Materials and methods for profiling microRNAs
The present invention provides materials and methods for detecting, quantifying, and/or high-throughput-profiling microRNAs. Advantageously, the present invention is more sensitive and specific than other currently-available miRNA qPCR assays. In addition, the present invention is convenient, easy-to-perform, and cost-effective. In one embodiment, the present invention provides a universal primer for reverse transcription of all miRNAs, a universal reverse primer for PCR amplification reaction, and universal probes. In another embodiment, the present invention provides assays that allow simultaneous detection and/or quantification of a plurality of target miRNAs using a single reverse transcription reaction.
1. A composition of matter comprising a universal probe for detecting, quantifying and/or profiling miRNA, wherein the universal probe comprises a sequence that is, or base-pairs with, at least part of the stem of a stem-looped universal adaptor sequence, wherein said universal probe comprises one or more locked nucleic acids and a detectable moiety, and wherein said sequence of said universal probe consists of ACC[A][T][C]A[A][T][C]G[T]G[T]G (SEQ ID NO: 16), wherein bracketed nucleotides are locked nucleic acids.
2. A probe comprising a sequence consisting of SEQ ID NO:16, wherein said probe comprises one or more locked nucleic acids.
3. The probe of claim 2 , wherein said probe further comprises a detectable moiety.
4. The probe of claim 3 , wherein said detectable moiety comprises a fluorophore.
5. The probe of claim 3 , wherein said probe further comprises a quencher molecule attached to a first end of said sequence, and wherein said detectable moiety is a fluorescent dye attached to a second end of said sequence.