Method for preparing tissue sections

Abstract

The present invention relates to methods for preparing RNA from preserved sample such as formalin-fixed, paraffin-embedded (FFPE) tissue sections. Through re-fixation of samples such as FFPE samples pursuant to processing, one improves recovery of RNA molecules or makes them accessible for probe detection. The present application relates to a method for preparing nucleic acid from formalin-fixed, paraffin-embedded tissue sections, comprising the following steps: (a) a fixation step of nucleic acid, which is obtained from formalin-fixed, paraffin-embedded tissue sections after deparaffinization and rehydration, with formaldehyde; (b) a nucleic acid retrieval step in a buffer; and (c) a permeabilization step using proteinase K and a vanadyl ribonucleoside complex.

Description

METHOD FOR PREPARING TISSUE SECTIONSBACKGROUND

[0001] Fixation of tissues with formalin and subsequent embedding in paraffine, so as to generate FFPE tissue sections, for example, is one of the most common methods for the preservation and stabilization of biological tissues so as to facilitate micro sectioning or histological examination under a microscope. Many clinical specimens are stored as FFPE- treated specimens. In some subsequent genetic analyses of the specimen, it is beneficial for the FFPE alterations of the specimen to be reversed, e.g., for extraction and subsequent analysis of sample DNA or RNA, such as by sequencing or by microarray. Such extracted nucleic acids facilitate personalized medicine, or comparison of nucleic acids to preserved proteins or other proteomics analysis. 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 methods that unmask the analyte.

[0002] For the generation of FFPE blocks, tissues are typically fixed for several hours (mostly 6-72 h) in formaldehyde of various concentrations (mostly 3,7%, 4% or 10%) at various temperatures (e.g., 4°C to room temperature). Afterwards, tissues are dehydrated with alcohol or other organic solvents and embedded in paraffin wax. This process leads to a cross-linking of as the sample so as to preserve proteins, RNA, and DNA molecules in the tissue, which is important for the preservation of tissue architecture and keeping the molecules in their physiological positions in the tissue. However, the progressive cross-linkage molecules makes it difficult to detect of RNA, DNA or protein via standard analytical methods, e.g., via genespecific probes (in-situ hybridization techniques) because binding sites of the nucleic acids such as RNA can be blocked by the cross-links. On the other hand, insufficient formaldehyde fixation can lead to a washout of RNA molecules from the tissue during the in-situ process which decreases sensitivity.

[0003] Numerous analytical detection methods have been established. Some use encoding techniques assigning a particular readable code to a specific first analyte which differs from a code assigned to a specific second analyte.

[0004] 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. Methodsl l(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 iseluted 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.

[0005] 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.

[0006] 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.

[0007] Xia et al. (2019) (https: / / doi.org / 10.1073 / pnas.1912459116) further increased the gene throughput of MERFISH and achieved 10,000 plex using 23 rounds of hybridization and 3 color channels. To reduce the impact of crowding and diffraction limited spots, this version of MERFISH uses expansion microscopy to increase the voxel space in which individual transcripts can be detected.

[0008] 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 “pseudocolors” used) per color-coding round. Each color-coding roundconsists 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.

[0009] A further development of this technology, termed “seqFISH+” (https: / / doi.org / 10.1038 / s41586-019-1049-y), used the same principle of pseudocolors, but encodes individual transcripts in one of three color channels separately to eliminate chromatic aberrations. To reduce the impact of crowding and diffraction limited spots, seqFISH+ dilutes signals into 4 color coding rounds with 20 serial hybridizations each in combination with subpixel localization of spots. Thereby, seqFISH+ achieves 10,000plex smRNA-FISH, but with very high false positive rates (FPR = 0.22).

[0010] All of these approaches for the detection of RNA in a sample are impeded by sample crosslinking such as that which occurs pursuant to FFPE sample generation. Similarly, protein and DNA detection is impeded by FFPE sample preservation. Therefore, finding compositions, methods and systems that facilitate analyte detection within FFPE samples, such as via genespecific probes is important, as is reliable analyte extraction from FFPE.SUMMARY

[0011] The technical problem underlying the present invention is to provide an efficient way to sufficiently fix nucleic acids, such as RNA and / or DNA of any kind and origin, in the tissue sections and at the same time to reach a high detection sensitivity. The present disclosure solves this technical problem.

[0012] The present disclosure describes the usage of the present method for preparing nucleic acid from formalin-fixed, paraffin-embedded tissue sections for the following detection of nucleic acids least two sets of labeled and unlabeled nucleic acid sequences for specific quantitative and / or spatial detection of different analytes 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.

[0013] Disclosed herein are methods of preparing a fixed sample for a hybridization assay. Various embodiments of the methods comprise one or more of refixing the sample, retrieving nucleic acid accessibility in the sample, permeabilizing the sample, refixing the sample subsequent to permeabilization, and blocking the sample from further refixing. Practice of various methods herein prepares a sample section to be assayed for the presence and position ofone, a few, 10s, 100s or 1,000s or more of nucleic acids such as DNA or RNA molecules or epitopes such as those found on proteins.

[0014] Counterintuitively, methods herein often comprise subjecting the sample, such as a FFPE sample or sample section, to one or more additional fixation steps. Without being bound by theory, such refixation steps may serve to stabilize the sample such that it may be permeabilized without loss of structure or loss of analytes to be detected.

[0015] Refixing is often performed using a fixative such as formaldehyde, often in PBS solution. A number of concentrations are consistent with the disclosure herein, such as 2%-6%, 3%-5%, about 4% or 4%, though concentrations outside of these ranges are also consistent with the disclosure. Fixation may occur above, at or below room temperature, such as no more than 15°C, 10°C or 4°C. Refixation may occur for at least 1, 5 or 10 minutes, or no more than 60, 30, 15 or 10 minutes. A preferred embodiment has a 10 minute incubation, though alternatives are also consistent with the disclosure herein.

[0016] Methods herein often comprise retrieving target analytes from the sample. Retrieving, as used herein, refers to rendering target analytes accessible to a probe, rather than removal of target analytes from a sample. In particular, retrieving often refers to rendering the specific local environment of the target analyte clear of fixative or fixation-related chemicals that may otherwise block the binding of a detection analyte such as a probe or antibody.

[0017] Retrieving often comprises chemically scavenging the fixative such as formaldehyde. This is effected in some cases by forming a Schoff base comprising the fixative, such as by contacting to a nucleophilic buffer. Exemplary buffers include Tris-EDTA, though others are consistent with the disclosure herein. Buffers are variously administered at a concentration of at least ImM, 5mM or lOmM, or no more than lOOmM, 50mM or lOmM. An exemplary concentration is lOmM, though alternatives are also consistent with the disclosure herein. Administration is of at or about pH9 is an exemplary pH, or a range spanning from pH 8.5 to 9.5, pH8 to pH 10, or pH7 to pH 11, or a pH outside of this range. Administration for 45 minutes is an exemplary embodiment, as are times in a range of at least 10, 20, 30 or no more than 120, or 60 minutes, though times outside of these ranges are also consistent with the disclosure herein. The temperature of retrieval is in various embodiments a no more than 100C, 90C, 80C or 70C, or at least 25C, 45C, 60C or 70C. An exemplary incubation temperature is 70C, though other temperatures are consistent with the disclosure herein.

[0018] Methods herein often comprise permeabilizing the sample, so as to render it porous or substantially more porous, so as to effect probe saturation or probe access to the interior of the sample. Retrieving and permeabilizing both serve to increase analyte accessibility, but while retrieving renders the analyte itself more accessible to a binding agent, permeabilizing rendersthe structural components of the sample more porous so as to allow the probes to access the analytes. A number of permeabilizing agents are consistent with the disclosure herein, such as proteases or other enzymes or protein degrading moieties. Serine proteases are in some cases preferred. Exemplary proteases include trypsin, bromelain, chymotrypsin, ficin, papain, serrapeptase, or proteinase K. In exemplary embodiments, the protease is proteinase K.

[0019] Permeabilization is often performed in the presence of a polypeptide or nonpolypeptide RNase inhibitor, such as a Vanadyl ribonucleoside complex, RNase inhibitor Hu, placental RNase inhibitor or other RNase inhibitor, alone or in combination with a protein degrading moiety such as proteinase K. A range of RNase inhibitor concentrations are consistent with the disclosure herein, such as at least lug / mL, 5ug / mL, or lOug / mL, or at most lOOug / mL, 50ug / mL. An exemplary RNase inhibitor is Vanadyl ribonucleoside complex, and an exemplary concentration is lOug / mL.

[0020] Fixation, retrieval and permeabilization are often performed consecutively, in order, following sample preparation such as sample drying, deparaffinization in xylene or other solvent and rehydration. Alternately, they may be performed out of order, individually or in tandem rather than all three steps being performed, iteratively, or with one or more intervening steps.

[0021] Optionally, one or more of Fixation, retrieval and permeabilization are often followed by a second fixation (or second refixation) step, and a blocking step so as to prevent further fixation or reaction of the fixative with subsequent processing reagents.

[0022] Practicing the methods herein results in a substantial improvement in target analyte detection. For example, refixation may in some cases increase target analyte counts by at least 5%, 10%, 20%, 30%, 40%, 50% or more than 50% over unrefixed samples. Retrieval steps may in some cases increase target analyte counts by at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, or more than lOx. Similarly, permeabilization using an enzyme such as proteinase K may increase target analyte counts by 50%, 100%, 200% or more than 200%.INCORPORATION BY REFERENCE

[0023] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrativeembodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0025] FIG. 1 shows a comparison of protocol steps of prior external state of art versus novel process, refixation is performed directly after deparaffinization and rehydration. FIG. 1 shows the differences of one embodiment of the novel process in comparison to the prior external state of art in a shortened fashion.

[0026] FIG. 2 shows the effects of the refixation step on various samples. Effects are measured as transcript counts per tile, in intervals of 10,000 ranging from 0 to 70,000 along the y-axis, for samples Mouse Heart, Mouse Brain, and Mouse Kidney, each presented along the x-axis in columns left, grey, without refixation, and right, black, with refixation. For all tissues assayed, refixation resulted in a substantial increase in transcripts per tile, ranging from about 20%, to about 40% to almost 100% in mouse kidney.

[0027] FIG. 3 shows an RNA retrieval buffer testing results for various retrieval buffers. Effects are measured as counts per tile, in intervals of 2,000 ranging from 0 to 16,000 along the y-axis, for buffers sodium citrate pH 6, alone or with Glycine, Tris-EDTA pH 7 and Tris-EDTA pH 9, from left to right along the X axis. One sees a substantial improvement using Tris-EDTA, of about 7x at pH 7 and over 14x at pH 9, over retrieval buffers in the art.

[0028] FIG. 4 shows RNA retrieval at different temperatures. Effects are measured as counts per tile, in intervals of 10,000 ranging from 0 to 60,000 along the y-axis, for 70°C and 90°C incubation temperatures. The results show a greater than 2.5x increase in counts per tile at 70°C relative to the higher temperature.

[0029] FIG. 5 shows the results of enzymatic permeabilization using proteinase K. Effects are measured as counts per tile, in intervals of 5,000 ranging from 0 to 40,000 along the y-axis, from PBS-R lacking proteinase K, and lOug / mL of proteinase K in PBS, PBS-R and Tris-EDTA from left to right along the x-axis. The results indicate that addition of a protease enzyme such as protease k increases counts per tile by 2.5x to over 3x.

[0030] FIG. 6 shows a bar graph displaying the count per tile (“counts / tile”) for each of the fixation times of 0 minutes, 5 minutes, 10 minutes, and 15 minutes across 4 different mouse heart tissue samples.

[0031] FIG. 7 shows a bar graph displaying the count per tile (“counts / tile”) for each of the mouse FFPE heart samples (Sample 1 and Sample 2) with each of the conditions, “Fixation after RNA retrieval” and “Fixation before and after RNA retrieval”.

[0032] FIG. 8 shows a bar graph displaying the count per tile (“counts / tile”) for each of the fixation conditions, “Fixation before RNA retrieval”, “Fixation after RNA retrieval”, and“Fixation before and after RNA retrieval” for each of the mouse liver samples (Sample 1 and Sample 2) and the average.

[0033] FIG. 9 shows a bar graph displaying the count per tile (“counts / tile”) for each of the tissue samples and fixation conditions, brain without re-fixation (“Brain (- re-fixation)”), brain with re-fixation (“Brain (+ re-fixation)”), kidney without re-fixation (“Kidney (- re-fixation)”), and kidney with re-fixation (“Kidney (+ re-fixation)”).DETAILED DESCRIPTION

[0034] According to the present invention, there is provided a method for preparing nucleic acids, such as RNA and / or DNA of any kind and from any origin, from formalin-fixed, paraffin- embedded tissue sections, comprising the following steps: (a) a fixation step of the nucleic acid, which is obtained from the formalin-fixed, paraffin-embedded (FFPE) tissue sections after deparaffinization and rehydration, with formaldehyde; (b) a nucleic acid retrieval step in a buffer; and (c) a permeabilization step using proteinase K and a vanadyl ribonucleoside complex.

[0035] It is pointed out that according to the present invention, the term “preparing nucleic acid” does not mean that the nucleic acid molecule itself is prepared, but rather that the nucleic acid molecule contained in a FFPE tissue section is further processed which as a result does also lead to prepared nucleic acid.

[0036] In the following, the invention is illustrated and explained by reference to RNA, but it is to be understood that the invention can equally be applied to DNA or other nucleic acid molecules as well in an analogous manner.

[0037] That is, in step (a) RNA can be used, which is provided from a FFPE tissue section after deparaffinization and rehydration of the FFPE tissue section. In one embodiment, the fixation step (a) can be carried out with an about 3 % to about 5 %, such as about 4 % solution of formaldehyde during about 5 minutes to about 30 minutes, such as about 10 minutes to about 20 minutes, in particular at a temperature between 1°C to 25°C, preferably 2°C to 15°C, more preferably 2-8°C, most preferably at 4°C.

[0038] The solvent of this solution can be a water-containing solvent, for example a buffer, such as phosphate-buffered saline (PBS). In a further embodiment, the buffer in step (b) can be a Tris- EDTA buffer having a pH of about 8 to 10, more preferably 8.5 to 9.5, most preferably 8.8 to 9.2, in particular having a pH of about 9. Step (b) can be carried out preferably at a temperature between 65°C to 75°C, more preferably 68°C-72°C, most preferably 70°C for a time between 30 min to 60 min, more preferably 35 min to 55 min, most preferably 40 min to 50 min, most preferably for 45 min.

[0039] In another embodiment, in step (c) the concentration of the proteinase K can be about 5 pg / ml to about 40 pg / ml, such as about 5 pg / ml to about 20pg / ml, for example about 10 pg / 1, dissolved in a buffer, for example PBS, containing about 5 to 15 mM, in particular about 10 mM of the vanadyl ribonucleoside complex.

[0040] In one embodiment, the following optional step is carried out: (d) a refixation step with formaldehyde, for with a solution of about 4 % formaldehyde, for example in PBS, can be used during about 10 to about 20 minutes at a temperature between 1°C to 25°C, preferably 2°C to 15°C, more preferably 2-8°C, most preferably at 4°C. It is pointed out that it is not required that step (d) is followed immediately after step (c). It is possible that between step (c) and (d) other method seps are carried out.

[0041] In a further embodiment, the following additional step after step (c) or (d) (if step (d) is employed) is carried out: (e) blocking step with glycine, for example wherein the concentration of glycine is about 50 to 150mM, in particular 80 to 120 mM, in particular about 100 mM. It is pointed out that it is not required that step (e) is followed immediately after step (d) or step (c) but it is possible that between step (c) or (d) and (e) other method steps are carried out. Step (e) can be carried out even if step (d) is not carried out. In particular the steps are carried out at a temperature between 2 and 8 °C.

[0042] The combination of three process steps (a), (b) and (c) leads to highly increased RNA detection in FFPE samples via the detection with gene-specific probes, for example via Spatial Transcriptomics like in situ hybridization, for example smFISH, in particular via a technique known as “Molecular Cartography”, described for example in WO 2020 / 254519 Al, WO 2021 / 255244 and WO 2021 / 255263.

[0043] Therefore, the present disclosure pertains to a method for detecting an analyte in a sample comprising i) preparing nucleic acid from formalin-fixed, paraffin-embedded tissue sections with a method for preparing nucleic acid from formalin-fixed, paraffin-embedded tissue sections according to the present disclosure; ii) detecting the analyte by spatial transcriptomics.

[0044] 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).

[0045] 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 useencoding techniques assigning a particular readable code to a specific first analyte which differs from a code assigned to a specific second analyte.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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 etal. (2016), High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization, Proc. Natl. Acad. Sci. USA. 113(39), p. 11046-11051.

[0050] 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.

[0051] EP 0611828 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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).

[0056] 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.

[0057] EP 2992115 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.

[0058] 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 included 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.

[0059] 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.

[0060] The method according to the present invention includes the fixation step (step (a) above) (for example with about 4% formaldehyde at about 4°C for about 10 minutes to about 20 minutes) directly after a deparaffinization and rehydration step of FFPE tissue sections. This step (a) is beneficial for fixation of RNA after insufficient fixation during the block preparation but also to re-fix RNA after the deparaffinization and rehydration step which might loosen the cross-linkage grid in the tissue. Furthermore, it leads to immobilization and better / flatter attachment of the tissue section to slide, increasing sensitivity during in-situ hybridization.

[0061] It has been found that performing this additional re-fixation step (a) right at the beginning of the method of the present invention is beneficial for various FFPE tissues (for example mouse FFPE tissue sections, experimentally confirmed) independent of the fixation time during the block preparation.

[0062] Step (b) of the method according to the present inventions (RNA retrieval step) can be a thermo-chemical step to retrieve the binding site / nucleotides of the cross-linked RNA for probe detection. This step can be performed in for example about 10 - about 25 mM Tris-EDTA buffer with for example a pH of about 9 for about 45 min at about 70°C. Tris-EDTA is an efficient quencher for crosslinks, due to its chemical ability to form a cyclic product upon reaction with formaldehyde. Tris-EDTA contains two nucleophiles (hydroxyl groups) that create a stable intramolecular five-membered ring after the formation of a Schiff base with formaldehyde. The propensity for Tris-EDTA to form these stable intramolecular products likely allows it to scavenge formaldehyde from other molecules and thereby facilitate crosslink reversal. This step can be carried out at about 70 °C. This step can also be performed at about 90 to about 100°C but it has been found that performing the step at lower temperatures increases the sensitivity of RNA detection in Molecular Cartography almost 3-fold.

[0063] In step (c) of the method according to the present invention, tissue is permeabilized for better probe accessibility using the enzyme Proteinase K (ProtK). This enzyme is a serine protease that hydrolyzes a variety of peptide bonds and is commonly used in the field. ProtK digestion can be performed with a concentration of about 10 pg / mL in for example PBS containing about 10 mM Vanadyl ribonucleoside complex (VRC = RNase Inhibitor) for instance during about 15 min at about 37°C. Usually, ProtK digestion is performed in Tris-EDTA buffer or PBS (concentration can vary from about 1 to about 20 pg / ml). However, it has been found a higher efficacy of the digestion in PBS-VRC buffer compared to Tris-EDTA buffer, but nevertheless Tris-EDTA as buffer sufficiently works in the method according to the present invention. Furthermore, the addition of the RNase-Inhibitor VRC to the Enzyme solution is beneficial.

[0064] The combination of the three steps (a), (b) and (c) as a preparation of FFPE tissue for in- situ hybridization yielded highly improved sensitivity in RNA detection. This combination of process steps for achieving this effect is not known from the prior art as will be evidenced by the following discussion of the prior art.

[0065] Formaldehyde fixation of RNA in in-situ hybridization techniques is commonly used. However, the time point of the re-fixation step according to the present invention is not known for FFPE tissues. Most of the literature or protocols are either suggesting no additional fixation of FFPE sections with formaldehyde (In situ hybridization (ISH) protocol | Abeam; MI-Protocol-RNAFISH-FFPETissue-Rev2.pdf (molecularinstruments.com; RNAscope2plex (mit.edu) or a formalin fixation step after enzymatic Retrieval (Fluorescence In situ Hybridization of Cells, Chromosomes, and Formalin-Fixed Paraffin-Embedded Tissues - PMC (nih.gov)). According to the method according to the present invention, there is also performed an additional re-fixation step after enzymatic retrieval. However, a highly increased sensitivity has been found when performing a short fixation step at the beginning of the protocol as well. It seems like RNA molecules are not sufficiently bound in the FFPE sections when using the prior external state processes.

[0066] Chemical / heat induced RNA retrieval (or decrosslinking) is also a step that is commonly used in protocols for RNA detection in FFPE tissues. Most common is either a boiling of the tissues sections in the microwave (MI-Protocol-RNAFISH-FFPETissue-Rev2.pdf (molecularinstruments.com)) or cooking at approximately 100 °C (RNAscope2plex (mit.edu)). There is literature where this step is performed similarly to our process step (TE buffer pH 9 at 70°C; CG000409_Demonstrated_Protocol_VisiumSpatialFFPE_Deparaffm_H_E_RevC.pdf (10xgenomics.com). However, this source does not combine the retrieval / decrosslinking step with a prior re-fixation step nor with the following enzymatic retrieval. When the retrieval step is carried out without prior re-fixation, the sensitivity was significantly reduced. It might be that RNA molecules are loosened from the formalin-grid and washed out during the harsh retrieval step at 70°C when the re-fixation is not done previously.

[0067] The last step (step (c) according to the method of the present inventions) to enable efficient RNA detection via probes is the enzymatic permeabilization of the tissue with proteinase K. This enzyme is often used in the field and the concentration can vary from 1-20 pg / ml. For efficient ProtK digestion, a buffer containing Tris-EDTA is particularly suitable and most in-situ hybridization protocols are using Tris-containing buffers for their digestion steps (In situ hybridization (ISH) protocol | Abeam). A much better permeabilization performance of ProtK has been found, resulting in higher RNA detection sensitivity, when using PBS or PBS+VRC buffer. There is also literature performing the Proteinase K digestion in PBS without the RNase-Inhibitor VRC (MI-Protocol-RNAFISH-FFPETissue-Rev2.pdf (molecularinstruments.com). However, it has been found that using RNase-inhibitor in the buffer is beneficial for certain tissues that might have a higher RNase-burden.

[0068] The combination of the three method steps (a), (b) and (c) of the method according to the present invention sufficiently fixes RNA in FFPE tissue and still makes them accessible to probes for RNA detection. A complete method starting with FFPE tissue sections to the probe detection is shown below in TABLE 1, wherein the following steps 7, 8, and 9 correspond tosteps (a), (b) and (c) of the method according to the present invention. Optional steps 10 and 11 correspond to optional steps (d) and (e) of the method according to the present invention.TABLE 1 - Method for Fixing FFPE Tissues

[0069] A “kit” is a combination of individual elements useful for carrying out the use and / or method of the disclosure, wherein the elements are optimized for use together in the methods. The kits may also contain additional reagents, chemicals, buffers, reaction vials etc. which may be useful for carrying out the method according to the disclosure. Such kits unify all essential elements required to work the method according to the disclosure, thus minimizing the risk of errors. Therefore, such kits also allow semi-skilled laboratory staff to perform the method according to the present disclosure.

[0070] 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. A quencher dye may be a fluorescent dye or non-fluore scent dye. When 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. Some embodiments of the present disclosure disclose signal oligonucleotides comprising a quencher and / or a quencher in combination with a signal element, and therefore the signal oligonucleotides is not detectable during imaging.

[0071] A “sample” as referred to herein is a composition in liquid or solid form suspected of comprising the analytes to be encoded. A sample described herein may be a biological sample. A sample may comprise biological tissue, biological cells, and / or extracts and / or part of cells, orany combination thereof. The sample may comprise eukaryotic or procaryotic cells. The sample may derive from any kind of animal (including Homo, rat, mouse, mammalia, birds, fish, insects, worms), plant, or fungi. In some embodiments, the sample may comprise a mammalian cell. In some embodiments, the sample may comprise a human cell. The sample can be selected from any organ, any tissue, any kind of culture, any specimen taken for diagnostic purposes (e.g. smear, liquid biopsy, tissue biopsy etc.). The sample may comprise DNA and / or RNA. Samples may taken from alive or dead organisms. Cells may not be complete and samples may contain only partial cells. The sample may be frozen, fixed or embedded. In some embodiments, the biological tissue, biological cells, extracts and / or part of cells are fixed. In some embodiments, the analytes are fixed in a permeabilized sample, such as a cell-containing sample.

[0072] The term “cell” as used herein is the smallest unit of life and comprises a number (for example, at least one, at least two, at least 5, at least 10, at least 20) genome elements that can be differentiated. The cell may be dead or alive. The cell may be eukaryotic or prokaryotic. The cell may not be complete and contain only a part of the cell (e.g., due to preparation or fixation of the sample or cell).

[0073] The term “genome” as used herein is a complete identity or a part of this. It can be DNA or RNA.

[0074] The term “transcription” is used herein for a process during which one strand of the genome sequence of the genome element is copied into a complementary RNA (e.g., an mRNA) strand. These single-stranded copies may be independent molecules (not covalently connected to another molecule) but may be connected for a certain time to the genome element by a non- covalently binding (e.g., by hydrogen bonding). The connection can be stabilized by a fixation method (e.g., methanol, formalin etc.).

[0075] The term “biological sample” is defined as a material that is derived from an organism and at least contains detectable nucleic acids, cells or parts of cells. These cells may originate from the same or different organs or even organisms.

[0076] The term “tissue” is used herein for any kind of a sample material that is formed by a certain number of cells of the same or different type with a meaningful structural relationship (or the lack thereof), and thus does comprise genome elements. The term “tissue section” is used herein for a thin section of a tissue favorably done by a cryotome or a microtome.

[0077] An “analyte” according to the disclosure may be any molecule (e.g., a biomolecule) of interest. Sometimes herein the term “analyte” is replaced by “target.” In some embodiments an analyte may comprise a biomolecule (e.g., a protein, a nucleic acid, a biomolecule, a lipid, or any combination thereof). In some embodiments, an analyte may be a nucleic acid (e.g., DNA, PNA, LNA, RNA, or any combination thereof). In some embodiments, an analyte may be aDNA molecule (e.g., genomic DNA, nuclear DNA, circular DNA, mitochondrial DNA, viral DNA, bacterial DNA, extra- or intracellular DNA, or any combination thereof). In some embodiments, an analyte may be an RNA molecule (e.g., mRNA, hnRNA, miRNA, viral RNA, bacterial RNA, extra- or intracellular RNA, circular mRNA, tRNA, siRNA, snRNA, rRNA, or any combination thereof). In some embodiments, an analyte may be an mRNA (e.g., a transcript). In some embodiments, there may be multiple analytes, e.g., at least two individual nucleic acids.

[0078] In some embodiments, an analyte may be a “coding sequence”, “encoding sequence”, “structural nucleotide sequence”, or “structural nucleic acid molecule” which refers to a nucleotide sequence that is translated into a polypeptide, e.g., via mRNA, when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'- terminus. A coding sequence can include, but is not limited to, genomic DNA, cDNA, EST, and recombinant nucleotide sequences.

[0079] As used in the present disclosure, “cell”, “cell line”, and “cell culture” can be used interchangeably and all such designations include progeny. Thus, the words “transformants” or “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progenies may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that has the same functionality as screened for in the originally transformed cell are included.

[0080] An “encoding scheme” may describe a set of code words that are associated with the analytes to be detected. Each code word refers to one of the analytes and can be distinguished from all other code words. A code word hereby is a sequence of signs provided by the detection cycles of the method. A sign within a code word is a detectable signal or the absence of a signal. A code word does not need to comprise of all different signals used in the method. The number of signs in a code word is defined by the number of detection cycles.

[0081] An “oligonucleotide” as used herein, refers to a nucleic acid molecule, such as DNA, PNA, LNA or RNA. The length of the oligonucleotides may be within the range of 10-10,000 nucleotides (nt), 10-15,000 nt, 10-10,000 nt, 10-5,000 nt, 10-2,000 nt, 10-1,500 nt, 10-1,000 nt, or 10-800 nt. In some embodiments, the length of oligonucleotides may be within the range of 100-1,500 nt, 100-1,200 nt, 100-1,000 nt, or 100-800 nts. In some embodiments, the length of oligonucleotides may be within the range of 400-1,500 nt, 400-1,200 nt, 400-1,000 nt, or 400- 800 nts. The nucleic acid molecule can be fully or partially single-stranded. The oligonucleotides may be linear or may comprise hairpin or loop structures. The oligonucleotidesmay comprise modifications such as biotin, labeling moieties, blocking moieties, or other modifications.

[0082] “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, e.g., perfect complementary sequences but also imperfect complementary sequences which, however, still have the capability to hybridize to each other under stringent conditions. Among experts it is well accepted that an “essentially complementary” sequence has at least 88% sequence identity to a fully or perfectly complementary sequence.

[0083] As disclosed herein, percent sequence identity (e.g., “percent identity,” “percent sequence identity,” or “sequence identity”) describes the similarity between two or more sequences (e.g., a nucleic acid sequence or an amino acid sequence). Sequence similarity calculations may be performed using the BLAST algorithm for sequence alignment, which is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ). Percent sequence identity compares a given sequence to a claimed or described sequence after alignment of the given sequence to be compared (the “Compared Sequence”) with the described or claimed sequence (the “Reference Sequence”). The percent identity is then determined according to the following formula: percent identity = 100*(l -(C / R)) wherein C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence, wherein (i) each base or amino acid in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Compared Sequence and (ii) each gap in the Reference Sequence and (iii) each aligned base or amino acid in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference, and (iv) the alignment has to start at position 1 of the aligned sequences; and R is the number of bases or amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid.

[0084] If an alignment exists between the Compared Sequence and the Reference Sequence for which the percent identity as calculated above is about equal to or greater than a specified minimum Percent Identity, then the Compared Sequence has the specified minimum percent identity to the Reference Sequence even though alignments may exist in which the herein above calculated percent identity is less than the specified percent identity.

[0085] As used herein, the term “about” in the context of a number in some cases refers to a range spanning 10% below to 10% above that number. In the context of a range, the term refers to an expanded range spanning 10% blow the listed lower limit to 10% above the listed upper limit. In cases where the number is small and cannot be subdivided, the term in some cases refers to a range spanning one unit below to one unit above the number referred to.EXAMPLES

[0086] The invention is further illustrated by the following non-limiting examples.EXAMPLE 1 - Refixation of Tissue After Deparaffinization

[0087] Fixation in formaldehyde is used to fix RNA molecules in the surrounding tissue. Performing the refixation step after deparaffinization and rehydration led to increased RNA detection sensitivity in the so-called Molecular Cartography technology. This effect was observed for different mouse tissues as shown in FIG. 2.

[0088] FIG. 2 shows the effects of a refixation step after deparaffinization on different mouse tissues (heart, brain, kidney) analyzed in Molecular Cartography. All tissues show significantly increased sensitivity, measured in transcript counts per tile, when the refixation step was performed. In all cases, a short fixation step after enzymatic permeabilization was also performed.EXAMPLE 2 - RNA-Retrieval Buffer

[0089] The refixation step probably binds RNA molecules that were previously insufficiently fixed in the tissue and prevents a wash out during the protocol steps. Furthermore, it might be that the cross-linkage grid generated during the block preparation is already loosened due to the deparaffinization and rehydration step. Therefore, a significantly higher number of RNA molecules can be detected via Molecular Cartography if the refixation step after the deparaffinization is added.

[0090] The refixation step is followed by a thermo-chemical RNA retrieval step. It is used to partly remove the formaldehyde cross-links from the RNA nucleotides to make them accessible for probe detection. Even though it seems illogical to remove crosslinks that were partly formed in the previous step, highly increased sensitivity has been found when combining the steps. For this step, Tris-EDTA buffer (pH 9) is used for 45 min at 70°C. Tris-EDTA has the ability to bind formaldehyde and is known as an efficient quencher for crosslinks. Different buffers for RNA retrieval have been tested and found Tris-EDTA to be the most efficient, as shown in FIG. 3.

[0091] FIG. 3 shows the testing of different buffers for RNA retrieval. Usage of Tris EDTA buffer at pH 9 resulted in the highest RNA detection rate, shown in transcript “counts per tile”.EXAMPLE 3 - RNA-Retrieval Temperature

[0092] RNA retrieval steps for FFPE sections using Tris-EDTA buffer are often found in literature. However, mostly this step was performed at much higher temperatures (e.g., boiling in microwave, or cooking at 100°C in buffer. Contrary, it has been found that using a higher temperature decreases the amount of detected RNA significantly, as shown in FIG. 4.

[0093] FIG. 4 shows the testing of different temperatures for RNA retrieval. Performing the retrieval step at 70°C resulted in much more efficient RNA detection compared to 90°C which is mostly suggested in literature.EXAMPLE 4 - Enzymatic Permeabilization Conditions

[0094] To make the FFPE tissue better accessible for RNA-detecting probes, an enzymatic permeabilization is performed after the RNA retrieval step. Proteinase K hydrolyzes peptide bonds and therefore permeabilizes the tissue. Proteinase K digestion is performed for 15 min at 37°C using concentrations of 10-15 pg / ml. Usage of Proteinase K after RNA retrieval resulted in a higher sensitivity as shown in FIG. 5. Furthermore, the selection of the buffer in which the digestion was performed had a significant effect on the results. Performing the enzymatic permeabilization in PBS-containing buffer seems to be more efficient compared to Tris-EDTA buffer which is commonly used for Proteinase K digestion in literature (FIG. 5).

[0095] FIG. 5 shows the enzymatic permeabilization using Proteinase K in different buffers. The graph shows that the usage of Proteinase K is needed for proper RNA detection since the control without Proteinase K resulted in the worst detection rate. Furthermore, the digestion seems to be more efficient in PBS containing buffers compared to Tris-EDTA buffer, the latter of which is mostly suggested in literature.EXAMPLE 5 - Fixation of Mouse Heart Samples

[0096] This example describes the fixation of mouse heart tissue samples with variable fixation times to evaluate the effect of fixation time on signal sensitivity of a spatial transcriptomic assay. Tissue sections from four mouse heart samples (FFPE) were immobilized on glass slides and deparaffinization was performed. After deparaffinization a 4% Formaldehyde solution was used to fix the samples for the indicated times at 4°C. Thereafter, the standard Molecular Cartography (e.g., a spatial transcriptomics assay) sample prep protocol was used to hybridize probes for the transcripts of 45 genes. After probe hybridization and specific washes, the slide was used for the analysis in the Molecular Cartography instrument. Thereafter, signals were decoded and counted. FIG. 6 shows a bar graph displaying the count per tile (“counts / tile”) for each of the fixation times of 0 minutes, 5 minutes, 10 minutes, and 15 minutes across 4 differentmouse heart tissue samples. As shown in FIG. 6 and TABLE 2, the longer fixation times increased the sensitivity of the spatial transcriptomics assay by increasing the counts per tile.TABLE 2 - Counts Per Tile in Variable Fixation Duration TiniesEXAMPLE 6 - Fixation of Mouse Heart Samples

[0097] This example describes the fixation of mouse heart tissue samples with fixation after an RNA retrieval step and fixation before and after an RNA retrieval step on signal sensitivity of a spatial transcriptomic assay. Tissue sections from two mouse heart samples (FFPE) were immobilized on glass slides and deparaffinization was performed. After deparaffinization either i) a 4% Formaldehyde solution was used to fix the samples for 10 minutes at 4°C after the RNA retireval step (Fixation after RNA retrieval) or ii) a 4% Formaldehyde solution was used to fix the samples for 10 minutes at 4°C before and after the RNA retireval step (Fixation before and after RNA retrieval). Thereafter, the standard Molecular Cartography (e.g., a spatial transcriptomics assay) sample prep protocol was used to hybridize probes for the transcripts of 45 genes. After probe hybridization and specific washes, the slide was used for the analysis in the Molecular Cartography instrument. Thereafter, signals were decoded and counted. FIG. 7 shows a bar graph displaying the count per tile (“counts / tile”) for each of the mouse FFPE heart samples (Sample 1 and Sample 2) with each of the conditions, “Fixation after RNA retrieval” and “Fixation before and after RNA retrieval”. As shown in FIG. 7 and TABLE 3, the additional 10 min fixation time prior to RNA retrival (Fixation before and after RNA retrieval) helps to increase the sensitivity in mouse heart FFPE samples.TABLE 3 - Counts Per Tile With / Without Additional Fixation Before RNA RetrievalEXAMPLE 7 - Fixation of Mouse Liver Samples

[0098] This example describes the fixation of mouse liver tissue samples with fixation before, after, or before and after an RNA retrieval step on signal sensitivity of a spatial transcriptomic assay. Tissue sections from two mouse liver samples (FFPE) were immobilized on glas slides and deparaffinization was performed. After deparaffinization a 4% Formaldehyde solution wasused to fix the samples for 10 minutes at 4°C i) before the RNA retrieval step (Fixation before RNA retrieval), ii) after the RNA retireval step (Fixation after RNA retrieval), or iii) before and after the RNA retireval step (Fixation before and after RNA retrieval). The samples were also compared to a treatment of fixation with formaldehyde for 72 hours prior to paraffin embedding that was also treated by a 4% Formaldehyde solution was used to fix the samples for 10 minutes at 4°C after enzymatic permeabilization (72 hours pre, after Prot K) as a control sample. Thereafter, the standard Molecular Cartography (e.g., a spatial transcriptomics assay) sample prep protocol was used to hybridize probes for the transcripts of 45 genes. After probe hybridization and specific washes, the slide was used for the analysis in the Molecular Cartography instrument. Thereafter, signals were decoded and counted. FIG. 8 shows a bar graph displaying the count per tile (“counts / tile”) for each of the fixation conditions, “Fixation before RNA retrieval”, “Fixation after RNA retrieval”, and “Fixation before and after RNA retrieval” for each of the mouse liver samples (Sample 1 and Sample 2) and the average. As shown in FIG. 8 and TABLE 4, the additional 10 min fixation time prior to RNA retrival (Fixation before RNA retrieval and Fixation before and after RNA retrieval) helps to increase the sensitivity in mouse liver FFPE samples.TABLE 4 - Counts Per Tile With / Without Additional Fixation Before / After RNA RetrievalEXAMPLE 8 - Fixation of Mouse Brain and Kidney Samples

[0099] This example describes the fixation of mouse brain and kidney tissue samples with and without an RNA retrieval step on signal sensitivity of a spatial transcriptomic assay. Tissue sections from mouse brain and kidney samples (FFPE) were immobilized on glas slides and deparaffinization was performed. After deparaffinization samples were optionally treated with a 4% Formaldehyde solution used to re-fix the samples for 10 minutes at 4°C (+ re-fixation). Thereafter, the standard Molecular Cartography (e.g., a spatial transcriptomics assay) sample prep protocol was used to hybridize probes for the transcripts of 45 genes. After probe hybridization and specific washes, the slide was used for the analysis in the Molecular Cartography instrument. Thereafter, signals were decoded and counted. FIG. 9 shows a bar graph displaying the count per tile (“counts / tile”) for each of the tissue samples and fixation conditions, brain without re-fixation (“Brain (- re-fixation)”), brain with re-fixation (“Brain (+re-fixation)”), kidney without re-fixation (“Kidney (- re-fixation)”), and kidney with re-fixation (“Kidney (+ re-fixation)”). As shown in FIG. 9 and TABLE 5, the additional 10 min re-fixation helps to increase the sensitivity in mouse brain and kidney FFPE samples.TABLE 5 - Counts Per Tile With / Without Additional Re-Fixation

[0100] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for preparing nucleic acid from formalin-fixed, paraffin-embedded tissue sections, comprising the following steps: a) a fixation step of nucleic acid, which is obtained from formalin-fixed, paraffin- embedded tissue sections after deparaffinization and rehydration, with formaldehyde;(b) a nucleic acid retrieval step in a buffer; and(c) a permeabilization step using proteinase K and a vanadyl ribonucleoside complex.

2. The method according to claim 1, wherein the fixation step (a) is carried out with an about 3 % to about 5 % solution of formaldehyde during about 5 to about 30 minutes at a temperature between 1°C to 25°C, preferably 2°C to 15°C, more preferably 2-8°C, most preferably at 4°C.

3. The method according to claim 1 or claim 2, wherein the buffer in step (b) is Tris-EDTA buffer having a pH of about 8 to 10, more preferably 8.5 to 9.5, most preferably 8.8 to 9.2, in particular having a pH of about 9.

4. The method according to any one of the preceding claims, wherein the step (b) is carried out at least about 70 °C during between 30 min to 60 min, more preferably 35 min to 55 min, most preferably 40 min to 50 min, most preferably for 45 min.

5. The method according to any one of the preceding claims, wherein a concentration of the proteinase K is about 5 pg / ml to about 40 pg / ml, such as about 5 pg / ml to about 20pg / ml, in particular about 10 pg / 1, dissolved in a buffer containing about 5 to 15 mM, in particular 10 mM of the vanadyl ribonucleoside complex.

6. The method according to any one of the preceding claims, wherein the following additional step is carried out:(d) a refixation step with formaldehyde.

7. The method according to claim 6, wherein step (d) is carried out with a solution of about 4 % formaldehyde during about 10 to about 20 minutes at about 4 °C.

8. The method according to claim 6 or 7, wherein the following additional step after step (d) is carried out:(e) blocking step with glycine.

9. The method according to claim 8, wherein step (d) is carried out with the concentration of glycine at about 100 mM or 200 mM during about 10 to about 20 minutes at about 20-25 °C.

10. A method of preparing a fixed sample for hybridization assay, the method comprising: refixing the sample, retrieving nucleic acid accessibility in the sample, and permeabilizing the sample.

11. The method of claim 10, wherein the sample is subjected to at least one step selected from the list of drying, baking, deparaffinization, and rehydration; wherein the at least one step is performed prior to the refixing.

12. The method of claim 10, wherein the refixing comprises contacting the sample to formaldehyde.

13. The method of claim 12, wherein the formaldehyde is in a PBS solution.

14. The method of claim 12 or claim 13, wherein the formaldehyde is at a concentration of 2%-6%.

15. The method of claim 12 or claim 13, wherein the formaldehyde is at a concentration of16. The method of claim 12 or claim 13, wherein the formaldehyde is at a concentration about 4%.

17. The method of claim 12, wherein the contacting the sample to formaldehyde occurs at below room temperature.

18. The method of claim 17, wherein the temperature is no greater than 15C.

19. The method of claim 17, wherein the temperature is no greater than 10C.

20. The method of claim 17, wherein the temperature is no greater than 4C.

21. The method of claim 12, wherein the refixing occurs over a duration of at least 1 minute.

22. The method of claim 12, wherein the refixing occurs over a duration of at least 5 minutes.

23. The method of claim 12, wherein the refixing occurs over a duration of at least 10 minutes.

24. The method of claim 12, wherein the refixing occurs over a duration of no more than 60 minutes.

25. The method of claim 12, wherein the refixing occurs over a duration of no more than 30 minutes.

26. The method of claim 12, wherein the refixing occurs over a duration of no more than 20 minutes.

27. The method of claim 10, wherein the retrieving nucleic acid accessibility comprises chemically scavenging fixative molecules from the sample.

28. The method of claim 27, wherein the fixative molecules comprise formaldehyde molecules.

29. The method of claim 28, wherein scavenging formaldehyde molecules from the sample comprises forming a Schiff base comprising formaldehyde.

30. The method of claim 27, wherein chemically scavenging fixative molecules from the sample comprises contacting the sample to a nucleophilic buffer.

31. The method of claim 30, wherein the nucleophilic buffer comprises a hydroxyl group.

32. The method of claim 30, wherein the nucleophilic buffer comprises Tris-EDTA.

33. The method of claim 32, wherein the Tris-EDTA is administered at a concentration of at least ImM.

34. The method of claim 32, wherein the Tris-EDTA is administered at a concentration of at least 5mM.

35. The method of claim 32, wherein the Tris-EDTA is administered at a concentration of at least lOmM.

36. The method of claim 32, wherein the Tris-EDTA is administered at a concentration of no more than 50mM.

37. The method of claim 32, wherein the Tris-EDTA is administered at a concentration of no more than 25 mM.

38. The method of claim 32, wherein the Tris-EDTA is administered at a pH of at least 7.

39. The method of claim 32, wherein the Tris-EDTA is administered at a pH of at least 8.

40. The method of claim 32, wherein the Tris-EDTA is administered at a pH of at least 9.

41. The method of claim 32, wherein the Tris-EDTA is administered at a pH of no more than11.

42. The method of claim 32, wherein the Tris-EDTA is administered at a pH of no more than 10.

43. The method of claim 32, wherein the Tris-EDTA is administered at a pH of no more than 9.

44. The method of claim 32, wherein the Tris-EDTA is administered for at least 10 minutes.

45. The method of claim 32, wherein the Tris-EDTA is administered for at least 30 minutes.

46. The method of claim 32, wherein the Tris-EDTA is administered for at least 45 minutes.

47. The method of claim 32, wherein the Tris-EDTA is administered for no more than 120 minutes.

48. The method of claim 32, wherein the Tris-EDTA is administered for no more than 60 minutes.

49. The method of claim 32, wherein the Tris-EDTA is administered for 45 minutes.

50. The method of claim 32, wherein the Tris-EDTA is administered at a temperature of no more than 100C.

51. The method of claim 32, wherein the Tris-EDTA is administered at a temperature of no more than 90C.

52. The method of claim 32, wherein the Tris-EDTA is administered at a temperature of no more than 80C.

53. The method of claim 32, wherein the Tris-EDTA is administered at a temperature of no more than 70C.

54. The method of claim 32, wherein the Tris-EDTA is administered at a temperature of at least 45C.

55. The method of claim 32, wherein the Tris-EDTA is administered at a temperature of at least 55C.

56. The method of claim 32, wherein the Tris-EDTA is administered at a temperature of at least 65C.

57. The method of claim 32, wherein the Tris-EDTA is administered at a temperature of at least 70C.

58. The method of claim 10, wherein permeabilizing the sample comprises enzymatic digestion.

59. The method of claim 58, wherein permeabilizing the sample comprises protease treatment.

60. The method of claim 58, wherein permeabilizing the sample comprises a serine protease treatment.

61. The method of claim 58, wherein permeabilizing the sample comprises trypsin treatment.

62. The method of claim 58, wherein permeabilizing the sample comprises bromelain treatment.

63. The method of claim 58, wherein permeabilizing the sample comprises chymotrypsin treatment.

64. The method of claim 58, wherein permeabilizing the sample comprises ficin treatment.

65. The method of claim 58, wherein permeabilizing the sample comprises papain treatment.

66. The method of claim 58, wherein permeabilizing the sample comprises serrapeptase treatment.

67. The method of claim 58, wherein permeabilizing the sample comprises proteinase K treatment.

68. The method of any one of claims 58 to 67, wherein permeabilizing the sample comprises contacting the sample to a nuclease inhibitor.

69. The method of any one of claims 58 to 67, wherein permeabilizing the sample comprises contacting the sample to an RNase inhibitor.

70. The method of any one of claims 58 to 67, wherein permeabilizing the sample comprises contacting the sample to Vanadyl ribonucleoside complex.

71. The method of any one of claims 58 to 67, wherein permeabilizing the sample comprises contacting the sample to RNase inhibitor Hu.

72. The method of any one of claims 58 to 67, wherein permeabilizing the sample comprises contacting the sample to placental RNase inhibitor.

73. The method of any one of claims 68 - 72, wherein the RNase is present at a concentration of at least lug / mL.

74. The method of any one of claims 68 - 72, wherein the RNase is present at a concentration of at least 5ug / mL.

75. The method of any one of claims 68 - 72, wherein the RNase is present at a concentration of at least lOug / mL.

76. The method of any one of claims 68 - 72, wherein the RNase is present at a concentration of at most lOOug / mL.

77. The method of any one of claims 68 - 72, wherein the RNase is present at a concentration of at most 50ug / mL.

78. The method of any one of claim 10 to claim 77, comprising a subsequent second refixing of the sample.

79. The method of any one of claim 10 to claim 77, comprising a subsequent blocking step.

80. A method of preparing a fixed sample for hybridization assay, the method comprising: refixing the sample.

81. A method of preparing a fixed sample for hybridization assay, the method comprising: retrieving nucleic acid accessibility in the sample.

82. A method of preparing a fixed sample for hybridization assay, the method comprising: permeabilizing the sample.

3. A method for detecting an analyte in a sample comprising i) preparing nucleic acid from formalin-fixed, paraffin-embedded tissue sections with a method of any one of claim 1 to claim 82; ii) detecting the analyte by spatial transcriptomics.

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