New miRNA labeling method for bead-based quantification.
BMC Mol Biol
; 11: 44, 2010 Jun 16.
Article
em En
| MEDLINE
| ID: mdl-20553585
ABSTRACT
BACKGROUND:
microRNAs (miRNAs) are small single-stranded non-coding RNAs that act as crucial regulators of gene expression. Different methods have been developed for miRNA expression profiling in order to better understand gene regulation in normal and pathological conditions. miRNAs expression values obtained from large scale methodologies such as microarrays still need a validation step with alternative technologies.RESULTS:
Here we have applied with an innovative approach, the Luminex xMAP technology validate expression data of differentially expressed miRNAs obtained from high throughput arrays. We have developed a novel labeling system of small RNA molecules (below 200 nt), optimizing the sensitive cloning method for miRNAs, termed miRNA amplification profiling (mRAP). The Luminex expression patterns of three miRNAs (miR-23a, miR-27a and miR-199a) in seven different cell lines have been validated by TaqMan miRNA assay. In all cases, bead-based meas were confirmed by the data obtained by TaqMan and microarray technologies.CONCLUSIONS:
We demonstrate that the measure of individual miRNA by the bead-based method is feasible, high speed, sensitive and low cost. The Luminex xMAP technology also provides flexibility, since the central reaction can be scaled up with additional miRNA capturing beads, allowing validation of many differentially expressed miRNAs obtained from microarrays in a single experiment. We propose this technology as an alternative method to qRT-PCR for validating miRNAs expression data obtained with high-throughput technologies.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sondas de Oligonucleotídeos
/
Análise de Sequência com Séries de Oligonucleotídeos
/
MicroRNAs
Limite:
Humans
Idioma:
En
Revista:
BMC Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Itália