Method for reducing nonspecific signals in in-SITU hybridization assays

Abstract

The application relates to a method for reducing nonspecific signals in in situ hybridization techniques by applying an oligonucleotide as a blocking reagent prior to hybridization of gene-specific probes to tissue to be investigated. Further, the use of an oligonucleotide in such a method is described.

Description

[0001] METHOD FOR REDUCING NONSPECIFIC SIGNALS IN IN-SITU HYBRIDIZATION ASSAYS

[0002] The present invention relates to a method for reducing nonspecific signals (and unwanted background) in in situ hybridization techniques and other hybridization approaches, and the use of oligonucleotides, for example oligo-dT, in such a method.

[0003] Prior Art

[0004] In situ hybridization (ISH) blockers are reagents for minimizing non-specific binding and unwanted background signals in such in situ assays. Examples include DNAfrom calf thymus, salmon sperm, and herring sperm, which are used in pre-hybridization and hybridization solutions. These DNA solutions block the non-specific binding of ISH probes to surfaces.

[0005] Bovine serum albumin (BSA) is used as a blocking agent to reduce background signal and thus improve the contrast of the probe (Choo (2008) Choo KHA. In situ hybridization protocols. Berlin: Springer Science & Business Media; 2008). BSA blocks nonspecific binding of probe molecules to nucleic acid binding sites on proteins within the tissue as it can saturate the binding sites prior to the introduction of the probe. The use of BSA as a blocking agent may be especially important when using antibody-based detection methods. BSA is generally used at a concentration of 1 mg / mL (PMID: 27383759) up to 10 mg / mL (PMID: 6961411).

[0006] Also, sheared salmon sperm DNA or tRNA from E. coli or yeast is usually included in the hybridization buffer. The purpose of competitive nucleic acids is also to saturate nonspecific binding sites for probes to reduce background. Additionally, the competitor tRNA may protect target mRNA molecules via nonspecific blocking of RNase molecules that may have contaminated the solution. The optimal concentration of tRNA within the hybridization buffer should be empirically determined as it may vary widely depending on the tissue sample and the probe (PMID: 25179474; PMID: 30590176).

[0007] There are several alternative hybridization buffer components that can be used to facilitate an optimal hybridization environment. Denhardt’s solution is a broad blocking reagent composed of BSA, Ficoll type 400 and polyvinylpyrrolidone that can be used in place of BSA alone. EDTA is a chelating agent that can be added to a final concentration of 10 mM to remove free divalent ions such as magnesium (PMID: 32219032).

[0008] Summary of the Invention

[0009] The technical problem underlying the present invention is to provide a method for reducing nonspecific signals (and unwanted background) in in situ hybridization techniques.

[0010] This technical problem is solved by the subject-matter of the independent claims. Further embodiments are described in the dependent claims.

[0011] Detailed Description of the Invention

[0012] According to the present invention, there is provided a method for reducing nonspecific signals in in situ hybridization techniques by applying oligonucleotides, for example oligo-dT, as a blocking reagent prior to hybridization of gene-specific probes to tissue to be investigated.

[0013] The term “reducing nonspecific signals” as used herein means that nonspecific signals are reduced when the method according to the present invention is applied compared to nonspecific signals produced without applying the method according to the present invention.

[0014] In situ hybridization (ISH, also hybridization in situ) is a molecular biological method for the detection of nucleic acids (RNA or DNA) in tissues, single cells or on metaphase chromosomes. In one embodiment, the in-situ hybridization technique is single molecule fluorescence in situ hybridization (smFISH).

[0015] The term „smFISH“ refers to single-molecule fluorescence in situ hybridization. It allows the detection and analysis of individual RNA molecules within cells, wherein the observation and quantification of gene expression at the level of individual RNA molecules is possible providing high sensitivity and resolution. Fluorescently labelled probes that emit light when bound to the target RNA molecules enable their visualization and detection. The technique is performed directly within the cells or tissue samples (in situ = in its original place) without the need for isolating or extracting the RNA molecules. Hybridization (binding / annealing of two complementary nucleic acid strands) occurs between the fluorescently labelled probes and the target RNA.

[0016] In one embodiment, the smFISH application is a technique known in this field as “Molecular Cartography”, described for example in WO 2020 / 254519 Al, WO 2021 / 255244 and WO 2021 / 255263, each of which is hereby incorporated by reference in its entirety.

[0017] The skilled person knows this method so that materials and methods for carrying it out are known as well. Briefly, Molecular Cartography is based on combinatorial single-molecule fluorescent in situ hybridization (smFISH). There are hybridized several tens of transcriptspecific probes per target RNA for analysis of up to 100 or even more different types of RNA per sample. Through a proprietary colorizing and de-colorizing chemistry during several imaging rounds, this technology accurately identifies individual transcripts by decoding the barcode that is specific to each transcript type. This approach enables Molecular Cartography to offer unprecedented specificity and sensitivity, with one spot corresponding to one transcript. The result is the visualization and identification of millions of individual transcripts per sample.

[0018] In some advantageous embodiments, the method according to the present invention is used for methods for detecting analytes by spatial transcriptomics. Typically, spatial transcriptomic analysis of biosafety samples is done after RNA or DNA is isolated. This is typical for a scenario using the Visium technology (lOx genomics) or the GeoMx system (Nanostring; https: / / www.nature. com / articles / s41467-021-21361-7).

[0019] The analysis and detection of small quantities of analytes in biological and non-biological samples has become a routine practice in the clinical and analytical environment. Numerous analytical methods have been established for this purpose. Some of them use encoding techniques assigning a particular readable code to a specific first analyte which differs from a code assigned to a specific second analyte.

[0020] One of the prior art techniques in this field is the so-called 'single molecule fluorescence in-situ hybridization' (smFISH) essentially developed to detect mRNA molecules in a sample. In Lubeck et al. (2014), Single-cell in situ RNA profiling by sequential hybridization, Nat. Methods 11(4), p. 360-361, the mRNAs of interest are detected via specific directly labeled probe sets. After one round of hybridization and detection, the set of mRNA specific probes is eluted from the mRNAs and the same set of probes with other (or the same) fluorescent labels is used in the next round of hybridization and imaging to generate gene specific color-code schemes over several rounds. The technology needs several differently tagged probe sets per transcript and needs to denature these probe sets after every detection round.

[0021] A further development of this technology does not use directly labeled probe sets. Instead, the oligonucleotides of the probe sets provide nucleic acid sequences that serve as initiator for hybridization chain reactions (HCR), a technology that enables signal amplification; see Shah et al. (2016), In situ transcription profiling of single cells reveals spatial organization of cells in the mouse hippocampus, Neuron 92(2), p. 342-357.

[0022] Another technique referred to as 'multiplexed error robust fluorescence in situ hybridization' (merFISH) is described by Chen et al. (2015), RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells, Science 348(6233):aaa6090. There, the mRNAs of interest are detected via specific probe sets that provide additional sequence elements for the subsequent specific hybridization of fluorescently labeled oligonucleotides. Each probe set provides four different sequence elements out of a total of 16 sequence elements. After hybridization of the specific probe sets to the mRNAs of interest, the so-called readout hybridizations are performed. In each readout hybridization, one out of the 16 fluorescently labeled oligonucleotides complementary to one of the sequence elements is hybridized. All readout oligonucleotides use the same fluorescent color. After imaging, the fluorescent signals are destroyed via illumination and the next round of readout hybridization takes place without a denaturing step. As a result, a binary code is generated for each mRNA species. A unique signal signature of 4 signals in 16 rounds is created using only a single hybridization round for binding of specific probe sets to the mRNAs of interest, followed by 16 rounds of hybridization of readout oligonucleotides labeled by a single fluorescence color.

[0023] A further development of this technology improves the throughput by using two different fluorescent colors, eliminating the signals via disulfide cleavage between the readout- oligonucleotides and the fluorescent label and an alternative hybridization buffer; see Moffitt et al. (2016), High-throughput single-cell gene-expression profiling with multiplexed error- robust fluorescence in situ hybridization, Proc. Natl. Acad. Sci. U S A. 113(39), p. 11046-11051. A technology referred to as 'intron seqFISH' is described in Shah et al. (2018), Dynamics and spatial genomics of the nascent transcriptome by intron seqFISH, Cell 117(2), p. 363-376. There, the mRNAs of interest are detected via specific probe sets that provide additional sequence elements for the subsequent specific hybridization of fluorescently labeled oligonucleotides. Each probe set provides one out of 12 possible sequence elements (representing the 12 ‘pseudo colors’ used) per color-coding round. Each color-coding round consists of four serial hybridizations. In each of these serial hybridizations, three readout probes, each labeled with a different fluorophore, are hybridized to the corresponding elements of the mRNA-specific probe sets. After imaging, the readout probes are stripped off by a 55% formamide buffer and the next hybridization follows. After 5 color-coding rounds with 4 serial hybridizations each, the color-codes are completed.

[0024] EP 0 611 828 discloses the use of a bridging element to recruit a signal generating element to probes that specifically bind to an analyte. A more specific statement describes the detection of nucleic acids via specific probes that recruit a bridging nucleic acid molecule. This bridging nucleic acids eventually recruit signal-generating nucleic acids. This document also describes the use of a bridging element with more than one binding site for the signal generating element for signal amplification like branched DNA.

[0025] Player et al. (2001), Single-copy gene detection using branched DNA (bDNA) in situ hybridization, J. Histochem. Cytochem. 49(5), p. 603-611, describe a method where the nucleic acids of interest are detected via specific probe sets providing an additional sequence element. In a second step, a preamplifier oligonucleotide is hybridized to this sequence element. This preamplifier oligonucleotide comprises multiple binding sites for amplifier oligonucleotides that are hybridized in a subsequent step. These amplifier oligonucleotides provide multiple sequence elements for the labeled oligonucleotides. This way a branched oligonucleotide tree is build up that leads to an amplification of the signal.

[0026] A further development of this method referred to as is described by Wang et al. (2012), RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues, J. Mol. Diagn. 14(1), p.22-29, which uses another design of the mRNA-specific probes. Here two of the mRNA-specific oligonucleotides have to hybridize in close proximity to provide a sequence that can recruit the preamplifier oligonucleotide. This way the specificity of the method is increased by reducing the number of false positive signals. Choi et al. (2010), Programmable in situ amplification for multiplexed imaging of mRNA expression, Nat. Biotechnol. 28(11), p. 1208-1212, disclose a method known as 'HCR- hybridization chain reaction'. The mRNAs of interest are detected via specific probe sets that provide an additional sequence element. The additional sequence element is an initiator sequence to start the hybridization chain reaction. Basically, the hybridization chain reaction is based on metastable oligonucleotide hairpins that self-assemble into polymers after a first hairpin is opened via the initiator sequence.

[0027] A further development of the technology uses so called split initiator probes that have to hybridize in close proximity to form the initiator sequence for HCR, similarly to the RNAscope technology, this reduces the number of false positive signals; see Choi et al. (2018), Third- generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145(12).

[0028] Mateo et al. (2019), Visualizing DNA folding and RNA in embryos at single-cell resolution, Nature Vol, 568, p. 49ff., disclose a method called 'optical reconstruction of chromatin structure (ORCA). This method is intended to make the chromosome line visible.

[0029] EP 2 992 115 Bl describes a method of sequential single molecule hybridization and provides technologies for detecting and / or quantifying nucleic acids in cells, tissues, organs or organisms through sequential barcoding.

[0030] Summary of the Disclosure

[0031] Spatial transcriptomics (or Spatial *omics) according to the present disclosure means any kind of analysis where data from the sample are derived in a spatial manner from in-situ samples of tissues or whole organisms. The in-situ sample may be a section of an organ or an organism. The in-situ sample may be not pretreated or pretreated in a way that is required for improving the result. Spatial*omics may include the detection of small molecules compounds of tissues or cells, proteins, DNA, and / or RNA. More preferentially, spatial*omics is restricted to proteins, DNA, and / or RNA. More preferentially, spatial*omics is restricted to DNA and / or RNA. Even more preferentially, spatial*omics is restricted to smFISH. Even more preferentially, spatial*omics is restricted to any kind of sequential smFISH. In particular, the spatial transcriptomics detecting comprises a multiplex method for detecting different analytes in a sample by sequential signal-encoding of said analytes as described in WO 2020 / 254519 Al, WO 2021 / 255244 and WO 2021 / 255263.

[0032] In one embodiment, the tissue to be investigated is tissue stemming from formalin-fixed paraffine embedded tissue.

[0033] Fixation of tissues with formalin and subsequent embedding in paraffine (FFPE tissue sections) is one of the most common methods for the preservation and stabilization of biological tissues prior to microsection and histological examination under a microscope. Many clinical specimens are stored as FFPE-treated specimens. In subsequent genetic analyses of the specimen, the FFPE alterations of the specimen must be reversed, e.g., for extraction and subsequent analysis of the DNA or RNA by sequencing or by microarray for purposes of personalized medicine or proteomics. Therefore, various extraction and renaturation methods have been developed to allow FFPE specimens to be used for other diagnostic procedures years later. These include the use of antigen unmasking methods.

[0034] Therefore, the method according to the present invention can be applied to the FFPE tissue sections immediately obtained after the forming of the tissue blocks. The method according to the present invention can also be applied to FFPE tissue blocks which are further processed, for example deparaffinization and / or rehydration, which are usual steps for processing FFPE tissue blocks.

[0035] For the generation of FFPE blocks, tissues can be fixed for several hours (mostly 6-72 h) in formaldehyde of various concentrations (mostly 3,7%, 4% or 10%) at various temperatures (4°C to room temperature). Afterwards, tissues can be dehydrated with alcohol and embedded in paraffin wax. This process leads to a cross-linking of proteins, RNA, and DNA molecules to proteins in the tissue, which is important for the preservation of tissue architecture and keeping the molecules in the physiological position in the tissue.

[0036] Oligonucleotides according to the present invention are DNA or RNA molecules, in particular single stranded molecules, which can have 5 to 100, for example 10 to 50 in particular 15 to 25 nucleotides. Oligo-dT as used herein means short single-stranded DNA sequences comprising or consisting of thymidine nucleotides. Alternate oligos comprise exclusively or predominantly pyrimidine bases, such as at least 75%, 80%, 85%, 90%, 95% or greater than 95% pyrimidines, or at least 75%, 80%, 85%, 90%, 95% or greater than 95% T. Alternately, in some cases oligos are selected to have reduced binding energy, such that the comprise at least at least 75%, 80%, 85%, 90%, 95% or greater than 95% AT bases.

[0037] Some blocking agents consistent with the disclosure herein comprise single stranded or double stranded nucleic acids such as foreign nucleic acids. A number of nucleic acid sources or types are consistent with the disclosure herein, such as those suitable for binding by nucleic acid binding moieties in the sample or in the materials through which the sample is bound or on which the sample is stabilized, such as a fixative or a slide surface.

[0038] Generally, a broad range of blocking nucleic acids are suitable for practice of the disclosure herein, such as salmon DNA or other readily available nontarget nucleic acids. Nucleic acids from such sources, particularly double stranded nucleic acids, are in some cases very effective at blocking samples or sample slides from binding by probes that may lead to off-target or generally higher background signal.

[0039] However, a risk of nucleic acids from such sources is that they may comprise genes or encode gene products which exhibit high similarity to one or target analytes in a sample. For example, some genes encoding proteins relating to highly conserved cellular functions, such as histones, ribosomal proteins, nucleic acid synthesis proteins, transcription or translation proteins, among others, exhibit a high degree of identity or similarity across even distantly related organisms, such as all vertebrates, all animals or all eukaryotes. In these cases, one may observe dramatic target or probe-specific variation in background signal, such that probes that target genes having a segment or segments with a high degree of similarity or identity to nucleic acids in the sample may exhibit probe specific high background rates, while probes targeting less conserved nucleic acids do not exhibit a similar degree of background. Accordingly, blocking nucleic acids comprising naturally occurring genomic DNA, for example, may exhibit dramatic probe specific variation in the extent to which they block background signal. This risk is particularly high when the blocking nucleic acids are single stranded. Accordingly, some blocking nucleic acids do not comprise sequences related to probes or to target nucleic acids, or do not comprise sequences arising from naturally occurring genomic sequence. Suitable blocking nucleic acids comprise sequence which does not occur or does not occur with any frequency in a target nucleic acid or probe sequence. Exemplary blocking nucleic acids comprise poly-T oligonucleotides, or at least 75%, 80%, 85%, 90%, 95% or greater than 95% T. Alternate oligos comprise exclusively or predominantly pyrimidine bases, such as at least 75%, 80%, 85%, 90%, 95% or greater than 95% pyrimidines.

[0040] Alternately, in some cases oligos are selected to have a low chance of binding to a sample nucleic acid and to have a reduced binding energy, such that the comprise at least at least 75%, 80%, 85%, 90%, 95% or greater than 95% AT bases.

[0041] According to the method of the present invention, in a first step the FFPE tissue section (optionally further processed as explained above) is bought into contact with an oligo such as oligo-dT. After this step a second step is carried out in which the smFISH application for detecting RNA is carried out with the tissue section obtained from the first step.

[0042] In one embodiment, the oligonucleotide, in particular the oligo-dT, can be applied to the tissue as an oligonucleotide (in particular oligo-dT)-solution, in which the oligonucleotide (in particular the oligo-dT) is dissolved in a solvent, prior to hybridization of gene-specific probes and / or wherein the oligonucleotide (in particular oligo-dT) is used in the hybridization buffer, e.g., that buffer, which is used in the in situ hybridization assay.

[0043] In a further embodiment, the oligonucleotide (or oligo-dT)-solution can be incubated with the tissue to be investigated during about 5 minutes to about 24 hours, for example during about 10 minutes to 5 hours, in particular about 30 minutes. This incubation can be carried out at about room temperature. Furthermore, it can be assured that a good contact between the components is achieved, for example by stirring and / or shaking the mixture.

[0044] In a further embodiment, the oligonucleotide (in particular oligo-dT)-solution can be applied to the tissue to be investigated after tissue preparation and before probe hybridization, that is the hybridization of the probe in the in situ hybridization assay. In one embodiment, the oligonucleotide (in particular oligo-dT)-solution can be obtained by dissolving the oligonucleotide / oligo-dT in buffer, which can be made from any type of buffer including Tris, phosphate, citrate buffers. The buffer may include other salts like NaCl, KC1, (NH4)C1, MgSO4, MgCh etc. The buffer can have a pH between 5-9. The buffer may include other components like BSA, RNasin, other macromolecules such as dextarnsulfate, PEG etc.) or complex binders (e.g., EDTA, EGTA, etc.).

[0045] In one embodiment, the concentration of the oligonucleotide (in particular oligo-dT) in the solution can be from about Ipg / mL to about 5 mg / mL, for example about 50 pg / mL to about 500 pg / mL, in particular about 100 pg / mL

[0046] The present invention further related to the use of oligonucleotides, in particular oligo-dT, for reducing nonspecific signals in in situ hybridization techniques, by applying the oligonucleotide (oligo-dT) as a blocking reagent prior to the hybridization of gene-specific probes to tissues to be investigated. Regarding the materials and the process steps for this use, it is referred to the above description of the method of the present invention in its entirety.

[0047] The present disclosure further relates to compositions consistent with the use above, such as compositions comprising samples such as FFPE or otherwise positionally preserved samples contacted to or comprising blocking nucleic acids as disclosed herein or otherwise effective to practice the disclosure herein.

[0048] A feature of some such compositions and practice of some such methods herein is that one observes a reduced level of background signal upon probing such samples or practicing such methods. One observes, for example, a level of background signal which is in various cases at least, no more than or about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, or less than 20% of the level observed for an untreated sample.

[0049] The present invention is further described in detail by referring to the figures. It is explicitly pointed out that it shall not be construed to restrict the invention to the figures.

[0050] Fig. 1 A and Fig. IB show the probable mechanism for reducing the nonspecific signals by the use of oligo-dT as a blocking agent without being bound to any theory. Figure 2: Molecular Cartography from human tumour cells without blocking (A) and with blocking by salmon sperm DNA (B) after hybridization during processing.

[0051] Figure 3: Molecular Cartography from mouse heart without blocking (A) and with blocking by oligo-dT oligonucleotides (B)

[0052] Figure 4: Strong binding of molecular probes to certain areas within tumour cells without blocking by nucleic acids.

[0053] Figure. 5: Strong nonspecific binding of molecular probes to the slide can be blocked by DNA especially single stranded DNA.

[0054] Briefly summarized, the subject-matter of the present invention relates to the use of Oligo-dT as a blocking reagent prior to the hybridization of gene-specific probes.

[0055] The present invention describes a method to block nonspecific signals in in-situ applications using oligonucleotides, in particular oligo-dT. Oligo-dT is a short, single-stranded oligonucleotide of repeating deoxythymidines. Commonly, oligo-dT is used as a primer sequence for cDNA synthesis and RT-PCR.

[0056] The positions, where the blocking DNA can be applied are during tissue preparation, during pre-blocking, during hybridization and / or during colorization.

[0057] In the past, there were issues with artifacts accumulating around nuclei in the analysis technique called Molecular Cartography. Since histone proteins in the nuclei are favorably binding DNA, it was justified to assume that the DNA probes used for RNA detection might bind to histone proteins to produce those artifacts. By using oligo-dT as a blocking reagent, it was possible to get rid of these artifacts presumably because oligo-dT (DNA molecules) were blocking potential binding sites at the histone proteins before the probe for RNA detection could bind.

[0058] This is shown in Fig. 2A and Fig. 2B. As can be taken from Fig. 2A, the probes for RNA detection, which are fluorescently labelled, can bind to the histone proteins in the nucleus causing nonspecific signals (and therefore unwanted) indicated in Fig. 2A as artefacts, which are herein also referred to as nonspecific signals. When according to the present invention oligo dT is applied (see Fig. 2B), the fluorescently labelled probes cannot bind to the histone proteins so that as a result no or at least less artefacts / nonspecific signals are produced.

[0059] The following options of reducing nonspecific signals are envisaged according to the present invention: a) Preblocking on tissue sections with Oligo-dT or other nucleic acid for in-situ hybridization b) Preblocking on tissue sections with Oligo-dT or other nucleic acid for smFISH analysis c) Preblocking on FFPE tissue sections with Oligo-dT or other nucleic acid for in-situ hybridization d) Preblocking on FFPE tissue sections with Oligo-dT or other nucleic acid for smFISH analysis

[0060] Examples

[0061] Example 1 :

[0062] The oligonucleotides used for blocking probably binds to the surface of the slide and biological sample (here: cultured cells), and especially to histones by non-specific interaction (as theorized in Fig. 1). These sites may be saturated and reduce the non-specific binding of probes. Therefore, the background and probably the nonspecific interaction to specific sites (e.g., histones) may be reduced. The plot profiles in areas covering the background on the slide and cells is reduced at least twofold from -1000 outside of cells) or 1300 (inside of cells) to -450 (outside of cells) or 500 (inside of cells) by using oligonucleotides for blocking nonspecific interaction.

[0063] In example, cells grown and fixed on glass slides were used for pre-blocking followed by Molecular Cartography. Pre-blocking was done prior to hybridization of transcript specific probes for 30 min at 37°C by a solution that contained 4xSSC, 10% Formamide, and denatured low molecular weight salmon sperm DNA at a concentration of 50 pg / ml.

[0064] The results are shown in Fig. 2. Example 2:

[0065] The oligonucleotides used for blocking probably binds to the surface of the slide and biological sample (here: mouse heart), and especially to histones by non-specific interaction. These sites may be saturated and reduce the non-specific binding of probes. Therefore, the background and probably the nonspecific interaction to specific sites (e.g., histones) may be reduced (see again Fig. 1). The plot profiles in areas covering the background on the slide and tissue is reduced from 5000 (inside of cells) to -3000 (inside of cells) by using oligo-dT oligonucleotides for blocking nonspecific interaction.

[0066] In example, mouse heart tissue sections that were fixed on glass slides, were used for preblocking followed by Molecular Cartography. Pre-blocking was done prior to hybridization of transcript specific probes for 30 min at 37°C by a solution that contained 4xSSC, 10% Formamide, and oligo-dT oligonucleotides at a concentration of 100 pg / ml.

[0067] The results are shown in Fig. 3.

[0068] Example 3 :

[0069] If no oligonucleotides are used for blocking, it may happen that probes accumulate in the nucleus and results in strong signals conglomerated in certain areas. An experiment using fixed cells from a cell culture showed probe binding to the nucleus that may be restricted to certain histones.

[0070] In example, cells grown and fixed on glass slides using methanol were used without preblocking followed by Molecular Cartography using 19 genes during hybridization and the standard Molecular Cartography protocol. Standard Molecular Cartography conditions were used for the experiment. Strong artefacts are visible, especially within areas of the nucleus.

[0071] The results are shown in Fig. 4. Example 4:

[0072] If no oligonucleotides are used for blocking, it may happen that probes bind to the slide surface. An experiment using fixed cells from a cell culture showed much higher background in cell free areas.

[0073] In example, cells grown and fixed on glass slides using methanol were used with and without pre-blocking followed by Molecular Cartography. For blocking, denatured salmon sperm, nondenatured salmon sperm, or oligo-dT oligonucleotides were used prior to the hybridization of gene-specific probes for 19 genes and the standard protocol for Molecular Cartography were used for the experiment. The background fluorescence on the glass slide could be reduced 2 - 4fold.

[0074] The results are shown in Fig. 5.

Claims

CLAIMS1. A method for reducing nonspecific signals in in situ hybridization techniques comprising applying an oligonucleotide as a blocking reagent prior to hybridization of genespecific probes to tissue to be investigated.

2. The method according to claim 1, wherein the oligonucleotide is oligo-dT.

3. The method according to claim 1 or 2, wherein the in-situ hybridization technique is single molecule fluorescence in situ hybridization.

4. The method according to any of the preceding claims, wherein the tissue is formalin- fixed paraffine embedded tissue.

5. The method according to any of the preceding claims, wherein the oligonucleotide is applied to the tissue as oligonucleotide-solution, in which the oligonucleotide is dissolved in a solvent, prior to hybridization of gene-specific probes and / or wherein the oligonucleotide is used in the hybridization buffer.

6. The method according to claim 5, wherein the oligonucleotide-solution is incubated with the tissue to be investigated during about 5 minutes to about 24 hours, for example during about 10 minutes to 5 hours, in particular about 30 minutes.

7. The method according to claim 5 or 6, wherein the oligonucleotide-solution is applied to the tissue to be investigated after tissue preparation and before probe hybridization.

8. The method according to claims 5 to 7, wherein the oligonucleotide-solution is obtained by dissolving the oligonucleotide in a buffer.

9. The method according to claims 5 to 8, wherein a concentration of the oligonucleotide in the oligonucleotide-solution is from about Ipg / mL to about 5 mg / mL, for example about 50 pg / mL to about 500 pg / mL, in particular about 100 pg / mL.

10. The method according to any of the preceding claims, wherein the length of the oligonucleotide is about 5 to about 100 nucleotides.

11. The method according to any of the preceding claims, wherein the in situ hybridization technique is Molecular Cartography.

12. A method for detecting an analyte in a sample comprising i) reducing nonspecific signals in in situ hybridization techniques by applying an oligonucleotide as a blocking reagent prior to hybridization of gene-specific probes to tissue to be investigated with a method of any one of claim 1 to claim 11; ii) Detecting the analyte by spatial transcriptomics.

13. The Use oligonucleotide for reducing nonspecific signals in in situ hybridization techniques, comprising applying oligo-dT as a blocking reagent prior to the hybridization of gene-specific probes to tissues to be investigated.

14. A composition comprising a positionally preserved sample and a first population of exogenous unlabeled nucleic acids provided to the sample, such that nonspecific binding by a labeled nucleic acid probe is reduced by at least 30%.

15. The composition of claim 14, wherein the positionally preserved sample is an FFPE sample.

16. The composition of claim 14, wherein the exogenous unlabeled nucleic acids are single stranded.

17. The composition of claim 14, wherein the exogenous unlabeled nucleic acids are oligos.

18. The composition of claim 17, wherein the oligos comprise a uniform population.

19. The composition of claim 14, wherein the exogenous unlabeled nucleic acids do not comprise a segment of at least 12 bases having at least 66% identity to a target analyte.

20. The composition of claim 14, wherein the exogenous unlabeled nucleic acids do not comprise a segment of at least 24 bases having at least 66% identity to a target analyte.

21. The composition of claim 18, wherein the oligos do not target a uniform target nucleic acid population in the sample.

22. The composition of claim 18 or claim 17, wherein the oligos comprise at least 80% pyrimidine bases.

23. The composition of claim 18 or claim 17, wherein the oligos consist of pyrimidine bases.

24. The composition of claim 18 or claim 17, wherein the oligos consist of T-bases.

25. The composition of any one of claims 14 to 24, further comprising a population of exogenous labeled oligos.

26. The composition of any one of claims 14 to 24, further comprising a second population of exogenous labeled oligos, wherein the second population comprises oligos reverse complementary to target nucleotides in the sample.

27. Amethod of reducing background signal from an in situ hybridization assay of a sample, comprising blocking nucleic acid binding proteins in the sample, and contacting the sample to a nucleic acid probe.

28. The method of claim 27, wherein the nucleic acid binding proteins comprise histones.

29. The method of claim 27, wherein the nucleic acid binding proteins comprise nucleosomes.

30. The method of claim 27, wherein the blocking comprises forming the composition of any one of claims 14 - 26.

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