Method and kit for characterizing rna in a composition

Abstract

The invention relates to a kit and a method for determining the sequence and / or quantity of a ribonucleic acid in a composition, comprising the steps of: . i. providing a composition comprising one or more ribonucleic acids molecules (RNA), . ii. hybridizing to said one or more RNAs, one or more two-part nucleic acid hybridization probes, wherein each probe comprises, . a. a first nucleic acid molecule with a 3'-tail wherein said tail does not hybridize to an RNA in the composition, . b. a second nucleic acid molecule with a 5'-tail wherein said tail does not hybridize to an RNA in the composition, . c. wherein said first and said second nucleic acid molecules when and if hybridized to their target RNA lie on one single stranded RNA molecule separated from each other by between 2 and 1000 nucleotides, . iii. covalently linking the hybridized 5'-tail of said first nucleic acid molecule to the hybridized 3'-tail of said second nucleic acid, wherein the linking is done by means of reverse transcription and subsequent ligation, . binding the RNA-DNA hybrids to duplex-specific antibodies, . iv. amplifying the linked molecules with primers that are specific for said first 3'-tail of said first nucleic acid molecule and said second 5'-tail of said second nucleic acid molecule and, . v. sequencing the amplification products by means of next generation sequencing. . The kit comprises primers, antibodies and ligase or reverse transcriptase.

Description

technical field [0001] The present invention relates to the fields of molecular biology, diagnostics and more particularly expression profiling. Background technique [0002] In recent years, research in the field of transcriptome analysis has evolved from candidate gene-based RNA detection using Northern blotting to high-throughput expression profiling driven by the advent of microarrays. Since 2006, next-generation sequencing technologies have revolutionized transcriptomics by providing opportunities for multidimensional examination of cellular transcriptomes in which high-throughput expression data are obtained at single-base resolution . Table 1 summarizes the gene expression profiling milestones. [0003] [0004] [0005] Table 1. Milestones of transcript analysis [0006] Many protocols and commercial kits have been developed for mRNA-seq by next-generation sequencing. In general, the flow chart of standard transcriptome analysis includes 11 steps (the numbe...

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Problems solved by technology

[0023] However, comprehensive analysis of complex transcriptomes, which includes data analysis and extraction of biologically and medically relevant information, will still be costly and labor-intensive

[0024] Additionally, the many work steps from RNA extraction to sequencing are time-consuming, error-prone, and make comparative studies difficult

Finally, many NGS machines do not have the capability to generate sufficient reads for an entire complex transcriptome within a single sequencing run

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