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Compositions and methods of RNA analysis

A technology of groups and substitutes, applied in the field of composition at the level of existence and expression, can solve problems such as inhibiting the extraction of important information, reducing RNA quality, and lagging gene expression profiles

Pending Publication Date: 2018-07-31
THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ability to characterize gene expression profiles in fixed tissue specimens (e.g., formalin-fixed paraffin-embedded tissue sections) has lagged because the fixation process degrades the quality of RNA in the sample, significantly inhibiting the generation of RNA from the sample. Ability to extract important information from samples

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  • Compositions and methods of RNA analysis
  • Compositions and methods of RNA analysis
  • Compositions and methods of RNA analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1: Development of in situ ligation hybridization sequencing (LISH-seq)

[0112] The optimal protocol to efficiently generate probe ligation products in FFPE sections was identified. A housekeeping gene probe set was used to explore the impact of various protocol differences on the signal (on-target probe ligation) and interference (off-target probe ligation) of the assay after a decade. The efficacy of LISH was assessed by direct comparison with RT-qPCR. High-throughput DNA sequencing of pooled LISH products ("LISH-seq") demonstrates the degree of multiplexing (eg, hundreds to thousands of transcripts per tissue section) afforded by this system. A batch of long-term stored specimens was analyzed by LISH-seq. This dataset demonstrates the extent to which LISH-seq unlocks archived large patient tumor specimens for highly multiplexed mRNA analysis. A strategy exists that uses LISH-seq to overcome limitations previously associated with the analysis of RNA extract...

Embodiment 2

[0139] Example 2: Maintaining spatial resolution of amplified LISH products: LISH-imprinting

[0140] If this spatial distribution of transcript levels is preserved, the value of multiplex gene expression analysis can be greatly enhanced. The technique is particularly useful for characterizing the complex interactions between tumor tissue and cells of the immune system. A platform capable of highly multiplexed, spatially resolved analysis of gene expression in FFPE tissue sections is transformable for cancer researchers and pathologists.

[0141]The utility of LISH is extended to methods to imprint LISH products on target surfaces and amplify to preserve spatial information ("LISH-imprinting"). This attempt is based on the imprinting of DNA molecules on replica high-density DNA microarrays. To locally amplify imprinted LISH products, bridge PCR is used, which has been revealed in the literature and is the basis of Illumina high-throughput DNA sequencing technology. After de...

Embodiment 3

[0162] Example 3: Measurement of RNA-Seq in FFPE

[0163] Reports in recent years have shown that RNA-templated probe ligation chemistry is sensitive, specific, and appropriate for large-scale multiplexing (Larman, H.B., et al. Nucleic acids Research 42, 9146-9157 (2014), incorporated herein by reference in its entirety). This pathway employs T4 RNA ligase 2 (Rnl2), an enzyme that ligates sets of adjacently annealed sequence-specific oligonucleotide probes to RNA "splints." In this context, Rn12 requires that the acceptor oligonucleotide be 3'-terminated by two ribonucleotides (herein referred to as "3'-diriboprobe"). In some examples, the acceptor oligonucleotide can have a 3' end cap of at least two RNA bases. Phosphorylated donor probes (referred to herein as "5'-phosphate probes") can be entirely DNA ( Figure 5A ). Thus, the linked 3'-diriboprobe set and 5'-phosphate probe set are DNA molecules containing internal diribonucleotide sequences.

[0164] No known DNA lig...

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Abstract

The present invention relates to compositions and methods of RNA analysis. In particular, the present invention provides a method of RNA analysis. The method comprises the steps of: obtaining a sample, applying one or more multi-partite probes to the sample, wherein each of the one or more multi-partite probes includes at least two sub-probes, annealing at least one of the applied one or more multi-partite probes to at least one target nucleic acid within the sample, and ligating the at least two sub-probes associated with the at least one annealed multi-partite probe to create a target nucleic acid proxy that can be detected.

Description

[0001] Cross References to Related Applications [0002] Based on 35 U.S.C. §119(e), this application claims priority to U.S. Provisional Patent Application No. 62 / 196,725, filed July 24, 2015, entitled "Compositions and Methods for RNA Analysis," which U.S. Provisional The patent application is hereby incorporated by reference in its entirety. Background technique [0003] Precision medicine relies on the ability of researchers and pathologists to molecularly characterize patient specimens, including resected tissue, tumor tissue, and biopsy material. The development of advanced technologies for DNA analysis such as high-throughput DNA sequencing (or next-generation DNA sequencing (NGS)) has contributed to the detection of cancer-associated genetic damage. However, the ability to characterize gene expression profiles within fixed tissue specimens (e.g., formalin-fixed paraffin-embedded tissue sections) has lagged because the fixation process degrades the quality of RNA in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2521/327C12Q2523/109C12Q2525/155C12Q2525/161C12Q2525/204C12Q2535/122C12Q2543/10C12Q2565/125C12Q2565/627C12Q2521/501C12Q1/68
Inventor H·B·拉尔曼J·克雷德尔
Owner THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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