Patent application EP19169263A (EP), filed 2019-04-15
Publication number: EP3725880A1
Title: METHOD AND KIT FOR THE PURIFICATION OF FUNCTIONAL RISC-ASSOCIATED SMALL RNAS
Abstract: The invention relates to methods and kits for the purification of functional RISC-associated small RNAs in organisms, organs, tissues, cells or biological fluids.
IPC codes: C12N  15/    10            A I
Claims: number: 1
text: Method for the purification of RISC-associated sRNAs, comprising the following steps:
number: 2
text: Method according to claim 1, wherein in step c), non-RISC associated nucleic acids are removed from the lysate by loading the lysate onto a column comprising a resin allowing the fixation of nucleic acids.
number: 3
text: Method according to claim 1, wherein in step d) RISCs are collected by applying an elution buffer to the column.
number: 4
text: Method according to claim 1, wherein the resin is an anion exchange resin.
number: 5
text: Method according to any of claims 1 to 4, wherein the column is a 96 well plate or a microfluidic chip.
number: 6
text: Method according to any of claims 1 to 5, wherein the specimen is from a human.
number: 7
text: Method according to any of claims 1 to 5, wherein the biological specimen is a RISC-containing sample generated by in vitro, in cellulo or in vivo RISC production.
number: 8
text: Method according to any of claims 1 to 7, further comprising:
number: 9
text: Method according to any of claims 1 to 8 for use in the diagnosis of a disease whose diagnosis or prognosis is characterized by the presence of particular sRNAs.
number: 10
text: Method for use according to claim 9, wherein the disease is a cancer, a metabolic disorder, an hereditary condition or an infectious disease.
number: 11
text: A kit for the purification of RISC-associated sRNAs, comprising:
number: 12
text: Kit according to claim 11, wherein the lysis buffer comprises 20 mM HEPES-KOH (pH 7.9), 10 to 20% (v/v) glycerol, 1.5 mM MgCI2, 0.2 mM EDTA, 1mM DTT, 100 mM CH3CO2K and 0.1% Triton X-100, with a measured conductivity from 7.5 to 8.5 mS/cm<2>.
number: 13
text: Kit according to claim 11, wherein the elution buffer comprises 20 mM HEPES-KOH (pH 7.9), 10 to 20% (v/v) glycerol, 1.5 mM MgCI2, 0.2 mM EDTA, 1mM DTT and a CH3CO2K concentration comprised between 400 and 800 mM, to reach a measured conductivity comprised between 30 and 50 mS/cm<2>.
number: 14
text: Kit according to claim 11, wherein the storage buffer comprises 20 mM HEPES-KOH (pH 7.9), 10 to 20% (v/v) glycerol, 1.5 mM MgCI2, 0.2 mM EDTA, 1mM DTT, 100 mM CH3CO2K, with a measured conductivity from 7.5 to 8.5 mS/cm<2>, and 2 mM NaN3.
Office action: search_report, dated 2020-10-21
Rejection reasons: novelty_epc_54
Prior art cited: ref_id: WO2010033652A1
ref_type: patent
severity: background
ref_id: AU2007202668A1
ref_type: patent
severity: background
ref_id: - FLORIAN BELLUTTI ET AL: "ABSTRACT", JOURNAL OF VIROLOGY., vol. 89, no. 3, 1 February 2015 (2015-02-01), US, pages 1608 - 1627, XP055600143, ISSN: 0022-538X, DOI: 10.1128/JVI.02336-14
ref_type: npl
severity: background
ref_id: AU2007202668
ref_type: patent
severity: novelty_destroying
ref_id: WO2010033652
ref_type: patent
severity: novelty_destroying
ref_id: - FLORIAN BELLUTTI ET AL, "ABSTRACT", JOURNAL OF VIROLOGY., US, (20150201), vol. 89, no. 3, doi:10.1128/JVI.02336-14, ISSN 0022-538X, pages 1608 - 1627, XP055600143 [X] 1 * page  1608  - page  1627 * [I] 2-14
ref_type: npl
severity: novelty_destroying
Final disposition: pending (2020-10-21)