A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA)

Castoldi, Mirco; Schmidt, Sabine; Beneš, Vladimír; Noerholm, Mikkel; Kulozik, Andreas E.; Hentze, Matthias W.; Muckenthaler, Martina U.

doi:10.1261/rna.2332406

Cited by 393 publications

(299 citation statements)

References 36 publications

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“…This is either done by altering the probe length (Goff et al 2005) or including nucleotide analogs (Castoldi et al 2006). In addition, different DNA surface attachment strategies have been published to reduce the influence of surface physics and chemistry on the hybridization behavior of probe/target pairs.…”

Section: Discussionmentioning

confidence: 99%

“…The strength of miRNA microarrays lay thus far in relative (ratio-based) measurements of known miRNAs in high throughput. In order to optimize the measurement of miRNAs, attempts have been made to adjust the melting temperature or the free energy (DG) of probe/target pairs by altering the individual probe length or including nucleotide analogs that increase duplex stability, such as 29OMe modified nucleotides or LNA residues (Castoldi et al 2006). However, while these approaches narrow the melting temperature differences of miRNA/ probe pairs, other effects distorting the microarray signal-such as the labeling efficiency or RNA folding-are not considered.…”

Section: Introductionmentioning

confidence: 99%

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Absolute quantification of microRNAs by using a universal reference

Bissels¹,

Wild²,

Tomiuk³

et al. 2009

RNA

180

132

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MicroRNAs (miRNAs) are a species of small RNAs ;21-23-nucleotides long that have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous PCR-, sequencing-, or hybridization-based methods have been established to identify and quantify miRNAs. Their short length results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray-based approach for global and absolute quantification of miRNAs. The method relies on the parallel hybridization of the sample of interest labeled with Cy5 and a universal reference of 954 synthetic miRNAs in equimolar concentrations that are labeled with Cy3 on a microarray slide containing probes for all human, mouse, rat, and viral miRNAs (miRBase 12.0). Each single miRNA is quantified with respect to the universal reference canceling biases related to sequence, labeling, or hybridization. We demonstrate the accuracy of the method by various spike-in experiments. Furthermore, we quantified miRNA copy numbers in liver samples and CD34(+)/ CD133(À) hematopoietic progenitor cells.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Absolute quantification of microRNAs by using a universal reference

Bissels¹,

Wild²,

Tomiuk³

et al. 2009

RNA

180

132

View full text Add to dashboard Cite

show abstract

“…Briefly, a poly(A) tailing reaction was performed and capture sequences were ligated to the poly(A) products. Tagged miRNA derived from both samples were then purified, pooled, and hybridized to a MiRCURY locked nucleic acid (LNA) microarray (Exiqon, Vedbaek, Denmark) containing 454 LNA-modified oligonucleotide probes for human, mouse, and rat miRNA as annotated in the miRBase release 8.0 (23,24).…”

Section: Methodsmentioning

confidence: 99%

Altered expression of MicroRNA in synovial fibroblasts and synovial tissue in rheumatoid arthritis

Stańczyk

Pedrioli

Brentano

et al. 2008

Arthritis & Rheumatism

758

624

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Objective. MicroRNAs (miRNA) have recently emerged as a new class of modulators of gene expression. In this study we investigated the expression, regulation, and function of miR-155 and miR-146a in rheumatoid arthritis (RA) synovial fibroblasts (RASFs) and RA synovial tissue.Methods. Locked nucleic acid microarray was used to screen for differentially expressed miRNA in RASFs treated with tumor necrosis factor ␣ (TNF␣). TaqMan-based real-time polymerase chain reaction was applied to measure the levels of miR-155 and miR-146a. Enforced overexpression of miR-155 was used to investigate the function of miR-155 in RASFs.Results

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“…LNA-modified arrays can 55 be designed to have a uniform and high melting temperature for all probes, increasing the sensitivity of the array and giving consistent quantitation across the full range of miRNAs. 23 Quantitative real-time PCR is another diagnostic approach that can be improved by LNA technology. In 5'-nuclease assay PCR, 60 a fluorescent probe oligonucleotide is degraded by the Taq polymerase.…”

mentioning

confidence: 99%