preparation of labelled miRNAs from mouse CNS tissue for microarray analysis. Labelled miRNAs were prepared either by a procedure involving linear amplification of miRNAs (labelled-aRNA) or using a direct labelling strategy (labelled-cDNA) and analysed using a custom miRNA microarray platform. Our aim was to develop a rapid, sensitive methodology to profile miRNAs that could be adapted for use on limited amounts of tissue.


RESULTS
We demonstrate the detection of an equivalent set of miRNAs from mouse CNS tissues using both amplified and non-amplified labelled miRNAs. Validation of the expression of these miRNAs in the CNS by multiplex real-time PCR confirmed the reliability of our microarray platform. We found that although the amplification step increased the sensitivity of detection of miRNAs, we observed a concomitant decrease in specificity for closely related probes, as well as increased variation introduced by dye bias.


CONCLUSIONS
The data presented in this investigation identifies several important sources of systematic bias that must be considered upon linear amplification of miRNA for microarray analysis in comparison to directly labelled miRNA.
Saba
Reuben
R
Division of Host Genetics & Prion Diseases, National Microbiology Laboratory, 1015 Arlington Street, Public Health Agency of Canada, Winnipeg, MB, R3E 3R2, Canada. Reuben_Saba@phac-aspc.gc.ca <Reuben_Saba@phac-aspc.gc.ca>
Booth
Stephanie A
SA
D000818:Animals; D001921:Brain / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D018390:Gene Targeting / Q000379:methods*; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015347:RNA Probes / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
D015347:RNA Probes