WO2024256715A1 - Réactif pour le contrôle qualité de l'hybridation in situ - Google Patents

Réactif pour le contrôle qualité de l'hybridation in situ Download PDF

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WO2024256715A1
WO2024256715A1 PCT/EP2024/066706 EP2024066706W WO2024256715A1 WO 2024256715 A1 WO2024256715 A1 WO 2024256715A1 EP 2024066706 W EP2024066706 W EP 2024066706W WO 2024256715 A1 WO2024256715 A1 WO 2024256715A1
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sequence
signal
monitoring
probe
analyte
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Seiichiro Watanabe
Lawrence Greenfield
Christian Korfhage
Cheng Frank Zhong
Frank Reinecke
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Resolve Biosciences GmbH
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Resolve Biosciences GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification

Definitions

  • RNAscope® reference ⁇ guide: ⁇ Guidelines ⁇ and ⁇ Protocols ⁇ for ⁇ Obtaining ⁇ Optimized ⁇ RNA ⁇ in ⁇ situ ⁇ Hybridization ⁇ Results ⁇ with ⁇ Any ⁇ Tissue ⁇ Type ⁇ from ⁇ Any ⁇ Species. ⁇ ACD.
  • the ⁇ disclosure ⁇ pertains ⁇ to ⁇ monitoring ⁇ probes comprising ⁇ at ⁇ least ⁇ one ⁇ identifier ⁇ element (t) ⁇ comprising ⁇ a ⁇ nucleotide ⁇ sequence ⁇ which ⁇ is ⁇ essentially ⁇ complementary ⁇ to ⁇ at ⁇ least ⁇ a ⁇ section ⁇ of ⁇ the ⁇ unique ⁇ set ⁇ identifier ⁇ sequence ⁇ of ⁇ an ⁇ identifier ⁇ element ⁇ (T) ⁇ of ⁇ a ⁇ corresponding ⁇ decoding ⁇ element.
  • ⁇ In ⁇ one ⁇ embodiment ⁇ the ⁇ nucleotide ⁇ acid ⁇ sequence of ⁇ the ⁇ monitoring ⁇ probe is ⁇ attached ⁇ to ⁇ a ⁇ particle ⁇ selected ⁇ from ⁇ a ⁇ non-magnetic ⁇ bead ⁇ particle, ⁇ magnetic ⁇ bead ⁇ particle, ⁇ a ⁇ streptavidin ⁇ coated ⁇ particle, ⁇ a ⁇ biotin ⁇ coated ⁇ particle, ⁇ and/or ⁇ any ⁇ combination ⁇ thereof.
  • In ⁇ yet ⁇ a ⁇ further ⁇ embodiment ⁇ the ⁇ monitoring ⁇ probe is ⁇ immobilized ⁇ on ⁇ a ⁇ slide ⁇ surface through ⁇ a ⁇ poly-lysine ⁇ coating ⁇ of ⁇ the ⁇ slide ⁇ surface.
  • the ⁇ monitoring ⁇ probe is ⁇ immobilized ⁇ to ⁇ the ⁇ poly-lysine ⁇ coated ⁇ slide ⁇ surface ⁇ using ⁇ homobifunctional ⁇ or ⁇ heterobifunctional ⁇ cross-linking ⁇ reagents ⁇ via ⁇ a ⁇ reactive ⁇ group.
  • In ⁇ yet ⁇ a ⁇ further ⁇ embodiment ⁇ the ⁇ monitoring ⁇ probe or ⁇ the ⁇ slide ⁇ surface comprise ⁇ a ⁇ reactive ⁇ group ⁇ consisting ⁇ of ⁇ an ⁇ alkyne ⁇ or ⁇ azide, ⁇ and ⁇ the ⁇ other ⁇ contact ⁇ surface ⁇ comprises ⁇ the ⁇ partner ⁇ azide or ⁇ alkyne, ⁇ such ⁇ that ⁇ the ⁇ immobilization ⁇ can ⁇ occur ⁇ using ⁇ click ⁇ chemistry.
  • In ⁇ yet ⁇ a ⁇ further ⁇ embodiment ⁇ the ⁇ size ⁇ of ⁇ the ⁇ monitoring ⁇ probe is ⁇ 250 ⁇ nm ⁇ or ⁇ less; ⁇ 225 ⁇ nm ⁇ or ⁇ less; ⁇ 210 ⁇ nm ⁇ or ⁇ less; ⁇ but ⁇ at ⁇ least ⁇ 90 ⁇ nm, ⁇ at ⁇ least ⁇ 130 ⁇ nm ⁇ at ⁇ least ⁇ 180 ⁇ nm, ⁇ in ⁇ one ⁇ embodiment ⁇ the ⁇ size ⁇ of ⁇ the ⁇ monitoring ⁇ probe is ⁇ about ⁇ 200 ⁇ nm.
  • the method ⁇ to ⁇ monitor ⁇ an ⁇ in ⁇ situ hybridization ⁇ multiplex ⁇ reaction ⁇ comprises ⁇ at ⁇ least ⁇ one ⁇ monitoring ⁇ probe as ⁇ described ⁇ herein, ⁇ wherein ⁇ the ⁇ in ⁇ situ hybridization ⁇ multiplex ⁇ reaction ⁇ is ⁇ characterized ⁇ by ⁇ comprising: ⁇ a.) a ⁇ binding ⁇ element which ⁇ is ⁇ essentially ⁇ complementary ⁇ to ⁇ at ⁇ least ⁇ a ⁇ section ⁇ of ⁇ an ⁇ analyte ⁇ nucleotide ⁇ sequence ⁇ to ⁇ be ⁇ detected ⁇ and ⁇ an ⁇ identifier ⁇ element ⁇ (T) ⁇ comprising ⁇ a ⁇ nucleotide ⁇ sequence ⁇ which ⁇ is ⁇ unique ⁇ to ⁇ the ⁇ analyte ⁇ nucleotide ⁇ sequence; ⁇ and/or b.) a ⁇ decoding ⁇ element comprising ⁇ an ⁇ identifier ⁇ element ⁇ (t) ⁇ comprising ⁇ a ⁇ nucleotide ⁇ sequence ⁇ which ⁇ is ⁇ essentially ⁇ comp
  • the in-situ hybridization ⁇ multiplex ⁇ reaction ⁇ is ⁇ described ⁇ in ⁇ more ⁇ details ⁇ in ⁇ this ⁇ description, ⁇ however, ⁇ it ⁇ should ⁇ be ⁇ mentioned ⁇ in ⁇ a ⁇ first ⁇ overview, ⁇ that ⁇ the ⁇ monitoring ⁇ probe ⁇ of ⁇ the ⁇ present ⁇ RES-PA18-PCT ⁇ invention ⁇ is ⁇ designed ⁇ to ⁇ allow ⁇ the ⁇ monitoring ⁇ of ⁇ the ⁇ correct ⁇ in ⁇ situ hybridization ⁇ multiplex ⁇ reaction ⁇ by ⁇ being ⁇ part ⁇ of ⁇ the ⁇ reaction, ⁇ thereby ⁇ being ⁇ exposed ⁇ to ⁇ the ⁇ same ⁇ reactants ⁇ and ⁇ reactions ⁇ steps ⁇ as ⁇ used ⁇ in ⁇ the ⁇ in ⁇ situ hybridization ⁇ multiplex ⁇ reaction.
  • In ⁇ one ⁇ embodiment ⁇ the ⁇ method/ ⁇ monitoring ⁇ probe comprises ⁇ a ⁇ set ⁇ of ⁇ monitoring ⁇ probes, ⁇ wherein ⁇ for ⁇ each ⁇ of ⁇ the ⁇ different ⁇ identifier ⁇ elements ⁇ (t) ⁇ of ⁇ the ⁇ decoding ⁇ elements the set
  • the ⁇ disclosure ⁇ encompasses ⁇ a method, ⁇ wherein ⁇ the monitoring ⁇ probe as ⁇ disclosed ⁇ herein, ⁇ can ⁇ be ⁇ detected ⁇ by ⁇ a ⁇ signal ⁇ element ⁇ with ⁇ a ⁇ fluorophore ⁇ being ⁇ the ⁇ same ⁇ or ⁇ being ⁇ different ⁇ from ⁇ the ⁇ fluorophore ⁇ used ⁇ in ⁇ the ⁇ detection ⁇ of ⁇ the ⁇ target ⁇ nucleic ⁇ acid ⁇ sequence ⁇ that ⁇ permits ⁇ normalizing ⁇ the ⁇ signal ⁇ of ⁇ the ⁇ immobilized ⁇ monitoring ⁇ probe.
  • the ⁇ delta ⁇ G ⁇ value ⁇ of ⁇ the ⁇ monitoring ⁇ probes is ⁇ adjusted ⁇ by ⁇ the ⁇ sequence ⁇ length ⁇ or ⁇ sequence ⁇ composition.
  • ⁇ In ⁇ yet ⁇ a ⁇ further ⁇ embodiment ⁇ the ⁇ delta ⁇ G ⁇ value ⁇ of ⁇ the ⁇ monitoring ⁇ probes is ⁇ adjusted ⁇ by ⁇ using ⁇ modified ⁇ bases ⁇ which ⁇ have ⁇ higher ⁇ stability ⁇ than ⁇ standard ⁇ bases.
  • temperature ⁇ fluctuation ⁇ can ⁇ be ⁇ read-out ⁇ by ⁇ specific ⁇ change ⁇ in ⁇ the ⁇ fluorescence ⁇ and/or ⁇ color ⁇ facilitated ⁇ by ⁇ the ⁇ direct ⁇ and/or ⁇ indirect ⁇ interaction ⁇ of ⁇ the ⁇ monitoring ⁇ probe ⁇ with ⁇ the signal ⁇ oligonucleotide.
  • the ⁇ kit ⁇ according ⁇ is ⁇ adapted for ⁇ monitoring ⁇ a ⁇ tissue ⁇ sample ⁇ preparation.
  • the control ⁇ slide ⁇ can ⁇ be ⁇ used for ⁇ assessing ⁇ in-situ ⁇ hybridization ⁇ instrument installations.
  • the ⁇ present ⁇ disclosure pertains ⁇ to a ⁇ reagent ⁇ (monitoring ⁇ probe ⁇ or ⁇ monitoring ⁇ probe ⁇ set) ⁇ for ⁇ methods ⁇ and ⁇ kits ⁇ for ⁇ detection ⁇ of ⁇ a ⁇ polymorphic analyte ⁇ in ⁇ a ⁇ sample ⁇ by ⁇ specific ⁇ signal-encoding ⁇ of ⁇ said ⁇ analyte by ⁇ in-situ ⁇ hybridization.
  • ⁇ the ⁇ detected ⁇ signal ⁇ may ⁇ label ⁇ a ⁇ gene ⁇ family, ⁇ a ⁇ single ⁇ gene, ⁇ a ⁇ specific ⁇ genetic ⁇ locus, ⁇ a ⁇ benign ⁇ or ⁇ malign ⁇ genetic ⁇ variation, ⁇ genetic ⁇ specificity ⁇ or ⁇ biomarker ⁇ in ⁇ a ⁇ cluster ⁇ of ⁇ patients, ⁇ a ⁇ virus-genome, ⁇ a ⁇ certain ⁇ subset ⁇ of ⁇ nucleic ⁇ acid ⁇ variation ⁇ (e.g. ⁇ a ⁇ group ⁇ of ⁇ SNPs, ⁇ a ⁇ mutation, ⁇ a ⁇ virus-variant, ⁇ a ⁇ gene-variant, ⁇ a ⁇ transcript, ⁇ isoforms, ⁇ splice-variants, ⁇ etc.).
  • the ⁇ technology ⁇ allows ⁇ the ⁇ high-throughput ⁇ detection ⁇ of ⁇ nucleic ⁇ acids ⁇ with ⁇ variable ⁇ sequences ⁇ of ⁇ at ⁇ least ⁇ 60%, ⁇ at ⁇ least ⁇ 65%, ⁇ at ⁇ least ⁇
  • this ⁇ disclosure ⁇ provides in ⁇ vitromethods for ⁇ diagnosis ⁇ of ⁇ a ⁇ disease ⁇ in ⁇ plants ⁇ selected ⁇ from ⁇ the ⁇ group ⁇ comprising: ⁇ diseases ⁇ caused ⁇ by ⁇ biotic ⁇ stress, ⁇ preferably ⁇ by ⁇ infectious ⁇ and/or ⁇ parasitic ⁇ origin, ⁇ or ⁇ diseases ⁇ caused ⁇ by ⁇ abiotic ⁇ stress, ⁇ preferably ⁇ caused ⁇ by ⁇ nutritional ⁇ deficiencies ⁇ and/or ⁇ unfavourable environment, ⁇ said ⁇ method ⁇ comprising ⁇ the ⁇ use ⁇ of ⁇ the ⁇ multiplex ⁇ method ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure.
  • ⁇ this ⁇ disclosure ⁇ relates to ⁇ optical ⁇ multiplexing ⁇ systems ⁇ suitable ⁇ for ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the present ⁇ disclosure, ⁇ comprising ⁇ at ⁇ least: - at ⁇ least ⁇ one ⁇ reaction ⁇ vessel ⁇ for ⁇ containing ⁇ the ⁇ kits ⁇ or ⁇ part ⁇ of ⁇ the ⁇ kits ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure; - a ⁇ detection ⁇ unit ⁇ comprising ⁇ a ⁇ microscope, ⁇ in ⁇ particular ⁇ a ⁇ fluorescence ⁇ microscope - a ⁇ camera - a ⁇ liquid ⁇ handling ⁇ device.
  • ⁇ the ⁇ unique ⁇ tags are ⁇ design ⁇ as ⁇ follow: - No ⁇ cross-hybridization ⁇ between ⁇ all ⁇ oligonucleotides ⁇ of ⁇ the ⁇ process ⁇ (probes, ⁇ decoders, ⁇ readout), ⁇ so ⁇ that ⁇ all ⁇ tag ⁇ sequences ⁇ are ⁇ usable ⁇ together ⁇ (compatible) - No ⁇ cross-hybridization ⁇ between ⁇ connector ⁇ elements ⁇ (bridges) ⁇ of ⁇ different ⁇ unique ⁇ tags - Stability ⁇ of ⁇ hybridization ⁇ of ⁇ the ⁇ unique ⁇ tags ⁇ should ⁇ be ⁇ in ⁇ a ⁇ narrow ⁇ range: ⁇ as ⁇ stable ⁇ as ⁇ possible ⁇ (fast ⁇ hybridization, ⁇ i.e., short ⁇ cycle ⁇ times) ⁇ but ⁇ significantly ⁇ different ⁇ (in ⁇ this ⁇ case ⁇ less ⁇ stable) ⁇ than ⁇ the ⁇ primary ⁇ probe ⁇ (for ⁇ differential ⁇ denaturation, ⁇ without ⁇ removing ⁇ primary ⁇ probes).
  • the ⁇ present ⁇ description ⁇ pertains ⁇ in ⁇ particular ⁇ to ⁇ the ⁇ usage ⁇ of ⁇ a ⁇ set ⁇ of ⁇ labeled ⁇ and ⁇ unlabeled ⁇ nucleic ⁇ acid ⁇ sequences ⁇ for ⁇ specific ⁇ quantitative ⁇ and/or ⁇ spatial ⁇ detection ⁇ of ⁇ different ⁇ analytes ⁇ in ⁇ parallel ⁇ via ⁇ specific ⁇ hybridization.
  • the ⁇ so ⁇ called ⁇ “multi-decoders” ⁇ allows ⁇ the ⁇ recruiting ⁇ of ⁇ more ⁇ than ⁇ just ⁇ one ⁇ signal ⁇ oligonucleotide ⁇ and ⁇ therefore ⁇ can ⁇ generate ⁇ new ⁇ signal ⁇ types ⁇ by ⁇ utilizing ⁇ the ⁇ combination ⁇ of ⁇ two ⁇ or ⁇ more ⁇ different ⁇ signal-oligonucleotides ⁇ without ⁇ decreasing ⁇ the ⁇ brightness ⁇ of ⁇ the ⁇ signals.
  • due ⁇ to ⁇ the ⁇ use ⁇ of ⁇ a ⁇ first ⁇ set ⁇ of ⁇ analyte-specific ⁇ probes ⁇ according ⁇ to ⁇ step ⁇ A1 i.e. ⁇ the ⁇ transcript ⁇ plexity ⁇ of ⁇ A1 ⁇ which ⁇ is ⁇ at ⁇ least ⁇ 10 ⁇ times ⁇ higher in ⁇ numbers than ⁇ the ⁇ number ⁇ of ⁇ probes
  • Fig. ⁇ 2 Embodiment ⁇ where ⁇ the ⁇ analyte ⁇ is ⁇ a ⁇ protein, and ⁇ the ⁇ probe ⁇ set ⁇ comprises ⁇ proteins ⁇ (here: ⁇ antibodies) ⁇ specifically ⁇ binding ⁇ to ⁇ the ⁇ analyte. ⁇ The ⁇ probes ⁇ comprise ⁇ a ⁇ unique ⁇ identifier ⁇ sequence ⁇ allowing ⁇ hybridization ⁇ of ⁇ decoding ⁇ oligonucleotides.
  • Fig. ⁇ 3 Flowchart ⁇ of ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ disclosure.
  • Fig. ⁇ 4 Alternative ⁇ options ⁇ for ⁇ the ⁇ application ⁇ of ⁇ decoding ⁇ and ⁇ signal ⁇ oligonucleotides.
  • Fig. ⁇ 5 Example ⁇ for ⁇ signal ⁇ encoding ⁇ of ⁇ three ⁇ different ⁇ nucleic ⁇ acid ⁇ sequences ⁇ by ⁇ two ⁇ different ⁇ signal ⁇ types ⁇ and ⁇ three ⁇ detection ⁇ rounds; ⁇ in ⁇ this ⁇ example, ⁇ the ⁇ encoding ⁇ scheme ⁇ includes ⁇ error ⁇ detection.
  • Fig. ⁇ 6 Number ⁇ of ⁇ generated ⁇ code ⁇ words ⁇ (logarithmic ⁇ scale) ⁇ against ⁇ number ⁇ of ⁇ detection ⁇ cycles.
  • Fig. ⁇ 7 Calculated ⁇ total ⁇ efficiency ⁇ of ⁇ a ⁇ 5-round ⁇ encoding ⁇ scheme ⁇ based ⁇ on ⁇ single ⁇ step ⁇ efficiencies.
  • Fig. ⁇ 8 Comparison ⁇ of ⁇ relative ⁇ transcript ⁇ abundances ⁇ between ⁇ different ⁇ experiments.
  • Fig. ⁇ 9 Correlation ⁇ of ⁇ relative ⁇ transcript ⁇ abundances ⁇ between ⁇ different ⁇ experiments.
  • Fig. ⁇ 10 Comparison ⁇ of ⁇ intercellular ⁇ distribution ⁇ of ⁇ signals.
  • Fig. ⁇ 11 Comparison ⁇ of ⁇ intracellular ⁇ distribution ⁇ of ⁇ signals.
  • Fig. ⁇ 12 Distribution ⁇ pattern ⁇ of ⁇ different ⁇ cell ⁇ cycle ⁇ dependent ⁇ transcripts.
  • the ⁇ number ⁇ of ⁇ codewords ⁇ for ⁇ merFISH ⁇ does ⁇ not ⁇ exponentially ⁇ increase ⁇ with ⁇ the ⁇ number ⁇ of ⁇ detection ⁇ cycles ⁇ but ⁇ gets ⁇ less ⁇ effective ⁇ with ⁇ each ⁇ added ⁇ round. ⁇ In ⁇ contrast, ⁇ the ⁇ number ⁇ of ⁇ codewords ⁇ for ⁇ intronSeqFISH, ⁇ the ⁇ method ⁇ of ⁇ the ⁇ present ⁇ disclosure ⁇ without ⁇ using ⁇ multi-decoders, ⁇ the method ⁇ with ⁇ multi-decoders ⁇ increases ⁇ exponentially. ⁇ The ⁇ slope ⁇ of ⁇ the ⁇ curve ⁇ for ⁇ the ⁇ method ⁇ using ⁇ multi-decoders ⁇ is ⁇ much ⁇ higher ⁇ than ⁇ that ⁇ of ⁇ the ⁇ prior ⁇ invention, ⁇ leading ⁇ to ⁇ more ⁇ than ⁇ 20 ⁇ 000 ⁇ 000 ⁇ times ⁇ more ⁇ code ⁇ words ⁇ usable ⁇ after ⁇ 20 ⁇ rounds ⁇ of ⁇ detection.
  • Fig. ⁇ 19 ⁇ Visual ⁇ representation ⁇ of ⁇ mRNA ⁇ and ⁇ control ⁇ particle.
  • Fig ⁇ 20 ⁇ Particle ⁇ attachment ⁇ to ⁇ microscope ⁇ slights.
  • Fig. ⁇ 21 ⁇ Assay ⁇ for ⁇ parameter ⁇ optimization.
  • Fig. ⁇ 22 ⁇ Example ⁇ for ⁇ the ⁇ use ⁇ of ⁇ ZEN’s ⁇ DFIs.
  • Fig. ⁇ 23 ⁇ Results ⁇ of ⁇ MC ⁇ Run ⁇ of ⁇ 1-plex ⁇ particle
  • Fig. ⁇ 24 ⁇ Results ⁇ of ⁇ MC ⁇ Run ⁇ of ⁇ 1-plex ⁇ particle, ⁇ 6-plex ⁇ particle, ⁇ 12-plex ⁇ particle
  • Fig. ⁇ 25 ⁇ MC ⁇ Run ⁇ of ⁇ 19-plex ⁇ particles
  • Fig.26 ⁇ Embodiments ⁇ of ⁇ the ⁇ monitoring ⁇ probe ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure. ⁇ A) ⁇ Monitoring ⁇ probes ⁇ (1) ⁇ attached ⁇ to ⁇ a ⁇ particle ⁇ (3) ⁇ like ⁇ a ⁇ magnetic ⁇ bead, ⁇ which ⁇ is ⁇ immobilized ⁇ to ⁇ a ⁇ slide ⁇ surface ⁇ (2). ⁇ On ⁇ the ⁇ right ⁇ a ⁇ tissue ⁇ sample ⁇ (4) ⁇ is ⁇ place ⁇ on ⁇ top ⁇ of ⁇ the ⁇ particles. ⁇ B) ⁇ shows ⁇ a ⁇ specific ⁇ embodiment ⁇ of ⁇ the ⁇ particle, ⁇ wherein ⁇ the ⁇ monitoring ⁇ pro
  • D) ⁇ shows ⁇ a ⁇ specific ⁇ embodiment ⁇ of ⁇ the ⁇ particle, ⁇ wherein ⁇ the ⁇ monitoring ⁇ probe ⁇ comprises ⁇ a ⁇ target ⁇ nucleic ⁇ acid ⁇ sequence ⁇ (1c) ⁇ allowing ⁇ the ⁇ binding/hybridization ⁇ of ⁇ a ⁇ binding ⁇ element ⁇ (7).
  • ⁇ the ⁇ present ⁇ disclosure ⁇ pertains ⁇ to ⁇ oligo ⁇ attached ⁇ beads ⁇ (monitoring ⁇ probes) ⁇ and ⁇ uses ⁇ thereof ⁇ to ⁇ create ⁇ ground ⁇ truth.
  • Altered ⁇ delta ⁇ G ⁇ values ⁇ can ⁇ be ⁇ accomplished ⁇ by ⁇ selecting ⁇ the ⁇ base ⁇ composition ⁇ of ⁇ the ⁇ sequence, ⁇ altering ⁇
  • ⁇ customer ⁇ samples ⁇ can ⁇ be ⁇ applied ⁇ directly ⁇ to ⁇ slides ⁇ containing ⁇ attached ⁇ control ⁇ beads.
  • ⁇ In ⁇ these ⁇ cases, ⁇ the ⁇ control ⁇ bead ⁇ will: 1. Serve ⁇ as ⁇ an ⁇ internal ⁇ control ⁇ to ⁇ monitor ⁇ the ⁇ entire ⁇ workflow ⁇ used ⁇ by ⁇ the ⁇ customer ⁇ in ⁇ running ⁇ their ⁇ sample. 2. Monitor ⁇ the ⁇ efficiency ⁇ of ⁇ the ⁇ tissue ⁇ preparation ⁇ process ⁇ in ⁇ permeabilizing ⁇ the ⁇ tissue ⁇ sufficiently ⁇ to ⁇ allow ⁇ diffusion ⁇ of ⁇ the ⁇ assay ⁇ oligonucleotides ⁇ into ⁇ and ⁇ through ⁇ the ⁇ tissues.
  • the ⁇ present ⁇ disclosure ⁇ also ⁇ describes ⁇ the ⁇ usage ⁇ of ⁇ at ⁇ least ⁇ one set ⁇ of ⁇ nucleic ⁇ acid ⁇ sequences ⁇ for ⁇ specific ⁇ quantitative ⁇ and/or ⁇ spatial ⁇ detection ⁇ of ⁇ different ⁇ analytes ⁇ in ⁇ parallel ⁇ via ⁇ specific ⁇ hybridization.
  • the ⁇ technology allows ⁇ the ⁇ discrimination ⁇ of ⁇ more ⁇ different ⁇ analytes ⁇ than ⁇ different ⁇ detection ⁇ signals ⁇ are ⁇ available.
  • the ⁇ discrimination ⁇ may ⁇ be ⁇ realized ⁇ via ⁇ sequential ⁇ signal-coding ⁇ of ⁇ the ⁇ analytes ⁇ achieved ⁇ by ⁇ several ⁇ cycles ⁇ of ⁇ specific ⁇ hybridization, ⁇ detection ⁇ of ⁇ signals ⁇ and ⁇ selective ⁇ elution ⁇ of ⁇ the ⁇ hybridized ⁇ nucleic ⁇ acid ⁇ sequences.
  • an ⁇ “slide” ⁇ is ⁇ any ⁇ surface ⁇ which ⁇ can ⁇ be ⁇ used ⁇ for ⁇ in-situ ⁇ hybridization. ⁇ In ⁇ some ⁇ embodiments ⁇ the ⁇ slide ⁇ is ⁇ a ⁇ microscope ⁇ slide, ⁇ in ⁇ some ⁇ embodiments ⁇ made ⁇ out ⁇ of ⁇ glass.
  • thee ⁇ cell ⁇ is ⁇ a ⁇ prokaryotic ⁇ cells ⁇ or ⁇ a ⁇ eukaryotic ⁇ cell, ⁇ in ⁇ particular ⁇ a ⁇ mammalian ⁇ cell, ⁇ in ⁇ particular ⁇ a ⁇ human ⁇ cell.
  • the ⁇ biological ⁇ tissue, ⁇ biological ⁇ cells, ⁇ extracts ⁇ and/or ⁇ part ⁇ of ⁇ cells ⁇ are ⁇ fixed.
  • In ⁇ particular, ⁇ the ⁇ analytes ⁇ are ⁇ fixed ⁇ in ⁇ a ⁇ permeabilized ⁇ sample, ⁇ such ⁇ as ⁇ a ⁇ cell-containing ⁇ sample.
  • an ⁇ "oligonucleotide ⁇ as ⁇ used ⁇ herein, ⁇ refers ⁇ to ⁇ s ⁇ short ⁇ nucleic ⁇ acid ⁇ molecule, ⁇ such ⁇ as ⁇ DNA, ⁇ PNA, ⁇ LNA ⁇ or ⁇ RNA.
  • the ⁇ length ⁇ of ⁇ the ⁇ oligonucleotides ⁇ is ⁇ within ⁇ the ⁇ range ⁇ 4-200 ⁇ nucleotides ⁇ (nt), ⁇ preferably ⁇ 6-80 ⁇ nt, ⁇ more ⁇ preferably ⁇ 8-60 ⁇ nt, ⁇ more ⁇ preferably ⁇ 10-50 ⁇ nt, ⁇ more ⁇ preferably ⁇ 12 ⁇ to ⁇ 35 ⁇ depending ⁇ on ⁇ the ⁇ number ⁇ of ⁇ consecutive ⁇ sequence ⁇ elements.
  • the ⁇ oligonucleotides ⁇ may ⁇ be ⁇ linear ⁇ or ⁇ may ⁇ comprise ⁇ hairpin ⁇ or ⁇ loop ⁇ structures.
  • the ⁇ oligonucleotides ⁇ may ⁇ comprise ⁇
  • the ⁇ "analyte-specific ⁇ probe" ⁇ consists ⁇ of ⁇ at ⁇ least ⁇ one, ⁇ preferably ⁇ at ⁇ least ⁇ two ⁇ elements, ⁇ namely ⁇ the ⁇ so-called ⁇ binding ⁇ element ⁇ (S) ⁇ which ⁇ specifically ⁇ interacts ⁇ with ⁇ one ⁇ of ⁇ the ⁇ analytes, ⁇ and ⁇ a ⁇ so-called ⁇ identifier ⁇ element ⁇ (T) ⁇ comprising ⁇ the ⁇ 'unique ⁇ identifier ⁇ sequence'.
  • the ⁇ binding ⁇ element ⁇ (S) ⁇ may ⁇ be ⁇ a ⁇ nucleic ⁇ acid ⁇ such ⁇ as ⁇ a ⁇ hybridization ⁇ sequence ⁇ or ⁇ an ⁇ aptamer, ⁇ or ⁇ a ⁇ peptidic ⁇ structure ⁇ such ⁇ as ⁇ an ⁇ antibody.
  • the ⁇ “monitoring ⁇ probe” ⁇ consists ⁇ of ⁇ at ⁇ least ⁇ one ⁇ element, ⁇ namely ⁇ the ⁇ so-called ⁇ identifier ⁇ element ⁇ (T) ⁇ comprising ⁇ the ⁇ 'unique ⁇ identifier ⁇ sequence'.
  • the ⁇ monitoring ⁇ probe ⁇ is ⁇ attached ⁇ to ⁇ a ⁇ surface, ⁇ selected ⁇ from ⁇ a ⁇ particle ⁇ (e.g., ⁇ a ⁇ magnetic ⁇ or ⁇ nonmagnetic ⁇ bead), ⁇ a ⁇ functional ⁇ group ⁇ (e.g., streptavidin, ⁇ biotin, ⁇ a ⁇ hybridization ⁇ sequence ⁇ or ⁇ an ⁇ aptamer, ⁇ or ⁇ a ⁇ peptidic ⁇ structure ⁇ such ⁇ as ⁇ an ⁇ antibody) ⁇ and ⁇ a ⁇ surface ⁇ (e.g., ⁇ a ⁇ slide), ⁇ as ⁇ well ⁇ as ⁇ any ⁇ combination ⁇ thereof.
  • the ⁇ monitoring ⁇ probe ⁇ may ⁇ also ⁇ comprise ⁇ a ⁇ so-called ⁇ binding
  • the ⁇ "unique ⁇ identifier ⁇ sequence" ⁇ as ⁇ comprised ⁇ by ⁇ the ⁇ analyte-specific ⁇ probe ⁇ and ⁇ the ⁇ monitoring ⁇ probe ⁇ is ⁇ unique ⁇ in ⁇ its ⁇ sequence ⁇ compared ⁇ to ⁇ other ⁇ unique ⁇ identifiers.
  • "Unique" ⁇ in ⁇ this ⁇ context ⁇ means ⁇ that ⁇ it ⁇ specifically ⁇ identifies ⁇ only ⁇ one ⁇ analyte, ⁇ such ⁇ as ⁇ Cyclin ⁇ A, ⁇ Cyclin ⁇ D, ⁇ Cyclin ⁇ E ⁇ etc., ⁇ or, ⁇ alternatively, ⁇ it ⁇ specifically ⁇ identifies ⁇ only ⁇ a ⁇ group ⁇ of ⁇ analytes, ⁇ independently ⁇ whether ⁇ the ⁇ group ⁇ of ⁇ analytes ⁇ comprises ⁇ a ⁇ gene ⁇ family ⁇ or ⁇ not. ⁇ Therefore, ⁇ the ⁇ analyte ⁇ or ⁇ a ⁇ group ⁇ of ⁇ analytes ⁇ to ⁇ be ⁇ encoded ⁇ by ⁇ this ⁇ unique ⁇ identifier ⁇ can ⁇ be ⁇ distinguished ⁇ from ⁇
  • a ⁇ "decoding ⁇ oligonucleotide” ⁇ or ⁇ an ⁇ “adapter” ⁇ or ⁇ a ⁇ /adapter ⁇ segment” ⁇ consists ⁇ of ⁇ at ⁇ least ⁇ one, ⁇ preferably ⁇ at ⁇ least ⁇ two ⁇ sequence ⁇ elements. ⁇ One ⁇ sequence ⁇ element ⁇ that ⁇ can ⁇ specifically ⁇ bind ⁇ to ⁇ a ⁇ unique ⁇ identifier ⁇ sequence, ⁇ referred ⁇ to ⁇ as an ⁇ “identifier ⁇ connector ⁇ element ⁇ “(t) ⁇ or "first ⁇ connector ⁇ element” ⁇ (t), ⁇ and ⁇ a ⁇ second ⁇ sequence ⁇ element ⁇ specifically ⁇ binding ⁇ to ⁇ a ⁇ signal ⁇ oligonucleotide, ⁇ referred ⁇ to ⁇ as ⁇ "translator ⁇ element” ⁇ (c). ⁇ The ⁇ length ⁇ of ⁇ the ⁇ sequence ⁇ elements ⁇ is ⁇ within ⁇ the ⁇ range ⁇ 8- 60 ⁇ nt, ⁇ preferably ⁇ 12-40 ⁇ nt, ⁇ more ⁇ preferably ⁇ 14-20 ⁇ nt, ⁇ depending ⁇ on ⁇ the
  • ⁇ the ⁇ decoding ⁇ oligonucleotide ⁇ in ⁇ the ⁇ kits ⁇ and/or ⁇ methods ⁇ of ⁇ the ⁇ present ⁇ disclosure ⁇ may ⁇ be ⁇ a ⁇ “multi-decoder”.
  • ⁇ the ⁇ decoding ⁇ oligonucleotide ⁇ is ⁇ a ⁇ multi-decoder ⁇ comprising ⁇ - an ⁇ identifier ⁇ connector ⁇ element ⁇ (t) ⁇ comprising ⁇ a ⁇ nucleotide ⁇ sequence ⁇ which ⁇ is ⁇ essentially ⁇ complementary ⁇ to ⁇ at ⁇ least ⁇ a ⁇ section ⁇ of ⁇ the ⁇ unique ⁇ identifier ⁇ sequence ⁇ of ⁇ the ⁇ identifier ⁇ element ⁇ (T) ⁇ of ⁇ the ⁇ corresponding ⁇ analyte-specific ⁇ probe ⁇ set, ⁇ and - at ⁇ least ⁇ one, ⁇ preferably ⁇ at ⁇ least ⁇ two translator ⁇ elements ⁇ (c) ⁇ comprising ⁇ each ⁇ a ⁇ nucleotide ⁇ sequence ⁇ allowing ⁇ a ⁇ specific ⁇ hybridization ⁇ of ⁇ a ⁇ different ⁇ signal ⁇ oligonucleotide.
  • ⁇ the ⁇ first ⁇ translator ⁇ element ⁇ binds ⁇ a ⁇ different ⁇ signal ⁇ oligonucleotide ⁇ as ⁇ the ⁇ second ⁇ translator ⁇ element.
  • ⁇ the ⁇ signal ⁇ oligonucleotides ⁇ differ ⁇ in ⁇ the ⁇ signal ⁇ element ⁇ comprised ⁇ in ⁇ the ⁇ signal ⁇ oligonucleotide, ⁇ e.g. ⁇ in ⁇ the ⁇ kind ⁇ of ⁇ the ⁇ fluorophore.
  • In ⁇ a ⁇ monitoring ⁇ probe ⁇ set ⁇ the ⁇ monitoring ⁇ probes ⁇ are ⁇ identical ⁇ in ⁇ the ⁇ binding ⁇ element ⁇ (S) ⁇ but ⁇ may ⁇ comprise ⁇ a ⁇ different ⁇ identifier ⁇ element ⁇ (T) ⁇ for ⁇ specifically ⁇ interacting ⁇ with ⁇ the ⁇ surface ⁇ on ⁇ which ⁇ they ⁇ are ⁇ immobilized, ⁇ but ⁇ for ⁇ specifically ⁇ interacting ⁇ with ⁇ different ⁇ decoding ⁇ elements.
  • “Selective ⁇ denaturation” may ⁇ be ⁇ the ⁇ process ⁇ of ⁇ eliminating ⁇ bound ⁇ decoding ⁇ oligonucleotides ⁇ and ⁇ signal ⁇ oligonucleotides ⁇ with ⁇ highest ⁇ efficiency ⁇ while ⁇ at ⁇ the ⁇ same ⁇ time ⁇ the ⁇ target ⁇ specific ⁇ probes ⁇ have ⁇ to ⁇ stay ⁇ hybridized ⁇ with ⁇ the ⁇ highest ⁇ efficiency.
  • the ⁇ total ⁇ efficiency ⁇ of ⁇ these ⁇ two ⁇ combined ⁇ events ⁇ may ⁇ to ⁇ be ⁇ at ⁇ least ⁇ 0.22 ⁇ for ⁇ two ⁇ detection ⁇ cycles, ⁇ 0.37 ⁇ for ⁇ three ⁇ detection ⁇ cycles, ⁇ 0.47 ⁇ for ⁇ four ⁇ detection ⁇ cycles, ⁇ 0.55 ⁇ for ⁇ five ⁇ detection ⁇ cycles, ⁇ 0.61 ⁇ for ⁇ six ⁇ detection ⁇ cycles, ⁇ 0.65 ⁇ for ⁇ seven ⁇ detection ⁇ cycles, ⁇ 0.69 ⁇ for ⁇ eight ⁇ detection ⁇ cycles, ⁇ 0.72 ⁇ for ⁇ nine ⁇ det
  • a ⁇ single ⁇ set refers ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ oligonucleotides.
  • An ⁇ "analyte ⁇ specific ⁇ probe ⁇ set" refers ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ moieties ⁇ or ⁇ sub-jects, ⁇ e.g., analyte-specific ⁇ probes ⁇ that ⁇ are ⁇ different ⁇ from ⁇ each ⁇ other ⁇ and ⁇ bind ⁇ to ⁇ independent ⁇ regions ⁇ of ⁇ the ⁇ analyte.
  • a "decoding ⁇ oligonucleotide ⁇ set” refers ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ decoding ⁇ oligonucleotides ⁇ specific ⁇ for ⁇ a ⁇ certain ⁇ unique ⁇ identifier ⁇ needed ⁇ to ⁇ realize ⁇ the ⁇ encoding ⁇ independent ⁇ of ⁇ the ⁇ length ⁇ of ⁇ the ⁇ code ⁇ word. ⁇ Each ⁇ and ⁇ all ⁇ of ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ included ⁇ in a ⁇ "decoding ⁇ oligonucleotide ⁇ set” ⁇ bind ⁇ to ⁇ the ⁇ same ⁇ unique ⁇ identifier ⁇ element ⁇ (T) ⁇ of ⁇ the ⁇ analyte-specific ⁇ probe.
  • the ⁇ codewords ⁇ for ⁇ each ⁇ analyte may ⁇ be ⁇ assigned ⁇ sequentially, ⁇ or ⁇ may ⁇ be ⁇ assigned ⁇ at ⁇ random. ⁇ For ⁇ instance, ⁇ a ⁇ first ⁇ analytemay ⁇ be ⁇ assigned ⁇ to ⁇ 101, ⁇ while ⁇ a ⁇ second ⁇ nucleic ⁇ acid ⁇ target ⁇ may ⁇ be ⁇ assigned ⁇ to ⁇ 110. ⁇
  • ⁇ the ⁇ codewords ⁇ may ⁇ be ⁇ assigned ⁇ using ⁇ an ⁇ error- detection ⁇ system ⁇ or ⁇ an ⁇ error- correcting ⁇ system, ⁇ such ⁇ as a ⁇ Hamming ⁇ system, ⁇ a ⁇ Golay ⁇ code, ⁇ or ⁇ an ⁇ extended ⁇ Hamming ⁇ system ⁇ (or ⁇ a ⁇ SECDED ⁇ system, ⁇ i.e., ⁇ single ⁇ error ⁇ correction, ⁇ double ⁇ error ⁇ detection).
  • an ⁇ error- detection ⁇ system ⁇ or ⁇ an ⁇ error- correcting ⁇ system such ⁇ as a ⁇ Hamming ⁇ system, ⁇ a ⁇ Golay ⁇ code, ⁇ or ⁇
  • Essentially ⁇ complementary ⁇ means, ⁇ when ⁇ referring ⁇ to ⁇ two ⁇ nucleotide ⁇ sequences, ⁇ that ⁇ both ⁇ sequences ⁇ can ⁇ specifically ⁇ hybridize ⁇ to ⁇ each ⁇ other ⁇ under ⁇ stringent ⁇ conditions, ⁇ thereby ⁇ forming ⁇ a ⁇ hybrid ⁇ nucleic ⁇ acid ⁇ molecule ⁇ with ⁇ a ⁇ sense ⁇ and ⁇ an ⁇ antisense ⁇ strand ⁇ connected ⁇ to ⁇ each ⁇ other ⁇ via ⁇ hydrogen ⁇ bonds ⁇ (Watson-and-Crick ⁇ base ⁇ pairs).
  • ⁇ essentially ⁇ complementary ⁇ includes ⁇ not ⁇ only ⁇ perfect ⁇ base-pairing ⁇ along ⁇ the ⁇ entire ⁇ strands, ⁇ i.e., perfect ⁇ complementary ⁇ sequences ⁇ but ⁇ also ⁇ imperfect ⁇ complementary ⁇ sequences ⁇ which, ⁇ however, ⁇ still ⁇ have ⁇ the ⁇ capability ⁇ to ⁇ hybridize ⁇ to ⁇ each ⁇ other ⁇ under ⁇ stringent ⁇ conditions.
  • the ⁇ decoding ⁇ oligonucleotides ⁇ of ⁇ a ⁇ particular ⁇ set ⁇ differ ⁇ from ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ of ⁇ another ⁇ set. ⁇ This ⁇ means, ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ of ⁇ set ⁇ 1 ⁇ bind ⁇ to ⁇ the ⁇ analyte-specific ⁇ probes ⁇ of ⁇ above ⁇ set ⁇ 1 ⁇ of ⁇ analyte-specific ⁇ probes, ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ of ⁇ set ⁇ 2 ⁇ binds ⁇ to ⁇ the ⁇ analyte-specific ⁇ probes ⁇ of ⁇ above ⁇ set ⁇ 2 ⁇ of ⁇ analyte-specific ⁇ probes, ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ of ⁇ set ⁇ 3 ⁇ bind ⁇ to ⁇ the ⁇ analyte-specific ⁇ probes ⁇ of ⁇ above ⁇ set ⁇ 3 ⁇ of ⁇ analyte-specific ⁇ probes, ⁇ etc. ⁇ It ⁇ is ⁇ also ⁇ understood ⁇ that
  • the ⁇ decoding ⁇ oligonucleotides ⁇ of ⁇ a ⁇ particular ⁇ set ⁇ differ ⁇ from ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ of ⁇ another ⁇ set.
  • the ⁇ term ⁇ "quencher” ⁇ or ⁇ "quencher ⁇ dye” ⁇ or ⁇ "quencher ⁇ molecule refers ⁇ to ⁇ a ⁇ dye ⁇ or ⁇ an ⁇ equivalent ⁇ molecule, ⁇ such ⁇ as ⁇ nucleoside ⁇ guanosine ⁇ (G) ⁇ or ⁇ 2'-deoxyguanosine ⁇ (dG), ⁇ which ⁇ is ⁇ capable ⁇ of ⁇ reducing ⁇ the ⁇ fluorescence ⁇ of ⁇ a ⁇ fluorescent ⁇ reporter ⁇ dye ⁇ or ⁇ donor ⁇ dye.
  • the ⁇ quencher ⁇ is ⁇ a ⁇ fluorescent ⁇ dye ⁇ its ⁇ fluorescence ⁇ wavelength ⁇ is ⁇ typically ⁇ substantially ⁇ different ⁇ from ⁇ that ⁇ of ⁇ the ⁇ reporter ⁇ dye ⁇ and ⁇ the ⁇ quencher ⁇ fluorescence ⁇ is ⁇ usually ⁇ not ⁇ monitored ⁇ during ⁇ an ⁇ assay. ⁇
  • the ⁇ method ⁇ is ⁇ particularly ⁇ qualified ⁇ to ⁇ monitor, ⁇ encode, ⁇ identify, ⁇ detect, ⁇ count, or ⁇ quantify ⁇ the ⁇ in ⁇ situ ⁇ analysis ⁇ of ⁇ analytes ⁇ or ⁇ single ⁇ analyte ⁇ molecules ⁇ in ⁇ a ⁇ biological ⁇ sample, ⁇ i.e., such ⁇ as ⁇ a ⁇ sample ⁇ which ⁇ contains ⁇ nucleic ⁇ acids ⁇ or ⁇ proteins ⁇ as ⁇ said ⁇ analytes.
  • the ⁇ biological ⁇ sample may ⁇ be ⁇ in ⁇ a ⁇ form ⁇ as ⁇ it ⁇ is ⁇ in ⁇ its ⁇ natural ⁇ environment ⁇ (i.e., liquid, ⁇ semi-liquid, ⁇ solid ⁇ etc.), ⁇ or ⁇ processed, ⁇ e.g., as ⁇ a ⁇ dried ⁇ film ⁇ on ⁇ the ⁇ surface ⁇ of ⁇ a ⁇ device ⁇ which ⁇ may ⁇ be ⁇ re-liquefied ⁇ before ⁇ the ⁇ method ⁇ is ⁇ carried ⁇ out. ⁇ In ⁇ another ⁇ embodiment ⁇ of ⁇ the ⁇
  • ⁇ in ⁇ some ⁇ embodiments, ⁇ the ⁇ cell ⁇ and/or ⁇ the ⁇ tissue ⁇ is ⁇ fixed ⁇ prior ⁇ to ⁇ introducing ⁇ the ⁇ probes, ⁇ e.g., ⁇ to ⁇ preserve ⁇ the ⁇ positions ⁇ of ⁇ the ⁇ analytes ⁇ like ⁇ nucleic ⁇ acids ⁇ within ⁇ the ⁇ cell.
  • ⁇ a ⁇ cell ⁇ may ⁇ be ⁇ fixed ⁇ using ⁇ chemicals ⁇ such ⁇ as ⁇ formaldehyde, ⁇ paraformaldehyde, ⁇ glutaraldehyde, ⁇ ethanol, ⁇ methanol, ⁇ acetone, ⁇ acetic ⁇ acid, ⁇ or ⁇ the ⁇ like.
  • This ⁇ measure ⁇ has ⁇ the ⁇ advantage ⁇ that ⁇ the ⁇ analytes ⁇ to ⁇ be ⁇ encoded, ⁇ e.g., the ⁇ nuclei ⁇ acids ⁇ or ⁇ proteins, ⁇ are ⁇ immobilized ⁇ and ⁇ cannot ⁇ escape. ⁇ In ⁇ doing ⁇ so, ⁇ the ⁇ analytes ⁇ then ⁇ prepared ⁇ for ⁇ a ⁇ better ⁇ detection ⁇ or ⁇ encoding ⁇ by ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ disclosure. ⁇ In ⁇ yet ⁇ a ⁇ further ⁇ embodiment ⁇ within ⁇ the ⁇ set ⁇ of ⁇ analyte-specific ⁇ probes ⁇ the ⁇ individual ⁇ analyte- specific ⁇ probes ⁇ comprise ⁇ binding ⁇ elements ⁇ (S1, ⁇ S2, ⁇ S3, ⁇ S4, ⁇ S5) ⁇ which ⁇ specifically ⁇ interact ⁇ with ⁇ different ⁇ sub-structures ⁇ of ⁇ one ⁇ of ⁇ the ⁇ analytes ⁇ to ⁇ be ⁇ encoded.
  • the ⁇ present ⁇ disclosure ⁇ pertains ⁇ to ⁇ kit ⁇ for ⁇ multiplex ⁇ analyte ⁇ encoding, ⁇ comprising: (A1) at ⁇ least ⁇ a ⁇ first ⁇ set of ⁇ analyte-specific ⁇ probes ⁇ for ⁇ encoding ⁇ different ⁇ analytes, ⁇ each ⁇ set ⁇ of ⁇ analyte-specific ⁇ probes ⁇ interacting ⁇ with ⁇ a ⁇ different ⁇ analyte, ⁇ wherein ⁇ if ⁇ the ⁇ analyte ⁇ is ⁇ a ⁇ nucleic ⁇ acid ⁇ each ⁇ set ⁇ of ⁇ analyte-specific ⁇ probes ⁇ comprises ⁇ analyte-specific ⁇ probes ⁇ which ⁇ specifically ⁇ interact ⁇ with ⁇ different ⁇ sub-structures ⁇ of ⁇ the ⁇ same ⁇ analyte, ⁇ each ⁇ analyte- specific ⁇ probe ⁇ comprising ⁇ (aa) ⁇ a ⁇ binding ⁇ element ⁇ (S) ⁇ that ⁇ specifically ⁇ interacts ⁇ with ⁇ one ⁇ of ⁇ the ⁇ different ⁇
  • RES-PA18-PCT ⁇ A ⁇ multiplex ⁇ method ⁇ or ⁇ assay ⁇ allow ⁇ the ⁇ simultaneously ⁇ measurement ⁇ of ⁇ multiple ⁇ analytes ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure it ⁇ may ⁇ be ⁇ used ⁇ to ⁇ determine ⁇ the ⁇ presence ⁇ or ⁇ absence ⁇ of ⁇ a ⁇ plurality ⁇ of ⁇ predetermined ⁇ (known) ⁇ analytes ⁇ like ⁇ nucleic ⁇ acid ⁇ target ⁇ sequences ⁇ in ⁇ a ⁇ sample. ⁇ An analyte ⁇ may ⁇ be “predetermined” ⁇ in ⁇ that ⁇ its ⁇ sequence ⁇ is known ⁇ to ⁇ design ⁇ a ⁇ probe ⁇ that ⁇ binds ⁇ to ⁇ the that ⁇ target.
  • the ⁇ unique ⁇ tag ⁇ can ⁇ be ⁇ identified ⁇ by ⁇ various ⁇ techniques, ⁇ including ⁇ hybridization, ⁇ e.g., with ⁇ labelled probes, ⁇ directly ⁇ or ⁇ indirectly ⁇ or ⁇ by ⁇ sequencing ⁇ (by ⁇ synthesis, ⁇ ligation).
  • the ⁇ kit ⁇ does ⁇ not ⁇ comprise ⁇ sets ⁇ of ⁇ analyte-specific ⁇ probes ⁇ as ⁇ defined ⁇ under ⁇ item ⁇ A1) and ⁇ A2).
  • ⁇ if ⁇ the ⁇ analyte ⁇ in ⁇ the ⁇ kits ⁇ or ⁇ methods ⁇ according ⁇ to ⁇ the ⁇ present disclosure is ⁇ a ⁇ nucleic ⁇ acid
  • ⁇ the ⁇ present ⁇ disclosure is ⁇ generally ⁇ directed ⁇ to ⁇ a ⁇ methods ⁇ including ⁇ acts ⁇ of ⁇ exposing ⁇ a ⁇ sample ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ analyte-specific ⁇ probes; ⁇ for ⁇ each ⁇ of ⁇ the ⁇ analyte-specific ⁇ probes, ⁇ determining ⁇ binding ⁇ of ⁇ the ⁇ analyte-specific ⁇ probes ⁇ within ⁇ the ⁇ sample; ⁇ creating ⁇ codewords ⁇ based ⁇ on ⁇ the binding ⁇ of ⁇ the ⁇ analyte-specific ⁇ probes, ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ and ⁇ the ⁇ signal ⁇ oligonucleotides; ⁇ and ⁇ for ⁇ at ⁇ least ⁇ some ⁇ of ⁇ the ⁇ codewords, ⁇ matching ⁇ the ⁇ codeword ⁇ to ⁇ a ⁇ valid ⁇ codeword.
  • To ⁇ create ⁇ such ⁇ a ⁇ zero ⁇ (0) ⁇ in ⁇ a ⁇ codeword ⁇ for ⁇ an ⁇ individual ⁇ analyte ⁇ the ⁇ kit ⁇ may ⁇ comprise: ⁇ (D) at ⁇ least ⁇ a ⁇ set ⁇ of ⁇ non-signal ⁇ decoding ⁇ oligonucleotides ⁇ for ⁇ binding ⁇ to ⁇ a ⁇ particular ⁇ identifier ⁇ element ⁇ (T) ⁇ of ⁇ analyte-specific ⁇ probes, ⁇ wherein ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ in ⁇ the ⁇ same ⁇ set ⁇ of ⁇ non-signal ⁇ decoding ⁇ oligonucleotides ⁇ interacting ⁇ with ⁇ the ⁇ same ⁇ different ⁇ identifier ⁇ element ⁇ (T), wherein ⁇ each ⁇ non-signal ⁇ decoding ⁇ oligonucleotide ⁇ comprises ⁇ an ⁇ identifier ⁇ connector ⁇ e
  • ⁇ the ⁇ different ⁇ sets ⁇ of ⁇ decoding ⁇ oligonucleotides ⁇ may ⁇ be ⁇ comprised ⁇ in ⁇ a ⁇ pre-mixture ⁇ of ⁇ different ⁇ sets ⁇ of ⁇ decoding ⁇ oligonucleotides ⁇ or ⁇ exist ⁇ separately.
  • ⁇ the ⁇ different ⁇ sets ⁇ of ⁇ analyte-specific ⁇ probes ⁇ may ⁇ be ⁇ comprised ⁇ in ⁇ a ⁇ pre-mixture ⁇ of ⁇ different ⁇ sets ⁇ of ⁇ analyte-specific ⁇ probes ⁇ or ⁇ exist ⁇ separately.
  • As ⁇ mentioned ⁇ above ⁇ the ⁇ analyte ⁇ to ⁇ be ⁇ encoded ⁇ may ⁇ be a ⁇ nucleic ⁇ acid, ⁇ preferably ⁇ DNA, ⁇ PNA ⁇ or ⁇ RNA, ⁇ in ⁇ particular mRNA, ⁇ a peptide, ⁇ polypeptide, ⁇ a ⁇ protein, ⁇ and/or ⁇ mixures thereof.
  • the binding ⁇ element ⁇ (S) ⁇ may ⁇ comprise ⁇ moieties ⁇ which ⁇ are ⁇ affinity ⁇ moieties ⁇ from ⁇ affinity ⁇ substances ⁇ or ⁇ affinity ⁇ substances ⁇ in ⁇ their ⁇ entirety ⁇ selected ⁇ from ⁇ the ⁇ group ⁇ consisting ⁇ of ⁇ antibodies, ⁇ antibody ⁇ fragments, ⁇ anticalin ⁇ proteins, ⁇ receptor ⁇ ligands, enzyme ⁇ substrates, ⁇ lectins, ⁇ cytokines, ⁇ lymphokines, ⁇ interleukins, ⁇ angiogenic ⁇ or ⁇ virulence ⁇ factors, ⁇ allergens, ⁇ peptidic ⁇ allergens, ⁇ recombinant ⁇ allergens, ⁇ allergen-idiotypical ⁇ antibodies, ⁇ autoimmune-provoking ⁇ structures, ⁇ tissue-rejection-inducing ⁇ structures, ⁇ immunoglobulin ⁇ constant ⁇ regions ⁇ and ⁇ combinations ⁇ thereof.
  • ⁇ the ⁇ binding ⁇ element ⁇ (S) ⁇ may ⁇ comprise ⁇ or ⁇ is ⁇ an ⁇ antibody ⁇ or ⁇ an ⁇ antibody ⁇ fragment ⁇ selected ⁇ from ⁇ the ⁇ group ⁇ consisting ⁇ of ⁇ Fab, ⁇ scFv; ⁇ single ⁇ domain, ⁇ or ⁇ a ⁇ fragment ⁇ thereof, ⁇ bis ⁇ scFv, ⁇ F(ab)2, ⁇ F(ab)3, ⁇ minibody, ⁇ diabody, ⁇ triabody, ⁇ tetrabody ⁇ and ⁇ tandab.
  • ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure ⁇ comprises ⁇ selectively ⁇ removing ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ and ⁇ signal ⁇ oligonucleotides ⁇ from ⁇ the ⁇ sample, ⁇ thereby ⁇ essentially ⁇ maintaining ⁇ the ⁇ specific ⁇ binding ⁇ of ⁇ the ⁇ analyte-specific ⁇ probes ⁇ to ⁇ the ⁇ analyte ⁇ to ⁇ be ⁇ encoded.
  • all ⁇ steps ⁇ are ⁇ performed ⁇ sequentially.
  • some ⁇ steps ⁇ may ⁇ be ⁇ performed ⁇ simultaneously, ⁇ in ⁇ particular ⁇ the ⁇ contacting ⁇ steps ⁇ A) ⁇ to ⁇ C), ⁇ in ⁇ particular ⁇ B) ⁇ and ⁇ C).
  • said ⁇ encoding ⁇ scheme ⁇ may ⁇ be predetermined ⁇ and ⁇ allocated ⁇ to ⁇ the ⁇ analyte ⁇ to ⁇ be ⁇ encoded.
  • ⁇ the ⁇ code ⁇ words ⁇ obtained ⁇ for ⁇ the ⁇ individual ⁇ analytes ⁇ in ⁇ the ⁇ performed ⁇ cycles ⁇ comprise ⁇ the ⁇ detected ⁇ signals ⁇ and ⁇ additionally ⁇ at ⁇ least ⁇ one ⁇ element ⁇ corresponding ⁇ to ⁇ no ⁇ detected ⁇ signal like ⁇ 0,1 ⁇ or ⁇ 0,1,2 ⁇ etc. ⁇ (see ⁇ also ⁇ Fig. ⁇ 13 ⁇ and ⁇ Fig. ⁇ 14).
  • In ⁇ this ⁇ cycle ⁇ the ⁇ position ⁇ has ⁇ the ⁇ value ⁇ zero ⁇ (0).
  • ⁇ at ⁇ least ⁇ for ⁇ one ⁇ individual ⁇ analyte ⁇ a ⁇ position ⁇ of ⁇ the ⁇ code ⁇ word ⁇ is ⁇ zero ⁇ (0).
  • the ⁇ ⁇ code ⁇ word ⁇ zero ⁇ (0) ⁇ is ⁇ generated ⁇ ⁇ by ⁇ using ⁇ no ⁇ decoding ⁇ oligonucleotides ⁇ ⁇ having ⁇ an ⁇ identifier ⁇ connector ⁇ element ⁇ (t) ⁇ comprising ⁇ a ⁇ nucleotide ⁇ sequence ⁇ which ⁇ is ⁇ essentially ⁇ complementary ⁇ to ⁇ at ⁇ least ⁇ a ⁇ section ⁇ of ⁇ the ⁇ unique ⁇ identifier ⁇ sequence ⁇ of ⁇ the ⁇ identifier ⁇ element ⁇ (T) ⁇ of ⁇ a ⁇ corresponding ⁇ analyte-specific ⁇ probe ⁇ for ⁇ an ⁇ individual ⁇ analyte.
  • oligonucleotides having ⁇ an ⁇ identifier ⁇ connector ⁇ element ⁇ (t) ⁇ comprising ⁇ a ⁇ nucleotide ⁇ sequence ⁇ which ⁇ is ⁇ essentially ⁇ complementary ⁇ to ⁇ at ⁇ least ⁇ a ⁇ section ⁇ of ⁇ the ⁇ unique ⁇ identifier ⁇ sequence ⁇ of ⁇ the ⁇ identifier ⁇ element ⁇ (T) ⁇ of ⁇ a ⁇ corresponding ⁇ analyte-specific ⁇ probe ⁇ for ⁇ an ⁇ individual ⁇ analyte ⁇ are ⁇ used.
  • the ⁇ sample ⁇ is ⁇ contacted ⁇ with ⁇ at ⁇ least ⁇ one, ⁇ preferably ⁇ at ⁇ least ⁇ two different ⁇ sets ⁇ of ⁇ signal ⁇ oligonucleotides, ⁇ wherein ⁇ the ⁇ signal ⁇ oligonucleotides ⁇ in ⁇ each ⁇ set ⁇ comprise ⁇ a ⁇ different ⁇ signal ⁇ element ⁇ and ⁇ comprise ⁇ a ⁇ different ⁇ connector ⁇ element ⁇ (C).
  • the ⁇ different ⁇ sets ⁇ of ⁇ non-signal ⁇ oligonucleotides may ⁇ be ⁇ comprised ⁇ in ⁇ a ⁇ pre- mixture ⁇ of ⁇ different ⁇ sets ⁇ of ⁇ non-signal ⁇ oligonucleotides ⁇ or ⁇ exist ⁇ separately.
  • the ⁇ decoding ⁇ oligonucleotides in ⁇ a ⁇ particular ⁇ set ⁇ of ⁇ decoding ⁇ oligonucleotides ⁇ interacts ⁇ with ⁇ identical ⁇ identifier ⁇ elements ⁇ (T) ⁇ which ⁇ are ⁇ unique ⁇ to ⁇ a ⁇ particular ⁇ analyte.
  • ⁇ the ⁇ different ⁇ sets ⁇ of ⁇ decoding ⁇ oligonucleotides ⁇ may ⁇ be ⁇ comprised ⁇ in ⁇ a ⁇ pre- mixture ⁇ of ⁇ different ⁇ sets ⁇ of ⁇ decoding ⁇ oligonucleotides ⁇ or ⁇ exist ⁇ separately as ⁇ well ⁇ as ⁇ the ⁇ different ⁇ sets ⁇ of ⁇ analyte-specific ⁇ probes ⁇ may ⁇ be ⁇ comprised ⁇ in ⁇ a ⁇ pre-mixture ⁇ of ⁇ different ⁇ sets ⁇ of ⁇ analyte- specific ⁇ probes ⁇ or ⁇ exist ⁇ separately as ⁇ well the ⁇ different ⁇ sets ⁇ of ⁇ signal ⁇ oligonucleotides ⁇ may ⁇ be ⁇ comprised ⁇ in ⁇ a ⁇ pre-mixture ⁇ of ⁇ different ⁇ sets ⁇ of ⁇ signal ⁇ oligonucleotides ⁇ or ⁇ exist ⁇ separately.
  • ⁇ the ⁇ analyte ⁇ specific ⁇ probes ⁇ may ⁇ be incubated ⁇ with ⁇ the ⁇ sample, ⁇ thereby ⁇ allowing ⁇ a ⁇ specific ⁇ binding ⁇ of ⁇ the ⁇ analyte ⁇ specific ⁇ probes ⁇ to ⁇ the ⁇ analytes ⁇ to ⁇ be ⁇ encoded, ⁇ further ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ may ⁇ be incubated ⁇ with ⁇ the ⁇ sample, ⁇ thereby ⁇ allowing ⁇ a ⁇ specific ⁇ hybridization ⁇ of ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ to ⁇ identifier ⁇ elements ⁇ (T) ⁇ of ⁇ the ⁇ respective ⁇ analyte-specific ⁇ probes, ⁇ further the ⁇ signal oligonucleotides ⁇ may ⁇ be incubated ⁇ with ⁇ the ⁇ sample, ⁇ thereby ⁇ allowing ⁇ a ⁇ specific ⁇ RES-PA18-PCT ⁇ hybridization ⁇ of ⁇ the ⁇ signal ⁇ oli
  • ⁇ the ⁇ analyte ⁇ to ⁇ be ⁇ encoded ⁇ may ⁇ be a ⁇ nucleic ⁇ acid, ⁇ preferably ⁇ DNA, ⁇ PNA, ⁇ RNA, ⁇ in ⁇ particular ⁇ mRNA, ⁇ a ⁇ peptide, ⁇ polypeptide, ⁇ a ⁇ protein or ⁇ combinations ⁇ thereof. Therefore, ⁇ the ⁇ binding ⁇ element ⁇ (S) ⁇ may ⁇ comprise ⁇ an ⁇ amino ⁇ acid ⁇ sequence ⁇ allowing ⁇ a ⁇ specific ⁇ binding ⁇ to ⁇ the ⁇ analyte ⁇ to ⁇ be ⁇ encoded.
  • the ⁇ binding ⁇ element ⁇ (S) ⁇ is ⁇ an ⁇ antibody ⁇ or ⁇ an ⁇ antibody ⁇ fragment ⁇ selected ⁇ from ⁇ the ⁇ group ⁇ consisting ⁇ of ⁇ Fab, ⁇ scFv; ⁇ single ⁇ domain, ⁇ or ⁇ a ⁇ fragment ⁇ thereof, ⁇ bis ⁇ scFv, ⁇ Fab ⁇ 2, ⁇ Fab ⁇ 3, ⁇ minibody, ⁇ diabody, ⁇ triabody, ⁇ tetrabody ⁇ and ⁇ tandab.
  • ⁇ the ⁇ signal ⁇ caused ⁇ by ⁇ the ⁇ signal ⁇ element therefore ⁇ in ⁇ particular ⁇ the ⁇ binding ⁇ of ⁇ the ⁇ signal ⁇ oligonucleotides ⁇ to ⁇ the ⁇ decoding ⁇ oligonucleotides, ⁇ interacting ⁇ with ⁇ the ⁇ corresponding ⁇ analyte ⁇ probes, ⁇ bound ⁇ to ⁇ the ⁇ respective ⁇ analyte ⁇ is ⁇ determined ⁇ by: (a) Imaging ⁇ at ⁇ least ⁇ a ⁇ portion ⁇ of ⁇ the ⁇ sample; ⁇ and/or (b) Using ⁇ an ⁇ optical ⁇ imaging ⁇ technique; ⁇ and/or (c) Using ⁇ a ⁇ fluorescence ⁇ imaging ⁇ technique; ⁇ and/or (d) Multi-color ⁇ fluorescence ⁇ imaging ⁇ technique; ⁇ and/or ⁇ (e) Super-resolution ⁇ fluorescence ⁇ imaging ⁇ technique.
  • the ⁇ kits ⁇ and ⁇ method ⁇ according to ⁇ the ⁇ present ⁇ disclosure ⁇ may ⁇ be ⁇ used ⁇ ideally ⁇ for ⁇ in ⁇ vitromethods ⁇ for ⁇ diagnosis ⁇ of ⁇ a ⁇ disease ⁇ selected ⁇ from ⁇ the ⁇ group ⁇ comprising ⁇ cancer, ⁇ neuronal ⁇ diseases, ⁇ cardiovascular ⁇ diseases, ⁇ inflammatory ⁇ diseases, ⁇ autoimmune ⁇ diseases, ⁇ diseases ⁇ due ⁇ to ⁇ a ⁇ viral ⁇ or ⁇ bacterial ⁇ infection, ⁇ skin ⁇ diseases, ⁇ skeletal ⁇ muscle ⁇ diseases, ⁇ dental ⁇ diseases, and ⁇ prenatal ⁇ diseases.
  • ⁇ the ⁇ kits ⁇ and ⁇ method ⁇ according to ⁇ the ⁇ present ⁇ disclosure ⁇ may ⁇ be ⁇ used ⁇ also ⁇ ideally ⁇ for ⁇ in ⁇ vitromethods ⁇ for ⁇ screening, ⁇ identifying ⁇ and/or ⁇ testing ⁇ a ⁇ substance ⁇ and/or ⁇ drug ⁇ comprising: (a) contacting ⁇ a ⁇ test ⁇ sample ⁇ comprising ⁇ a ⁇ sample ⁇ with ⁇ a ⁇ substance ⁇ and/or ⁇ drug ⁇ (b) detecting ⁇ different ⁇ analytes ⁇ in ⁇ a ⁇ sample ⁇ by ⁇ sequential ⁇ signal-encoding ⁇ of ⁇ said ⁇ analytes ⁇ with ⁇ a ⁇ method ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure.
  • optical ⁇ multiplexing ⁇ system ⁇ suitable ⁇ for ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure comprising ⁇ at ⁇ least: - a reaction ⁇ vessel ⁇ for ⁇ containing ⁇ the ⁇ kits ⁇ or ⁇ part ⁇ of ⁇ the ⁇ kits ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure; - a ⁇ detection ⁇ unit ⁇ comprising ⁇ a ⁇ microscope, ⁇ in ⁇ particular ⁇ a ⁇ fluorescence ⁇ microscope; - a ⁇ camera; - a ⁇ liquid ⁇ handling ⁇ device.
  • optical ⁇ multiplexing ⁇ system ⁇ may ⁇ comprise ⁇ further ⁇ a ⁇ heat ⁇ and ⁇ cooling ⁇ device and/or ⁇ a ⁇ robotic ⁇ system.
  • ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure ⁇ encodes a ⁇ nucleic ⁇ acid ⁇ analyte, ⁇ such ⁇ as ⁇ an ⁇ mRNA, ⁇ e.g., such ⁇ an ⁇ mRNA ⁇ coding ⁇ for ⁇ a ⁇ particular ⁇ protein.
  • the ⁇ disclosure is ⁇ now ⁇ further ⁇ explained ⁇ by ⁇ means ⁇ of ⁇ embodiments ⁇ resulting ⁇ in ⁇ additional ⁇ features, ⁇ characteristics ⁇ and ⁇ advantages ⁇ of ⁇ the ⁇ disclosure.
  • the ⁇ technology ⁇ allows ⁇ distinguishing ⁇ a ⁇ higher ⁇ number ⁇ of ⁇ analytes ⁇ than ⁇ different ⁇ signals ⁇ are ⁇ available.
  • the ⁇ process ⁇ preferably ⁇ includes ⁇ at ⁇ least ⁇ one, ⁇ preferably ⁇ at ⁇ least ⁇ two consecutive ⁇ rounds ⁇ of ⁇ specific ⁇ binding, ⁇ signal ⁇ detection ⁇ and ⁇ selective ⁇ denaturation ⁇ (if ⁇ a ⁇ next ⁇ round ⁇ is ⁇ required), ⁇ eventually ⁇ producing ⁇ a ⁇ signal ⁇ code.
  • the ⁇ decoding ⁇ oligonucleotide ⁇ transcribes ⁇ the ⁇ information
  • ⁇ the ⁇ present ⁇ disclosure ⁇ pertains ⁇ to ⁇ a ⁇ method ⁇ of ⁇ assigning ⁇ an ⁇ analyte ⁇ to ⁇ a ⁇ position ⁇ in ⁇ an ⁇ image, ⁇ comprising ⁇ assigning ⁇ a ⁇ fluorescence ⁇ pattern ⁇ to ⁇ the ⁇ analyte, ⁇ observing ⁇ the ⁇ fluorescence ⁇ pattern ⁇ at ⁇ the ⁇ position ⁇ in ⁇ the ⁇ image, ⁇ and ⁇ assigning ⁇ the ⁇ analyte ⁇ to ⁇ the ⁇ position, ⁇ in ⁇ particular ⁇ wherein ⁇ observing ⁇ the ⁇ fluorescence ⁇ pattern ⁇ comprises ⁇ repeating ⁇ steps ⁇ of ⁇ labeling ⁇ the ⁇ position ⁇ using ⁇ a ⁇ fluorophore ⁇ tagged ⁇ oligo ⁇ drawn ⁇ from ⁇ a ⁇ re-accessible pool, ⁇ performing ⁇ a ⁇ single ⁇ excitation ⁇ at ⁇ the ⁇ position ⁇ in ⁇ the ⁇ image, ⁇ and ⁇ contacting ⁇ the ⁇ analyte ⁇ to ⁇ a ⁇ denaturant, ⁇ in ⁇ particular ⁇ wherein ⁇ observing ⁇ the ⁇ the ⁇
  • ⁇ the ⁇ present ⁇ disclosure ⁇ pertains ⁇ to ⁇ a ⁇ method ⁇ of ⁇ assigning ⁇ coded ⁇ fluorescence ⁇ patterns ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ target ⁇ analytes ⁇ in ⁇ a ⁇ cell, ⁇ comprising: ⁇ subjecting ⁇ the ⁇ cell ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ detection ⁇ rounds, ⁇ each ⁇ detection ⁇ round ⁇ comprising: ⁇ contacting ⁇ the ⁇ cell ⁇ to ⁇ representatives ⁇ of ⁇ the ⁇ same ⁇ at ⁇ least ⁇ one, ⁇ preferably ⁇ at ⁇ least ⁇ two populations ⁇ of ⁇ tagged ⁇ fluorescence ⁇ moieties, ⁇ and ⁇ removing ⁇ the ⁇ fluorescent ⁇ moieties ⁇ after ⁇ a ⁇ single ⁇ excitation ⁇ event, ⁇ in ⁇ particular - wherein ⁇ the ⁇ number ⁇ of ⁇ patterns ⁇ detectable ⁇ increases ⁇ exponentially ⁇ with ⁇ the ⁇ number ⁇ of ⁇ detection ⁇ rounds, ⁇ - wherein ⁇ the ⁇ fluorescence ⁇ moieties
  • ⁇ the ⁇ present ⁇ disclosure ⁇ pertains ⁇ to ⁇ a ⁇ method ⁇ of ⁇ assigning ⁇ coded ⁇ fluorescence ⁇ patterns ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ target ⁇ analytes ⁇ in ⁇ a ⁇ cell, ⁇ comprising: ⁇ contacting ⁇ a ⁇ target ⁇ to ⁇ a ⁇ bipartite ⁇ labeling ⁇ probe, ⁇ the ⁇ bipartite ⁇ labeling ⁇ probe ⁇ comprising ⁇ a ⁇ target- specific ⁇ moiety ⁇ and ⁇ a ⁇ fluorophore-specifying ⁇ moiety; ⁇ contacting ⁇ the ⁇ bipartite ⁇ labeling ⁇ probe ⁇ to ⁇ a ⁇ first ⁇ aliquot ⁇ of ⁇ a ⁇ fluorophore ⁇ reservoir ⁇ comprising ⁇ no ⁇ more ⁇ than ⁇ two ⁇ populations ⁇ of ⁇ fluorophores; ⁇ replacing
  • ⁇ replacing ⁇ the ⁇ fluorophore ⁇ specifying ⁇ moiety ⁇ in ⁇ the ⁇ bipartite ⁇ probe ⁇ comprises ⁇ denaturing ⁇ a ⁇ binding ⁇ between ⁇ a ⁇ target-specific ⁇ moiety ⁇ and ⁇ a ⁇ fluorophore-specifying ⁇ moiety ⁇ after ⁇ subjecting ⁇ the ⁇ bipartite ⁇ labeling ⁇ probe ⁇ bound ⁇ to ⁇ a ⁇ RES-PA18-PCT ⁇ fluorophore ⁇ of ⁇ the ⁇ fluorophore ⁇ to ⁇ excitation ⁇ energy.
  • ⁇ replacing ⁇ the ⁇ fluorophore ⁇ specifying ⁇ moiety ⁇ in ⁇ the ⁇ bipartite ⁇ probe ⁇ comprises ⁇ drawing ⁇ from ⁇ one ⁇ of ⁇ no ⁇ more ⁇ than ⁇ two ⁇ fluorophore ⁇ specifying ⁇ moiety ⁇ reservoirs.
  • Step ⁇ 1 ⁇ Applying ⁇ the ⁇ at ⁇ least ⁇ 20 ⁇ analyte- or ⁇ target-specific ⁇ probe ⁇ sets.
  • ⁇ a ⁇ probe ⁇ set ⁇ of ⁇ 5 ⁇ different ⁇ probes ⁇ is ⁇ shown, ⁇ each ⁇ comprising ⁇ a ⁇ sequence ⁇ element ⁇ complementary ⁇ to ⁇ an ⁇ individual ⁇ subsequence ⁇ of ⁇ the ⁇ target ⁇ nucleic ⁇ acid ⁇ sequence ⁇ (S1 ⁇ to ⁇ S5).
  • S1 ⁇ to ⁇ S5 ⁇ In ⁇ this ⁇ example, ⁇ the ⁇ regions ⁇ do ⁇ not ⁇ overlap.
  • Step ⁇ 5 ⁇ Hybridization ⁇ of ⁇ decoding ⁇ oligonucleotides.
  • the ⁇ decoding ⁇ oligonucleotides ⁇ are ⁇ hybridized ⁇ with ⁇ the ⁇ unique ⁇ identifier ⁇ sequences ⁇ of ⁇ the ⁇ probes ⁇ (T) ⁇ via ⁇ their ⁇ complementary ⁇ first ⁇ sequence ⁇ elements ⁇ (t). ⁇ After ⁇ incubation, ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ provide ⁇ the ⁇ translator ⁇ sequence ⁇ element ⁇ (c) ⁇ for ⁇ a ⁇ subsequent ⁇ hybridization ⁇ step.
  • Step ⁇ 6 Eliminating ⁇ the ⁇ excess ⁇ of ⁇ decoding ⁇ oligonucleotides. ⁇ After ⁇ hybridization, ⁇ the ⁇ unbound ⁇ decoding ⁇ oligonucleotides ⁇ are ⁇ eliminated, ⁇ e.g. ⁇ by ⁇ washing ⁇ steps.
  • Step ⁇ 7 ⁇ Applying ⁇ the ⁇ signal ⁇ oligonucleotide. ⁇ The ⁇ signal ⁇ oligonucleotides ⁇ are ⁇ applied. ⁇ The ⁇ signal ⁇ oligonucleotides ⁇ comprise ⁇ at ⁇ least ⁇ one ⁇ second ⁇ connector ⁇ element ⁇ (C) ⁇ that ⁇ is ⁇ essentially ⁇ complementary ⁇ to ⁇ the ⁇ translator ⁇ sequence ⁇ element ⁇ (c) ⁇ and ⁇ at ⁇ least ⁇ one ⁇ signal ⁇ element ⁇ that ⁇ provides ⁇ a ⁇ detectable ⁇ signal ⁇ (F).
  • Step ⁇ 8 ⁇ Hybridization ⁇ of ⁇ the ⁇ signal ⁇ oligonucleotides.
  • the ⁇ signal ⁇ oligonucleotides ⁇ are ⁇ hybridized ⁇ via ⁇ the ⁇ complementary ⁇ sequence ⁇ connector ⁇ element ⁇ (C) ⁇ to ⁇ the ⁇ translator ⁇ element ⁇ (c) ⁇ of ⁇ decoding ⁇ oligonucleotide.
  • Step ⁇ 9 ⁇ Eliminating ⁇ the ⁇ excess ⁇ of ⁇ signal ⁇ oligonucleotides. ⁇ After ⁇ hybridization, ⁇ the ⁇ unbound ⁇ signal ⁇ oligonucleotides ⁇ are ⁇ eliminated, ⁇ e.g. ⁇ by ⁇ washing ⁇ steps.
  • Step ⁇ 12 ⁇ Eliminating ⁇ the ⁇ denatured ⁇ decoding ⁇ oligonucleotides.
  • Step ⁇ 1 Target ⁇ nucleic ⁇ acids: ⁇ In ⁇ this ⁇ example ⁇ three ⁇ different ⁇ target ⁇ nucleic ⁇ acids ⁇ (A), ⁇ (B) ⁇ and ⁇ (C) ⁇ have ⁇ to ⁇ be ⁇ detected ⁇ and ⁇ differentiated ⁇ by ⁇ using ⁇ only ⁇ two ⁇ different ⁇ types ⁇ of ⁇ signal ⁇ oligonucleotides.
  • the ⁇ three ⁇ different ⁇ nucleic ⁇ acid ⁇ sequences ⁇ are ⁇ encoded ⁇ by ⁇ three ⁇ rounds ⁇ of ⁇ detection ⁇ with ⁇ three ⁇ different ⁇ signal ⁇ types ⁇ (1), ⁇ (2) ⁇ and ⁇ (1/2) ⁇ and ⁇ a ⁇ resulting ⁇ hamming ⁇ distance ⁇ of ⁇ 3 ⁇ to ⁇ allow ⁇ for ⁇ error ⁇ de
  • Step ⁇ 3 Hybridization ⁇ of ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ and ⁇ multi-decoders: ⁇ For ⁇ each ⁇ unique ⁇ identifier ⁇ present, ⁇ a ⁇ certain ⁇ decoding ⁇ oligonucleotide ⁇ or ⁇ multi-decoder ⁇ is ⁇ applied ⁇ specifically ⁇ hybridizing ⁇ to ⁇ the ⁇ corresponding ⁇ unique ⁇ identifier ⁇ sequence ⁇ by ⁇ its ⁇ first ⁇ RES-PA18-PCT ⁇ sequence ⁇ element ⁇ (here ⁇ (t1) ⁇ to ⁇ (T1), ⁇ (t2) ⁇ to ⁇ (T2) ⁇ and ⁇ (t3) ⁇ to ⁇ (T3)).
  • Each ⁇ of ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ or ⁇ multi-decoders ⁇ provides ⁇ a ⁇ translator ⁇ or ⁇ two ⁇ translator ⁇ elements ⁇ that ⁇ define ⁇ the ⁇ signals ⁇ that ⁇ will ⁇ be ⁇ generated ⁇ after ⁇ hybridization
  • Step ⁇ 4 ⁇ Hybridization ⁇ of ⁇ signal ⁇ oligonucleotides: ⁇ For ⁇ each ⁇ type ⁇ of ⁇ translator ⁇ element, ⁇ a ⁇ signal ⁇ oligonucleotide ⁇ with ⁇ a ⁇ certain ⁇ signal, ⁇ differentiable ⁇ from ⁇ signals ⁇ of ⁇ other ⁇ signal ⁇ oligonucleotides, ⁇ is ⁇ applied. ⁇ This ⁇ signal ⁇ oligonucleotide ⁇ can ⁇ specifically ⁇ hybridize ⁇ to ⁇ the ⁇ corresponding ⁇ translator ⁇ element. ⁇ The ⁇ illustration ⁇ in ⁇ Fig. ⁇ 16 ⁇ summarizes ⁇ steps ⁇ 7 ⁇ to ⁇ 9 ⁇ of ⁇ Fig. ⁇ 3 Step ⁇ 5: ⁇ Signal ⁇ detection ⁇ for ⁇ the ⁇ encoding ⁇ scheme: ⁇ The ⁇ different ⁇ signals ⁇ are ⁇ detected. ⁇ Note ⁇ that ⁇ in ⁇ this ⁇ example ⁇ the nucleic ⁇ acids ⁇ (A), ⁇ (B) ⁇ and ⁇ (C) ⁇ can ⁇ already ⁇ be ⁇ distinguished ⁇ after ⁇ the ⁇
  • Step ⁇ 6 ⁇ Selective ⁇ denaturation: ⁇ The ⁇ decoding ⁇ (and ⁇ signal) ⁇ oligonucleotides ⁇ and/or ⁇ multi- decoders ⁇ of ⁇ all ⁇ nucleic ⁇ acid ⁇ sequences ⁇ to ⁇ be ⁇ detected ⁇ are ⁇ selectively ⁇ denatured ⁇ and ⁇ eliminated ⁇ as ⁇ described ⁇ in ⁇ steps ⁇ 11 ⁇ and ⁇ 12 ⁇ of ⁇ Fig.3. Afterwards ⁇ the ⁇ unique ⁇ identifier ⁇ sequences ⁇ of ⁇ the ⁇ different ⁇ probe ⁇ sets ⁇ can ⁇ be ⁇ used ⁇ for ⁇ the ⁇ next ⁇ round ⁇ of ⁇ hybridization ⁇ and ⁇ detection.
  • This ⁇ way ⁇ three ⁇ different ⁇ nucleic ⁇ acids ⁇ can ⁇ be ⁇ distinguished in ⁇ three ⁇ detection ⁇ rounds ⁇ with ⁇ two ⁇ different ⁇ signals, ⁇ allowing ⁇ an ⁇ error ⁇ detection ⁇ and ⁇ correction.
  • Step ⁇ 2 ⁇ Hybridization ⁇ of ⁇ the ⁇ probe ⁇ sets. ⁇ For ⁇ each ⁇ target ⁇ nucleic ⁇ acid, ⁇ an ⁇ own ⁇ probe ⁇ set ⁇ is ⁇ applied, ⁇ specifically ⁇ hybridizing ⁇ to ⁇ the ⁇ corresponding ⁇ nucleic ⁇ acid ⁇ sequence ⁇ of ⁇ interest. ⁇ Each ⁇ probe ⁇ set ⁇ provides ⁇ a ⁇ unique ⁇ identifier ⁇ sequence ⁇ (T1), ⁇ (T2) ⁇ or ⁇ (T3). ⁇ This ⁇ way ⁇ each ⁇ different ⁇ target ⁇ nucleic ⁇ acid ⁇ is ⁇ uniquely ⁇ labeled. ⁇ In ⁇ this ⁇ example ⁇ sequence ⁇ (T) ⁇ is ⁇ labeled ⁇ with ⁇ (T1), ⁇ sequence ⁇ (B) ⁇ with ⁇ (T2) ⁇ and ⁇ sequence ⁇ (C) ⁇ with ⁇ (T3). ⁇ The ⁇ illustration ⁇ summarizes steps ⁇ 1 ⁇ to ⁇ 3 ⁇ of ⁇ Figure ⁇ 3.
  • Step ⁇ 3 ⁇ Hybridization ⁇ of ⁇ the ⁇ decoding ⁇ oligonucleotides.
  • each ⁇ unique ⁇ identifier ⁇ present ⁇ a ⁇ certain ⁇ decoding ⁇ oligonucleotide ⁇ is ⁇ applied ⁇ specifically ⁇ hybridizing ⁇ to ⁇ the ⁇ corresponding ⁇ unique ⁇ identifier ⁇ sequence ⁇ by ⁇ its ⁇ first ⁇ sequence ⁇ element ⁇ (here ⁇ (t1) ⁇ to ⁇ (T1), ⁇ (t2) ⁇ to ⁇ (T2) ⁇ and ⁇ (t3) ⁇ to ⁇ (T3)).
  • Each ⁇ of ⁇ the ⁇ decoding ⁇ oligonucleotides provides ⁇ a ⁇ translator ⁇ element ⁇ that ⁇ defines ⁇ the ⁇ signal ⁇ that ⁇ will ⁇ be ⁇ generated ⁇ after ⁇ hybridization ⁇ of ⁇ signal ⁇ oligonucleotides.
  • Step ⁇ 4 ⁇ Hybridization ⁇ of ⁇ signal ⁇ oligonucleotides. ⁇ For ⁇ each ⁇ type ⁇ of ⁇ translator ⁇ element, ⁇ a ⁇ signal ⁇ oligonucleotide ⁇ with ⁇ a ⁇ certain ⁇ signal ⁇ (2), ⁇ differentiable ⁇ from ⁇ signals ⁇ of ⁇ other ⁇ signal ⁇ oligonucleotides, ⁇ is ⁇ applied. ⁇ This ⁇ signal ⁇ oligonucleotide ⁇ can ⁇ specifically ⁇ hybridize ⁇ to ⁇ the ⁇ corresponding ⁇ translator ⁇ element. ⁇ The ⁇ illustration ⁇ summarizes ⁇ steps ⁇ 7 ⁇ to ⁇ 9 ⁇ of ⁇ Figure ⁇ 3.
  • Step ⁇ 6 ⁇ Selective ⁇ denaturation.
  • the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ disclosure combines ⁇ the ⁇ advantages ⁇ of ⁇ seqFISH (mainly ⁇ complete ⁇ freedom ⁇ concerning ⁇ the ⁇ encoding ⁇ scheme) ⁇ with ⁇ all ⁇ advantages ⁇ of ⁇ methods ⁇ using ⁇ only ⁇ one ⁇ specific ⁇ hybridization ⁇ event ⁇ while ⁇ eliminating ⁇ the ⁇ major ⁇ problems ⁇ of ⁇ such ⁇ methods.
  • the ⁇ efficiency ⁇ E ⁇ of ⁇ the ⁇ whole ⁇ encoding ⁇ process ⁇ can ⁇ be ⁇ described ⁇ by ⁇ the ⁇ following ⁇ equation: RES-PA18-PCT ⁇ Based ⁇ on ⁇ this ⁇ equation ⁇ the ⁇ efficiency ⁇ of ⁇ each ⁇ single ⁇ step ⁇ can ⁇ be ⁇ estimated ⁇ for ⁇ a ⁇ given ⁇ total ⁇ efficiency ⁇ of ⁇ the ⁇ method. ⁇ The ⁇ calculation ⁇ is ⁇ hereby ⁇ based ⁇ on ⁇ the ⁇ assumption, ⁇ that ⁇ each ⁇ process ⁇ has ⁇ the ⁇ same ⁇ efficiency. ⁇ The ⁇ total ⁇ efficiency ⁇ describes ⁇ the ⁇ portion ⁇ of ⁇ successfully ⁇ decodable ⁇ signals ⁇ of ⁇ the ⁇ total ⁇ signals ⁇ present.

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Abstract

Sonde de suivi contenant : une séquence nucléotidique correspondant à un traducteur (c) permettant l'hybridation d'un oligonucléotide signal ; un élément identificateur permettant l'hybridation d'un élément de décodage, l'identificateur comprenant une séquence sensiblement complémentaire d'une section de l'identificateur d'ensemble unique d'un élément identificateur (T) d'un décodeur correspondant ; une séquence oligonucléotidique cible permettant l'hybridation d'un élément de liaison. La sonde et la cible peuvent être immobilisées. La cible peut être obtenue à partir d'un échantillon tissulaire. La sonde peut être utilisée pour assurer le suivi des plages de températures ambiantes. La sonde supervise la qualité (QC) de réactifs et de kits fabriqués dans le domaine de l'hybridation in situ pendant toutes les étapes de fabrication. L'invention concerne également des kits et lames témoin contenant les sondes de suivi.
PCT/EP2024/066706 2023-06-16 2024-06-16 Réactif pour le contrôle qualité de l'hybridation in situ Pending WO2024256715A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025125442A1 (fr) * 2023-12-13 2025-06-19 Resolve Biosciences Gmbh Procédé multiplex à haute résolution pour détecter au moins deux cibles

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050272032A1 (en) 2004-05-04 2005-12-08 Ventana Medical Systems, Inc., A Corporation Of The State Of Delaware Internal control for in situ hybridization
US20200277663A1 (en) * 2018-12-10 2020-09-03 10X Genomics, Inc. Methods for determining a location of a biological analyte in a biological sample
WO2020254519A1 (fr) * 2019-06-18 2020-12-24 Apollo Life Sciences Gmbh Procédé de codage de signal d'analytes dans un échantillon
WO2021067246A1 (fr) * 2019-09-30 2021-04-08 Yale University Codification à barres déterministe pour un séquençage omique spatial
US20210189475A1 (en) * 2018-12-10 2021-06-24 10X Genomics, Inc. Imaging system hardware
WO2021168287A1 (fr) * 2020-02-21 2021-08-26 10X Genomics, Inc. Méthodes et compositions pour dosage spatial in situ intégré
WO2021255244A1 (fr) * 2020-06-18 2021-12-23 Resolve Biosciences Gmbh Procédé multiplex de détection d'analytes différents dans un échantillon
US20220042097A1 (en) * 2020-08-04 2022-02-10 The Broad Institute, Inc. In-situ spatial transcriptomics and proteomics
WO2023012272A1 (fr) * 2021-08-06 2023-02-09 Resolve Biosciences Gmbh Procédé de détection d'un analyte dans un échantillon comprenant un pathogène
WO2023086847A1 (fr) * 2021-11-10 2023-05-19 Encodia, Inc. Procédés de codage de macromolécules dans des cellules individuelles
WO2024003221A1 (fr) * 2022-06-30 2024-01-04 Resolve Biosciences Gmbh Procédé multiplex à haute résolution pour détecter au moins deux cibles avec une distance au-delà de la limite de diffraction dans un échantillon
US20240158836A1 (en) * 2022-06-30 2024-05-16 Resolve Biosciences Gmbh High resolution multiplex method for detecting at least two targets with a distance of beyond the diffraction limit in a sample

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050272032A1 (en) 2004-05-04 2005-12-08 Ventana Medical Systems, Inc., A Corporation Of The State Of Delaware Internal control for in situ hybridization
US20200277663A1 (en) * 2018-12-10 2020-09-03 10X Genomics, Inc. Methods for determining a location of a biological analyte in a biological sample
US20210189475A1 (en) * 2018-12-10 2021-06-24 10X Genomics, Inc. Imaging system hardware
WO2020254519A1 (fr) * 2019-06-18 2020-12-24 Apollo Life Sciences Gmbh Procédé de codage de signal d'analytes dans un échantillon
WO2021067246A1 (fr) * 2019-09-30 2021-04-08 Yale University Codification à barres déterministe pour un séquençage omique spatial
WO2021168287A1 (fr) * 2020-02-21 2021-08-26 10X Genomics, Inc. Méthodes et compositions pour dosage spatial in situ intégré
WO2021255244A1 (fr) * 2020-06-18 2021-12-23 Resolve Biosciences Gmbh Procédé multiplex de détection d'analytes différents dans un échantillon
US20220042097A1 (en) * 2020-08-04 2022-02-10 The Broad Institute, Inc. In-situ spatial transcriptomics and proteomics
WO2023012272A1 (fr) * 2021-08-06 2023-02-09 Resolve Biosciences Gmbh Procédé de détection d'un analyte dans un échantillon comprenant un pathogène
WO2023086847A1 (fr) * 2021-11-10 2023-05-19 Encodia, Inc. Procédés de codage de macromolécules dans des cellules individuelles
WO2024003221A1 (fr) * 2022-06-30 2024-01-04 Resolve Biosciences Gmbh Procédé multiplex à haute résolution pour détecter au moins deux cibles avec une distance au-delà de la limite de diffraction dans un échantillon
US20240158836A1 (en) * 2022-06-30 2024-05-16 Resolve Biosciences Gmbh High resolution multiplex method for detecting at least two targets with a distance of beyond the diffraction limit in a sample

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
"Methods in Enzymology", vol. 572, 1 January 2016, ELSEVIER, ACADEMIC PRESS, NL, ISBN: 978-0-12-805382-9, article J.R. MOFFITT ET AL: "RNA Imaging with Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH)", pages: 1 - 49, XP055693313, DOI: 10.1016/bs.mie.2016.03.020 *
ARALAR, AYUAN, YCHEN, KGENG, YVELEZ DOSINHA, MLAWRENCE, SMFRALEY, SI: "Improving Quantitative Power in Digital PCR through Digital High-Resolution Melting", J. CLIN. MICRO., vol. 58, no. 6, 2020, pages e00325 - 20
BUCKWALTER, SPSLOAN, LMCUNNINGHAM, SAESPY, MJUHL, JRJONES, MFVETTER, EAMANDREKAR, JCOCKERILL III, FRPRITT, BS: "Inhibition Controls for Qualitative Real-Time PCR Assays: Are They Necessary for All Specimen Matrices?", J. CLIN. MICRO., vol. 52, no. 6, 2014, pages 2139 - 2143
CHEN KOK HAO ET AL: "Spatially resolved, highly multiplexed RNA profiling in single cells", SCIENCE, vol. 348, no. 6233, 24 April 2015 (2015-04-24), US, XP055879252, ISSN: 0036-8075, DOI: 10.1126/science.aaa6090 *
ROSENSTRAUS, MWANG, ZCHANG, S-YDEBONVILLE, DSPADARO, JP: "An Internal Control for Routine Diagnostic PCR: Design, Properties, and Effect on Clinical Performance", J. CLIN. MICRO., vol. 36, no. 1, 1998, pages 191 - 197
TANG, ZGU, JTANG, GMEDEIROS, LJ.: "Quality Assurance/Quality Control of Fluorescence in Situ Hybridization Tests in Hematologic Malignancies", OBM GENETICS, vol. 2, no. 4, 2018
WILCOX, JN: "Fundamental Principles of In Situ Hybridization", J OF HISTOCHEM. CYTOCHEM., vol. 41, no. 12, 1993, pages 1725 - 1733
XIE, FTIMME, KAWOOD, JR: "Using Single Molecule mRNA Fluorescent in Situ Hybridization (RNA-FISH) to Quantify mRNAs in Individual Murine Oocytes and Embryos", SCI. REP., vol. 8, no. 1, 2018, pages 7930 - 7941
YOUNG, APJACKSON, DJWYETH, RC: "A Technical Review and Guide to RNA Fluorescence in situ Hybridization", PEER J, vol. 8, 19 March 2022 (2022-03-19), pages e8806
ZAMBENEDETTI, MRPAVONI, DPDALLABONA, ACDOMINGUEZ, ACPOERSCH, CDOPRAGOSO, SPKRIEGER, MA: "Internal Control for Real-Time Polymerase Chain Reaction Based on MS2 Bacteriophage for RNA Viruses Diagnostics", MEM INST OSWALDO CRUZ, RIO DE JANEIRO, vol. 112, no. 5, 2017, pages 339 - 347, XP055688300, DOI: 10.1590/0074-02760160380

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025125442A1 (fr) * 2023-12-13 2025-06-19 Resolve Biosciences Gmbh Procédé multiplex à haute résolution pour détecter au moins deux cibles

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