miChip: a microarray platform for expression profiling of microRNAs based on locked nucleic acid (LNA) oligonucleotide capture probes.
As key regulators of post-transcriptional gene expression, it is important to monitor the expression of microRNAs (miRNA) in diverse physiological and pathophysiological processes. Here, we describe a method for sensitive and accurate microarray-based expression profiling of miRNAs. The protocol focuses on the use of locked nucleic acid (LNA)-modified capture probes. LNAs are bicyclic nucleotide analogues that significantly increase the melting temperature (T(m)) of hybrids with miRNAs. Mixed LNA/DNA capture probes thus can be designed for equal T(m)s for all miRNAs, which naturally cover a range between 45 and 74 degrees C. The protocols established are easy to apply, as they do not require RNA size selection and/or amplification of miRNAs. Moreover, they enable high affinity hybridizations yielding accurate signals that discriminate between single nucleotide differences and hence closely related miRNA family members.
Castoldi
Mirco
M
Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Im Neuenheimer Feld 156, D-69210 Heidelberg, Germany.
Benes
Vladimir
V
Hentze
Matthias W
MW
Muckenthaler
Martina U
MU
D001483:Base Sequence; D020869:Gene Expression Profiling / Q000379:methods*; D035683:MicroRNAs / Q000235:genetics* / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics
D035683:MicroRNAs; D015345:Oligonucleotide Probes