Method and kit for characterizing rna in a composition

A composition and hybrid technology, applied in the field of expression profiling, can solve the problems of time-consuming, expensive, and error-prone comparative research in working steps

Inactive Publication Date: 2014-10-08
QIAGEN GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Method used

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  • Method and kit for characterizing rna in a composition
  • Method and kit for characterizing rna in a composition
  • Method and kit for characterizing rna in a composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] Example 1 - mRNA profile obtained by hybrid capture technology:

[0135] Specific DNA oligonucleotides containing universal adapters at the 5' and 3' ends are hybridized to the target mRNA and then captured with antibodies that bind the DNA / RNA hybrid. After magnetic isolation of DNA / RNA hybrid molecules, the purified probe library was enriched by PCR prior to sequencing ( figure 1 ).

[0136] DNA probes used to hybridize to target mRNAs are specifically designed to have comparable thermodynamic properties. Hybridization of RNA to excess oligonucleotides prior to purification of DNA / RNA hybrids allows quantification of target mRNA by determining the number of DNA probes via sequencing.

[0137] Selectivity for different mRNAs can be increased by placing probes at exon-exon junctions and adjusting appropriate hybridization conditions. In addition, it allows obtaining expression profiles of different splice variants of mRNA.

[0138] Example 1 - Experiment 1: Hybridi...

Embodiment 2

[0157] Example 2 - mRNA profiles obtained by ligation of oligonucleotide probes on RNA templates:

[0158] principle:

[0159] The probe consists of two tailed oligonucleotides. OligoB contains a universal 5' tail and a target-specific 3' sequence. OligoA consists of a target-specific 5' end and a general 3' tail. The two tails differ in their base composition. In addition, oligoA at the 5' end is phosphorylated. The two oligomers are matched in a gap-free, directly adjacent manner on their target RNA molecules, allowing the 3' end of oligoB to ligate to the phosphorylated 5' end of oligoA. After hybridization and ligation, the fusion oligo probes can be amplified via standard PCR using sequencer platform-specific enrichment primers ( Figure 9 ). Probes that do not hybridize in immediate proximity and / or in the correct order will not be amplified by enrichment PCR. Subsequently, the enriched probes can be sequenced. Probe design follows traditional primer design rules...

Embodiment 3

[0165] Example 3 - mRNA profiles obtained by hybridization, reverse transcriptase reaction and subsequent ligation of tailed oligonucleotide probes on RNA templates:

[0166] principle:

[0167] Probes were designed as in Example 2, but oligoA and oligoB were separated by a significant distance to match their RNA targets. Therefore, after hybridization, a polymerase step is required to shorten the gap between the two probe oligomers prior to ligation ( Figure 12 ).

[0168] Compared to the previous examples, this additional DNA synthesis step offers several advantages:

[0169] - Improved selectivity for generating probe fragments that can be amplified by PCR. Generation of PCR amplifiable fragments requires proper priming of the two probe oligomers, proper filling of the gap between the two oligomers and ligation of the target-specific oligomeric ends.

[0170] - The number of probes for the complete splicing profile of the target gene can be reduced, since one probe pai...

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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...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2522/101C12Q1/6804C12Q1/6874C12Q2531/113C12Q1/6806C12Q2525/161C12Q2521/501C12Q2521/107C12Q1/6869C12Q2525/155
Inventor 埃丽卡·韦德勒霍尔格·韦德勒
Owner QIAGEN GMBH
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