Evaluation of a new high-dimensional miRNA profiling platform

Julie M Cunningham,Pedro M Borralho,Betsy T Kren,Yan Zeng,Ann L Oberg,Liang Wang,Kevin At Silverstein,Christopher A Hilker,Brian M Bot,Stephen N Thibodeau,Jian-Bing Fan,Clifford J Steer,Bruce W Morlan,Anne-Francoise Lamblin,Amy J French,Rod Staggs

doi:10.1186/1755-8794-2-57

Julie M Cunningham, Pedro M Borralho + Show 14 more

Open Access

https://doi.org/10.1186/1755-8794-2-57

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Abstract

BackgroundMicroRNAs (miRNAs) are a class of approximately 22 nucleotide long, widely expressed RNA molecules that play important regulatory roles in eukaryotes. To investigate miRNA function, it is essential that methods to quantify their expression levels be available.MethodsWe evaluated a new miRNA profiling platform that utilizes Illumina's existing robust DASL chemistry as the basis for the assay. Using total RNA from five colon cancer patients and four cell lines, we evaluated the reproducibility of miRNA expression levels across replicates and with varying amounts of input RNA. The beta test version was comprised of 735 miRNA targets of Illumina's miRNA profiling application.ResultsReproducibility between sample replicates within a plate was good (Spearman's correlation 0.91 to 0.98) as was the plate-to-plate reproducibility replicates run on different days (Spearman's correlation 0.84 to 0.98). To determine whether quality data could be obtained from a broad range of input RNA, data obtained from amounts ranging from 25 ng to 800 ng were compared to those obtained at 200 ng. No effect across the range of RNA input was observed.ConclusionThese results indicate that very small amounts of starting material are sufficient to allow sensitive miRNA profiling using the Illumina miRNA high-dimensional platform. Nonlinear biases were observed between replicates, indicating the need for abundance-dependent normalization. Overall, the performance characteristics of the Illumina miRNA profiling system were excellent.

Highlights

MicroRNAs are a class of approximately 22 nucleotide long, widely expressed RNA molecules that play important regulatory roles in eukaryotes
Binding of the RISC leads to suppression of translation and possibly degradation of target mRNAs
These were chosen to provide examples of bias curves and variability observed among our test set, ranging from a low amount of scatter to a greater amount

Summary

Introduction

MicroRNAs (miRNAs) are a class of approximately 22 nucleotide long, widely expressed RNA molecules that play important regulatory roles in eukaryotes. First identified nearly 15 years ago [1], microRNAs (miRNAs) are a family of short RNA molecules that predominantly inhibit gene expression at the post-transcriptional level in eukaryotes [2,3]. Genes encoding primary miRNAs (pri-miRNA) are much longer than the mature form. These primary transcripts are processed by a nuclease (Drosha) and the double-stranded RNA binding protein, DGCR8, to short 60–70 nucleotide stem-loop structures (pre-miRNA). Mature miRNA are ~22 nucleotides in length and guide the RNA-induced silencing complex (RISC, the core components of which contain Argonaute proteins) to the target sites, usually located in the 3' untranslated region of gene transcripts[4]. Binding of the RISC leads to suppression of translation and possibly degradation of target mRNAs

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