New miRNA labeling method for bead-based quantification.
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.
Biscontin
Alberto
A
Department of Biology and CRIBI Biotechnology Centre, Università degli Studi di Padova, Via U. Bassi, 58/B, 35121 Padova, Italy.
Casara
Silvia
S
Cagnin
Stefano
S
Tombolan
Lucia
L
Rosolen
Angelo
A
Lanfranchi
Gerolamo
G
De Pittà
Cristiano
C
D045744:Cell Line, Tumor; D005456:Fluorescent Dyes; D057166:High-Throughput Screening Assays; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D015345:Oligonucleotide Probes
D005456:Fluorescent Dyes; D035683:MicroRNAs; D015345:Oligonucleotide Probes