WO2025099161A2 - Stockage à sec - Google Patents

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Publication number
WO2025099161A2
WO2025099161A2 PCT/EP2024/081517 EP2024081517W WO2025099161A2 WO 2025099161 A2 WO2025099161 A2 WO 2025099161A2 EP 2024081517 W EP2024081517 W EP 2024081517W WO 2025099161 A2 WO2025099161 A2 WO 2025099161A2
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rna
storage device
absorbent material
biological samples
impregnated
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WO2025099161A3 (fr
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Björn REINIUS
Antonio Lentini
Joyce NOBLE
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Sequrna AB
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Sequrna AB
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/99—Enzyme inactivation by chemical treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
    • C12Q2521/327—RNAse, e.g. RNAseH
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/125—Specific component of sample, medium or buffer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/127—Reactions demanding special reaction conditions the enzyme inhibitor or activator used

Definitions

  • the present invention relates to devices and methods for effective storage of biological samples, hereunder dry storage of RIMA samples, preventing RNA degradation.
  • nucleic acids rely on polymeric nucleic acids. Yet during their storage and use, nucleic acids encounter nucleases that degrade nucleic acid. For example, human skin is an abundant source of nucleases that can be transferred accidentally to surfaces and solutions, and biological samples analysed for nucleic acid content are themselves generally a source of them.
  • a ribonuclease (commonly abbreviated RNase), is a type of nuclease that catalyzes the degradation of RNA into smaller components.
  • RNA messenger RNA
  • mRNA serves as the critical conduit for genetic information, carrying the information that is ultimately utilized from protein synthesis by the cell.
  • the mRNA profiles of biological specimens be they tissue, liquid biopsies, or cell cultures, contain vital information reflective of the specimens' conditions, as do profiles of other types of RNA.
  • extracted RNA can be subjected to RNA-sequencing library preparation followed by high-throughput sequencing, allowing efficient and global characterization of the complex RNA profile contained in a biological sample.
  • RNA is notoriously labile and prone to degradation by ubiquitous ribonucleases (RNases) present in the environment and within biological samples themselves.
  • RNases ubiquitous ribonucleases
  • RNA degradation needs to be prevented during sample collection, storage, and in the following RIMA detection procedure.
  • the samples are preserved in liquid form through cryopreservation, typically requiring storage in specialized freezers at -80°C.
  • Ribonuclease inhibitor is a large 50 kDa protein present in the cytosol of mammalian cells. RI forms extremely tight complexes with certain RNases and controls the activity of RNases. Inhibitors of ribonuclease are useful in a variety of molecular biology applications where RNase contamination is a potential problem. Examples of these applications include mRNA isolation and purification, storage, reverse transcription of mRNA, RNA Sequencing (RNA-seq), and in situ RNA- sequencing.
  • RNA-seq RNA Sequencing
  • RNA-seq RNA Sequencing
  • DEPC and other similar chemicals are known carcinogens and require caution and training for their use. These chemicals also react quite readily with amine, thiol, and alcohol groups so some solutions (e.g., primary amine containing compounds such as Tris) cannot be treated with DEPC at all. Finally, DEPC must be inactivated by autoclaving post-treatment, but DEPC residues may still interfere with downstream enzymatic reactions such as reverse transcription (RT) and polymerase chain reaction (PCR).
  • RT reverse transcription
  • PCR polymerase chain reaction
  • RNA-seq RNA-sequencing
  • in vitro synthesized biological RNase inhibitors i.e. recombinant RI proteins
  • recombinant RI proteins RI proteins
  • the use of recombinant inhibitors is inconvenient due to its relatively high cost fraction to the library, but also due to its degradability; which may introduce batch variation in library yield and quality due to production lot, storage time, and temperature conditions for the inhibitor.
  • ribonuclease inhibitors for applications which require capture of intact RIMA. If thermostable RNase inhibitors could be identified, this would enable new and simplified workflows, and may increase reproducibility and throughput of RNA-seq applications.
  • ribonuclease inhibitors must not only be capable of preserving cellular RNA in the lysis buffer but should be fully compatible with each of the following library preparation steps that are universal for all RNA analysis of relevance, including reverse transcription and amplification by PCR, without introducing base errors or reducing sensitivity to detect RNA species in contained in the analysed sample material.
  • Protein-based RIs are considered specific for RNase whereas chemicals with RNase inhibitory do in general also affect or inhibit other enzymes which are critical in the molecular biology application, such as reverse transcriptase and DNA polymerase.
  • the inventors have found that certain chemicals are suitable for use in applications where inhibition of RNase activity is desirable such that certain specific concentrations do not negatively affect other enzymes.
  • RNA-seq Although there are potentially many chemical substances or treatments that may in principle inhibit RNase activity, these are generally expected to also negatively affect RNA-seq library yield as well as the quality and error rate in the final sequencing library. Thus, from a chemical compound being a potent RNase inhibitor it does not follow that the agent is also suitable RNase inhibitor in RNA-seq.
  • the present invention relates to a method of using chemical RNase inhibitors alone or in combinations inhibitors in devices for storage of biological samples comprising RNA as well as to such devices.
  • the devices and methods may be used in applications where inhibition of RNase activity is desired, including in RNA sequencing and storing of biological samples before such sequencing.
  • the present invention provides devices for dry storage of biological RNA samples, conserving RNA from degradation.
  • fibrous or synthetic sheet materials are pre-treated with sodium alginate (NaAIg), heparin, fucoidan, vinyl sulfonic acid (VSA), polyvinyl sulfonic acid (PVSA), 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid (HEPES), and/or dextran sulfate (DexSulf), which the inventors demonstrate prevents degradation of RNA in dry-storage devices.
  • NaAIg sodium alginate
  • VSA vinyl sulfonic acid
  • PVSA polyvinyl sulfonic acid
  • HPES 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid
  • DexSulf dextran sulfate
  • thermostability of chemical RNase inhibition means it need not be supplemented twice during RNA collection and cDNA library preparation (i.e. in the cell lysis or collection step and in the reverse transcription step), which enables new and simplified workflows, increasing reproducibility and throughput of RNA-seq.
  • stable premade sample collection buffers can be made, frozen, thawed, and kept at room-temperature for extended periods of time, or even subjected to high-temperature conditions before use.
  • PVSA polyfvinylsulfonic acid
  • an absorbent sheet material is pre-treated with one of the following agents: polyvinyl sulfonic acid (PVSA), vinyl sulfonic acid (VSA), sodium alginate (NaAIg), dextran sulfate (DexSulf), fucoidan, heparin, and/or 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic acid (HEPES), which the inventors demonstrate prevents degradation of RIMA in the storage device.
  • PVSA polyvinyl sulfonic acid
  • VSA vinyl sulfonic acid
  • NaAIg sodium alginate
  • DexSulf dextran sulfate
  • fucoidan fucoidan
  • heparin and/or 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic acid (HEPES)
  • RNA-storage methods such as cryopreservation or storage of RNA in bone-dry crystalline form.
  • the ability to store RNA in a dry, stabilized state, at ambient temperatures without compromising its integrity provides a multiple advantage in fields of molecular biology, biomedicine, and biotechnology.
  • a sample optionally in the form of a lysate may be applied to a storage device according to the invention without the need for additional steps such as preparing storage buffers and/or cooling or freezing the collected sample or samples.
  • Cost-Effective Storage Eliminating the need for ultra-low temperature storage equipment reduces energy consumption and operational costs.
  • the present invention obviates the need for continuous power supply and expensive refrigerants, making RNA storage more cost-effective and sustainable.
  • Dry storage vastly simplifies the handling and transportation of RNA samples. Samples can be shipped at ambient temperatures without the risk of thawing, which is a critical vulnerability of cryopreserved samples during transit.
  • Rapid Sample Preparation Dry storage potentially allows for more rapid reconstitution and preparation of RNA samples for downstream applications, as the rewarming and thawing steps inherent to cryopreservation are circumvented.
  • the dry storage method does not require the handling of hazardous materials such as liquid nitrogen, enhancing safety in laboratory settings. It also facilitates wider accessibility to RNA storage and transportation, including in resource-limited settings that lack specialized cryopreservation facilities.
  • the present invention minimizes the risk of sample crosscontamination that can occur with liquid storage systems.
  • Time range for storage The dry storage device and method provides for storage at ambient temparature and below for extended periods while maintaining RNA quality.
  • samples may be stored for 1, 2, 3, 4, 5, 6, 7 days, or more, such as ten days, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, a year, or two years, with an acceptable quality and yield.
  • the described invention fulfills the need for a robust method of RNA sample preservation that is practical, economical, and conducive to the widespread storage and distribution of RNA samples. It leverages the stabilization of RNA in a desiccated state achieved by chemical compounds which the inventors demonstrate to prevent RNA degradation, significantly improving upon the limitations associated with cryopreservation.
  • the benefits of this invention are not limited to the preservation of integrity alone but encompass logistical, economic, and environmental aspects, thereby representing a transformative approach to RIMA sample management.
  • the described innovation has multiple potential usage areas, e.g., in biological and medical research, as well as in clinical applications, in which biological samples are to be collected and stored until the RNA of the sample is analyzed.
  • analyses include, but are not limited to, characterizing the transcriptional state of the biological sample or the detection of specific RNA sequences within the sample, such as a molecular marker molecule, bacterial, or viral RNA.
  • a filter may capture incompletely lysed cell debris (e.g., mesh filter in spin-down column or compression syringe), as alternative to the cell debris removal by centrifugation.
  • polymer we include the meaning of any of a class of natural or synthetic substances that are multiples of simpler chemical units called monomers.
  • the polymer is a non-protein polymer.
  • the polymer is a non-biological polymer.
  • non- biolog ical we include the meaning of a molecule or agent not normally found in a biological system.
  • such polymers are vinyl polymers.
  • the relevant monomers i.e. sulfonated and/or carboxylated monomers, as described herein
  • vinyl polymers we include the meaning of products from the polymerization of vinyl monomers.
  • vinyl monomers we include the meaning of monomers containing vinyl groups, i.e. small molecules containing carbon-carbon double bonds.
  • Salt forms of any of the monomers, polymers and polysaccharides described herein may also be used in the methods of the present invention. Any salt form used should not comprise a cation which inhibits any part of the method of the invention, such as the PCR. reaction. Salts that may be used include acid addition salts and base addition salts. Examples of addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, potassium or calcium. In an embodiment, the salt form is a sodium salt.
  • the counter ion may be exchanged to another counter ion. Indeed, it is common knowledge in chemistry that a functional charged molecule can be paired with various counter ions.
  • sodium alginate (NaAIg) having sodium (Na + ) as counter ion could be replaced by potassium alginate (KAIg) having potassium (K + ) as counter ion.
  • sulfonated polymer we include the meaning of a repeated chain of molecules wherein a sulfonate residue appears at least once per unit in the chain.
  • the sulfonated polymer comprises one sulfonate group per unit.
  • carboxylated polymer we include the meaning of a repeated chain of molecules wherein a carboxylate residue appears at least once per unit in the chain.
  • the carboxylated polymer comprises one carboxyl group per unit.
  • sulfonated monomer we include the meaning of a compound that is non-repeating and that contains at least one sulfonate residue.
  • carboxylated monomer we include the meaning of a compound that is non-repeating and that contains at least one carboxylated residue.
  • polysaccharide we include the meaning of polymeric carbohydrates composed of repeating units, e.g. monosaccharides or disaccharides, linked together by glycosidic bonds. Polysaccharide compounds such as glycosaminoglycans are also included. Polysaccharides are known in the art and include but are not limited to, cellulose, amylose, dextran, and heparin. Native heparin has a molecular weight ranging from 3 to 30 kDa, although the average molecular weight of most commercial heparin preparations is in the range of 12 to 15 kDa. Dextrans are available in multiple molecular weights ranging from 3 kDa to 2 MDa. The molecular weight of amylose varies between several thousand and one-half million daltons with a degree of polymerization of 1000-10,000 glucose units.
  • the polysaccharide comprises one or more acidic group.
  • the functionalised polysaccharide is a sulfated and/or carboxylated polysaccharide.
  • sulfated polysaccharide we include the meaning of a chain of repeating units linked together by glycosidic bonds wherein a sulfate residue appears at least once per unit in the chain.
  • the repeating unit may be a monosaccharide or a disaccharide.
  • the sulfated polysaccharide comprises one sulfate group per monosaccharide.
  • carboxylated polysaccharide we include the meaning of a chain of repeating units linked together by glycosidic bonds wherein a carboxylate residue appears at least once per unit in the chain.
  • the repeating unit may be a monosaccharide or a disaccharide.
  • the carboxylated polysaccharide comprises one carboxyl group per monosaccharide.
  • non-ionic detergent surfactants containing no charged group, we include but are not limited to Triton X-100, nonyl phenoxypolyethoxylethanol (NP)-40, Tween-20, Tween-80, digitonin.
  • ionic detergent By ionic detergent, detergents have a hydrophilic head group that is charged and can be either negatively (anionic) or positively (cationic) charged, we include but are not limited to sodium dodecyl sulfate (SDS), sarkosyl, sodium deoxycholate.
  • SDS sodium dodecyl sulfate
  • sarkosyl sodium deoxycholate
  • zwitter-ionic detergent we include 3-((3-cholamidopropyl) dimethylammonio)-l -propanesulfonate (CHAPS).
  • Chaotropic agents that have the ability to disrupt hydrogen bonding and other non-covalent interactions between molecules, such as guanidinium thiocyanate, sodium iodide, and guanidinium hydrochloride, may also act as lysis agent and may replace detergent.
  • Triton X-100 we include Triton X-100 (C14H22O(C2H4O)n) is a nonionic surfactant that has a hydrophilic polyethylene oxide chain (on average it has 9.5 ethylene oxide units) and an aromatic hydrocarbon lipophilic or hydrophobic group.
  • the hydrocarbon group is a 4-(l, 1,3,3- tetramethylbutyl)-phenyl group.
  • the agent is selected from the group consisting of: sodium alginate, dextran sulfate, polyvinyl sulfonic acid, vinyl sulfonic acid, heparin, fucoidan, HEPES.
  • the agent inhibits RNase.
  • inhibit in the context of the activity of Rnase, we include the meaning that the activity of at least one RNase is reduced in a sample to which an agent of the invention is added, compared to the activity in an analogous sample to which the agent is not added. Inhibition is not limited to complete inhibition or inactivation of a given RNase. In a given application, it may be that some low level of RNase activity can be tolerated that will not have a detrimental effect on the outcome of the reaction, purification and/or assay being performed (in this case, preparation of a cDNA sequencing library).
  • the agent inhibits the activity of an RNase by at least 10%, such as at least 20%, 30%, 40% or 50% compared to the activity in an analogous sample to which the agent is not added. In an embodiment, the agent inhibits the activity of an RNase by at least 50%, such as at least 60%, 70%, 80% or 90%, such as by 95% compared to the activity in an analogous sample to which the agent is not added. It will be appreciated that the extent to which the polymer inhibits the activity of RNase depends on the concentration of the polymer, the RNase concentration and the conditions of the reaction.
  • Substantial inhibition is achieved when the RNase activity in a sample is below the level that is tolerable in a given application (i.e., the preparation of a cDNA sequencing library, or other applications where inhibition of RNase activity is desired). The level of inhibition that is substantial will then depend upon the application in which the inhibitory agents are employed. In contrast, the term inactivation is used when there is no detectable level of activity of a given RNase. An RNase that is inactivated need not be rendered irreversibly inoperative. Agents of this invention may exhibit inhibition of certain RNases and inactivation of other RNases.
  • RNases examples include eukaryotic RNases (e.g., mammalian RNases or fungal RNases) and prokaryotic RNases.
  • exemplary RNases include RNase A, RNase B, RNase C, RNase 1, RNase Tl, and bacterial RNase (e.g., those of E. coll) .
  • the agent reduces and/or prevents RNA degradation.
  • reduced and/or prevents RNA degradation we include the meaning that the degradation of RNA is reduced in a sample to which an agent of the invention is added, compared to the degradation of RNA in an analogous sample to which the agent is not added.
  • the agent reduces the degradation of RNA by at least 10%, such as at least 20%, 30%, 40% or 50% compared to the degradation of RNA in an analogous sample to which the agent is not added.
  • the agent reduces the degradation of RNA by at least 50%, such as at least 60%, 70%, 80% or 90%, such as by 95% compared to the degradation of RNA in an analogous sample to which the agent is not added.
  • agents of the invention can be used alone or in combination with other agents of the invention in the methods described herein, such as in a device comprising an absorbent material impregnated with one or more of such agents.
  • the RNase is selected from the group comprising
  • the agent i.e. RNase inhibitor
  • the agent does not inhibit or affect fidelity or processivity of modifying enzymes like reverse transcriptases, DNA polymerases, and/or transposases under the given reaction conditions.
  • RNA in a stored sample can be inspected by generating a full-length cDNA library using reverse transcription and PCR amplification of the full-length cDNA (e.g., using Smart-seq2 (Picelli 2023, Picelli 2014)). Yield of cDNA and cDNA size distribution of the samples can be inspected using gel electrophoresis, e.g., quantitatively using Agilent Bioanalyzer 2100 High sensitivity DNA chips.
  • full-length cDNA traces is known in the field to reflect the quality and integrity of the underlying mRNA sample (Trombetta 2014), and an intact library (satisfactory library) is expected to have a peak around ⁇ 2kb, reflecting the median length of full-length mRNA transcripts in human cells. Exact patterns of spikes in the cDNA traces can further be experiment- or cell-type specific, as abundant cell-type-specific or condition-specific RNA transcripts, for which one or a few transcripts account for a large proportion of total cellular transcripts lead to additional peaks on the Bioanalyzer trace.
  • Biological medium we include the meaning of any liquid in which a biological reaction or assay can be carried out or performed during the preparation of a cDNA library, which might be detrimentally affected by the presence of one or more active RNases.
  • biological medium includes any buffers (e.g. storage and lysis buffers) and reagents employed in the preparation of a cDNA sequencing library.
  • the inhibitory agents described herein can, for example, be added along with reagents (e.g., prior to, or simultaneous with reagents) to inactivate or inhibit RNases that might be present in a reaction mixtures.
  • the inhibitory agents can be bound to the internal and/or external surfaces (e.g., glass, plastic, or fiber material) of absorbent material.
  • the inhibitory agents can be bound in a material such as a membrane, for example a cotton or paper sheet pre-soaked in the RNase inhibitory agent, onto which a biological sample is added for storage and subsequent elusion and processing into an RNA-sequencing library.
  • agent-coated surfaces can be achieved using an in situ polymerization method or by incubating material or surfaces with the inhibitory agent, such as in an aqueous solution comprising the agent or agents.
  • the inhibitory agent such as in an aqueous solution comprising the agent or agents.
  • the method includes the use of an agent
  • the agent may already be included in a buffer that is used in the method or the agent may be added to one of the buffers used in the method before the method is carried out.
  • the agent may be present in a reaction vessel (such as a multi-well plate) prior to be method being carried out.
  • the method includes the addition of the agent.
  • the method includes the use and/or addition of an effective amount of the agent.
  • an agent we include the meaning of the amount of an agent or the combined amount of a mixture of agents which is used in or added to a biological medium containing one or more RNases to observe inhibition (as defined above) of at least one of the one or more RNases, whilst not substantially interfering with the biological reactions necessary for the generation of a cDNA sequencing library (e.g. first strand synthesis reaction and/or subsequent PCR reactions).
  • not substantially interfering we include that addition of the agent does not negatively affect the biochemical reactions necessary for the generation of a cDNA sequencing library (e.g. first strand synthesis reaction and/or subsequent PCR reactions) such that the final yield of cDNA is not substantially decreased and that original RIMA molecules are accurately recorded in the resulting sequencing library.
  • RNA purification may include e.g. RNA precipitation or purification that removes some or all of the one or more RNA inhibitors.
  • agents of this invention that are inhibitory toward a given RNase or mixture of RNases or which render one or more RNases inactive can be readily determined by one of ordinary skill in the art without undue experimentation in view of the teachings herein and in view of what is generally known in the art.
  • the purity and/or yield of RNA and cDNA retrieved in the presence of the agent can be measured using a spectrophotometer, fluorometer or Bioanalyzer/Fragment Analyzer and compared to the purity and/or yield of RNA retrieved in the absence of the agent.
  • the quality of a total RNA prep can be assessed for signs of degradation by running a portion on an agarose or acrylamide gel or by using an instrument such as the Agilent Bioanalyzer. Examples of methods for assessing the quantity of RIMA include using : UV absorbance, fluorescence, and an Agilent Bioanalyzer.
  • RNA and resulting cDNA can be analysed by fluorometry for quantification and the Bioanalyzer (or equivalent device) for quantification and RNA integrity evaluation.
  • a fluorometer such as Life Technologies' Qubit
  • the concentration of RNA and cDNA can also be estimated from a Bioanalyzer or Fragment Analyzer trace.
  • Another way to quantitate RNA is by measuring the absorbance at 260 nm. In case of full-length cDNA libraries, the size distribution of yielded cDNA after reverse transcription and PCR amplification provides an accurate readout of the underlying RNA integrity.
  • Mammalian mRNA and full-length cDNA samples should display a characteristic peak at approximately 2000 bp, reflecting the length distribution of mRNAs in mammalian cells. Degradation, due to failed RNase inhibition, display an mRNA and cDNA size distribution skewed towards shorter fragments.
  • agents of this invention can be used in combination or can be combined with any art-known RNase inhibitor (that are or are not agents of this invention) to achieve a desired inhibitory effect on or inactivation of one or more RNases.
  • RNA sequencing or "RNA-seq” we include the meaning of a genomic approach for the detection and quantitative analysis of RNA molecules in a biological sample by the readout of nucleotide sequences.
  • RNA-seq is a multipurpose methodology that is increasingly used in biological, biomedical and clinical settings. RNA-seq can for example be useful for studying cellular states and responses in vivo and in vitro by studying proteinencoding mRNA molecules as well as non-protein-coding RNAs (collectively termed the 'transcriptome').
  • RNA-seq is also a useful methodology to detect foreign biological material or infection in a sample, such as that of an RIMA virus or bacteria transcribing their nucleic acid. RNA-seq is furthermore a useful readout in various in vitro applications and synthetic biology utilizing RNA.
  • the method and devices of the invention may be used for bulk RNA sequencing or single-cell RNA sequencing methods.
  • bulk RNA sequencing we include the meaning of the sequencing of RNA isolated from pools of cells, including tissues, blood, secretions, tissue sections etc.
  • bulk RNA sequencing we also include the meaning of the sequencing of RNA from pools of cells, including tissues, blood, secretions, tissue setions etc.
  • single cell RNA sequencing or “scRNAseq” we include the meaning of the sequencing of RNA isolated from an individual cell which allows comparison of the transcriptomes of individual cells.
  • Single-cell RNA-seq methods can also be used to detect RNA of parts or sub-compartments of a cell.
  • the performance of scRNAseq methods can be characterized using single cells (generally containing 10-30 pg of total RNA in case of mammalian cells) or low amounts of input RNA, such as 10-100 pg of total RNA from an pool of RNA extracted from multiple cells.
  • the methods of the present invention can be used as part of any "multiomics" method, i.e., a method that combines preparing a cDNA sequencing library from one part or fraction of the sample material and measurement of another biological modality from another part or fraction of the same sample, such as for example a DNA or protein library.
  • the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes that possibility). Moreover, the method can be paused following one or more steps and resumed at a later stage, if technically appropriate to do so.
  • cDNA sequencing library (may also be termed “next generation sequencing (NGS) library”) we include the meaning of a collection of complementary DNA (cDNA) fragments, which together constitute some portion of the transcriptome of a single cell or a plurality of cells.
  • the collection of cDNA fragments in the library include a partial or complete sequencing platform adapter sequence at their termini useful for sequencing using a sequencing platform of interest.
  • the cDNA sequencing library can be subject to a full- length transcript, or 3'/5'-end sequencing protocol.
  • a “full-length transcript sequencing protocol” we include the meaning of methods that generates sequencing-read coverage across most of the length of the RNA transcripts, such as for example in Smart-seq (Ramskold, D. et al. Nat. Biotechnol. 30, 777-782 (2012)); Smart-seq2 (Picelli, S. et al.
  • 3'/5'-end sequencing protocol we include the meaning of methods that generates sequencing-read coverage in either 3' or 5' end of the RNA transcripts coverage across most of the length of the RNA transcripts, such as for example in STRT-seq-2i (Hochgerner, H. et al. Sci. Rep. 7, 16327 (2017)); SCRB-seq (Soumillon, M., Cacchiarelli, D., Semrau, S., van Oudenaarden, A. & Mikkelsen, T. S. Preprint at bioRxiv https://doi.org/10.1101/003236 (2014)).
  • sequencing platform adapter sequence or “sequencing platform adapter construct” we include the meaning of a nucleic acid construct that includes at least a portion of a nucleic acid domain (e.g., a sequencing platform adapter nucleic acid sequence) utilized by a sequencing platform of interest.
  • sequence tags to allow for multiplexing (known as barcodes or indices), unique molecular identifiers (UMIs), and/or a second sequence-priming site to allow for sequencing of the insert from the other side (known as paired-end sequencing).
  • a sequencing platform adapter sequence includes one or more nucleic acid domains selected from: a platform-dependent domain that specifically binds to a surface-attached sequencing platform oligonucleotide (e.g., the P5 or P7 oligonucleotides attached to the surface of a flow cell in an Illumina® sequencing system); a sequencing primer binding domain (e.g., a domain to which the Read 1 or Read 2 primers of the Illumina® platform may bind); a barcode domain (e.g., a domain that uniquely identifies the sample source of the nucleic acid being sequenced to enable sample multiplexing by marking every molecule from a given sample with a specific barcode or "tag"); a barcode sequencing primer binding domain (a domain to which a primer used for sequencing a barcode binds); a unique molecular identification domain (e.g., a molecular index tag, such as a randomized tag of 4, 6, or other number of nucleotides) for uniquely marking
  • a barcode domain e.g., sample index tag combination including a unique index or unique dual indexes (UDIs)
  • UMI unique molecular identifier
  • a sequencing platform adapter construct when present, may include one or more nucleic acid domains of any length and sequence suitable for the sequencing platform of interest.
  • the nucleic acid domains are from 4 to 200 nts in length.
  • the nucleic acid domains may be from 4 to 100 nts in length, such as from 6 to 75, from 8 to 50, or from 10 to 40 nts in length.
  • the sequencing platform adapter construct may include a nucleic acid domain that is from 2 to 8 nucleotides in length, such as from 9 to 15, from 16 to 22, from 23 to 29, or from 30 to 36 nts in length.
  • Such sequencing platform adapter constructs can be added to each end of the insert during the first- and/or second-strand synthesis steps.
  • the reverse transcriptase primer can contain an overhanging or nested sequence that does not anneal to the RIMA template but contains at least a portion of the adapter sequences.
  • the forward PCR primer can contain over-hanging sequences and therefore introduce such adapters.
  • adapters can be introduced via ligation. This approach is used in the Illumina TruSeq Small RNA kit, the NEBNext Small RNA prep kit, and in the SOLID RNA kits from Life Technologies. These kits use ligation procedures that allow two different adapters to be ligated onto each end of the target RIMA. These adapters are then used to prime the first- and second-strand synthesis reactions resulting in cDNAs terminated by the appropriate adapter sequences.
  • nucleotide sequences of nucleic acid domains useful for sequencing on a sequencing platform of interest may vary and/or change over time.
  • Adapter sequences are typically provided by the manufacturer of the sequencing platform (e.g., in technical documents provided with the sequencing system and/or available on the manufacturer's website).
  • sequence of any sequencing platform adapter domains such as the template switch oligonucleotide, first strand cDNA primer, amplification primers, and/or the like, may be designed to include all or a portion of one or more nucleic acid domains in a configuration that enables sequencing the nucleic acid insert (corresponding to the template RNA) on the platform of interest.
  • sequencing we include the meaning of high throughput sequencing.
  • high throughput sequencing we include the meaning of the simultaneous or near simultaneous sequencing of thousands of nucleic acid molecules. High throughput sequencing is sometimes referred to as “next generation sequencing (NGS)” or “massively parallel sequencing”.
  • Sequencing platforms of interest include, but are not limited to, sequencing platforms provided by Illumina® (e.g., the NextSeqTM, HiSeqTM, MiSeqTM, NovaSeqTM and/or Genome AnalyzerTM sequencing systems); Ion TorrentTM (e.g., the Ion PGMTM and/or Ion ProtonTM sequencing systems); Pacific Biosciences (e.g., the PACBIO RS II sequencing system); Life TechnologiesTM (e.g., a SOLID sequencing system); Roche (e.g., the 454 GS FLX+ and/or GS Junior sequencing systems); MGI Tech Co., Ltd. "MGI” (e.g., the DNBSEQ-T7TM, DNBSEQ-
  • the method comprises: i. preparing a lysate and thereby releasing a plurality of RIMA molecules from one or more cells, tissue, tissue extract or cell extract concomitant with or followed by application of the RNA molecules and/or lysate to a carrier impregnated with an agent wherein the agent is according to any embodiment disclosed herein; ii. eluting the RNA from the carrier iii. synthesizing a plurality of cDNA strands from the RNA molecules by reverse transcription; and iv. processing the cDNA strands to generate a cDNA sequencing library.
  • the time between in particular step i. and step ii. may be up to 30 days, such as up to 25 days, such as up to 20 days, such as up to 14 days such as up to 10 days, such as up to 7 days, such as up to 6 days, such as up to 5 days, such as up to 4 days, such as up to 3 days, such as up to 2 days, such as up to a day.
  • Releasing a plurality of RNA molecules from one or more cells or cell extract can be achieved by, for example, heating or freeze-thaw of cells, or by the use of detergents, chaotropic agents, mechanical methods, or other chemical methods, or by a combination of these, in the presence of an aqueous solution comprising the agent.
  • Mechanical methods for homogenizing tissues include using cryo-grinding with a mortar/pestle, shearing using a rotor-stator homogenizer or a Dounce homogenizer, sonication, or bead-beating. After homogenization two methods are commonly used to recover RNA from the cell lysate: (1) extraction with organic solvents; or (2) solid-phase extraction on silica.
  • releasing a plurality of RIMA molecules from one or more cells or cell extract, tissue or other sample, in the presence of an aqueous solution comprising the agent comprises contacting one or more cells or cell extract with an aqueous solution to release RNA molecules.
  • the RNA molecules are preferably poly(A) containing RNA molecules, such as mRNA molecules, and are typically present in and released from the cytoplasm of the lysed cell.
  • aqueous solution we include the meaning of any liquid solution that can be used in a method of liberating RNA from cells.
  • aqueous solutions include buffers such as sample collection buffers and lysis buffers. Examples of suitable buffers include, PBS, Tris, sodiumacetate, HEPES, MOPS.
  • the aqueous solution may be a sample collection buffer.
  • a sample collection buffer may not comprise a detergent and/or chaotropic agent.
  • a sample collection buffer could contain the bulk sample of intact cells without detergent, and the RNA can be extracted through another means, such as using Trizol, phenol, and/or a commercially available RNA extraction kit.
  • the aqueous solution may be a lysis buffer.
  • lysis buffer we include the meaning of a buffer used for the purpose of breaking open cells.
  • suitable lysis buffer to which the agent could be added are described herein and are described in known protocols for preparing a cDNA library.
  • the lysis buffer may comprise enzymes (e.g. Proteinase K), detergents (e.g. Triton X-100, SDS, NP-40/Igepal, Tween-20, sodium deoxycholate, and CHAPS) and/or chaotropic agent (i.e. (compounds that disrupt both hydrophobic and hydrogen-bond interactions, such as guanidine salts) together with the agent.
  • Triton X-100 could be used as a detergent when lysing cells.
  • Guanidinium is a strong protein denaturant capable of denaturing recalcitrant proteins such as RNases.
  • the buffer is a lysis buffer comprising 0.1-1% Triton X-100 and the agent.
  • the buffer is a lysis buffer comprising 0.1% Triton X-100.
  • a mild lysis procedure can advantageously be used to prevent the release of nuclear chromatin, thereby avoiding genomic contamination of the cDNA library, and to minimize degradation of mRNA. For example, heating the cells at 72°C for 2-10 minutes in the presence of mild detergent (together with the agent) is generally sufficient to lyse cells.
  • one or more cells refers to any number of (e.g. unlysed) cells desired to be analysed.
  • One or more cells may include at least 1 cell, at least 10 cells, or alternatively at least 25 cells, or alternatively at least 50 cells, or alternatively at least 100 cells, or alternatively at least 200 cells, or alternatively at least 500 cells, or alternatively at least 1000 cells, or alternatively 5,000 cells or alternatively 10,000 cells.
  • One or more cells may include from 10 to 100 cells, or alternatively from 50 to 200 cells, alternatively from 100 to 500 cells, or alternatively from 100 to 1000, or alternatively from 1,000 to 5,000 cells.
  • One or more cells may include 10,000 cells, 20,000 cells, 30,000 cells, 40,000 cells, 50,000 cells, 60,000 cells, 70,000 cells, 80,000 cells, 90,000 cells or alternatively 100,000 cells.
  • the "one or more cells or cell extract” comprises template RNA and may be derived from any sample of interest, including but not limited to, a single cell, a plurality of cells (e.g., cultured cells), a tissue, an organ, or an organism (e.g., bacteria, yeast, or higher eukaryotic organisms, such as a plant, or a mouse, or a worm, or the like).
  • the one or more cells or cell extract are derived from a tissue, organ, and/or the like of a mammal (e.g., a human, a rodent (e.g., a mouse), or any other mammal of interest).
  • the one or more cells or cell extract can be derived from live samples, non-conserved samples, preserved samples, embalmed samples and/or fixed samples.
  • the RIMA molecules are liberated into an aqueous solution comprising the agent from one or more cells in a fixed biological sample, e.g., formalin-fixed, formaldehyde/paraformaldehyde-fixed, paraffin-embedded (FFPE) tissue.
  • FFPE paraffin-embedded
  • RNA from one or more cells in FFPE tissue may be released using commercially available kits - such as the NucleoSpin® FFPE RNA kits by Clontech Laboratories, Inc. (Mountain View, CA).
  • samples from which one or more cells or cell extract can be derived from includes a cell culture sample, blood, serum, plasma, reticulocytes, lymphocytes, any product prepared from blood or lymph, bone marrow tissue, cerebrospinal fluid, sweat, tear, saliva, sputum, amniotic fluid, seminal fluid, vaginal excretion, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, or faecal samples), any type of tissue biopsy (e.g.
  • Suitable samples containing cells further comprise clinical samples (which are samples provided by a patient), biological swabs and biological washes. Suitable samples containing cells may be fresh or may have been stored (e.g. cryopreserved), such as at -80°C.
  • cells from any population can be used in the methods, such as a population of prokaryotic or eukaryotic single-celled organisms including bacteria or yeast.
  • the biological sample may comprise one or more viruses.
  • RNA-seq After obtaining an RIMA preparation that is suitable for RNA-seq (step 1) the RNA is typically converted to double-stranded complementary DNA (cDNA).
  • cDNA double-stranded complementary DNA
  • RNA In order to convert RNA to DNA the RNA must be used as a template for a DNA polymerase. Most DNA polymerases cannot use RNA as a template. However, retroviruses encode a unique type of polymerase known as reverse transcriptases, which are able to synthesize DNA using an RNA template.
  • RNA molecules we include the meaning of the template ribonucleic acid (RNA) liberated from the one or more cells or contained within the cell extract. It may be a polymer of any length composed of ribonucleotides, e.g., 10 nts or longer, 20 nts or longer, 50 nts or longer, 100 nts or longer, 500 nts or longer, 1000 nts or longer, 2000 nts or longer, 3000 nts or longer, 4000 nts or longer, 5000 nts or longer or more nts.
  • ribonucleotides e.g., 10 nts or longer, 20 nts or longer, 50 nts or longer, 100 nts or longer, 500 nts or longer, 1000 nts or longer, 2000 nts or longer, 3000 nts or longer, 4000 nts or longer, 5000 nts or longer or more nt
  • the template ribonucleic acid is a polymer composed of ribonucleotides, e.g., 10 nts or less, 20 nts or less, 50 nts or less, 100 nts or less, 500 nts or less, 1000 nts or less, 2000 nts or less, 3000 nts or less, 4000 nts or less, or 5000 nts or less, 10,000 nts or less, 25,000 nts or less, 50,000 nts or less, 75,000 nts or less, 100,000 nts or less.
  • ribonucleotides e.g., 10 nts or less, 20 nts or less, 50 nts or less, 100 nts or less, 500 nts or less, 1000 nts or less, 2000 nts or less, 3000 nts or less, 4000 nts or less, or 5000 nt
  • the template RNA may be any type of RNA (or sub-type thereof) including, but not limited to, a messenger RNA (mRNA), a microRNA (miRNA), a small interfering RNA (siRNA), a transacting small interfering RNA (ta-siRNA), a natural small interfering RNA (nat-siRNA), a ribosomal RNA (rRNA), a transfer RNA (tRNA), a small nucleolar RNA (snoRNA), a small nuclear RNA (snRNA), a long non-coding RNA (IncRNA), a non-coding RNA (ncRNA), a transfermessenger RNA (tmRNA), a precursor messenger RNA (pre-mRNA), or any combination of RNA types thereof or subtypes thereof.
  • mRNA messenger RNA
  • miRNA microRNA
  • siRNA small interfering RNA
  • ta-siRNA transacting small interfering RNA
  • nat-siRNA
  • the inhibitory agents can be bound to an absorbent material (e.g., fiber material, sponge, paper, etc) and so forms or is comprised in a device.
  • an absorbent material e.g., fiber material, sponge, paper, etc
  • the absorbent material is a fibrous material, such as paper.
  • Fibrous materials may comprise cotton fibre, glass fibre, polymer, cellulose, or a combination thereof. It will be appreciated that the fibrous material is suitable for binding RNA.
  • Chemical groups on paper surface e.g., hydroxyl and carboxyl groups
  • cellulose contains hydroxyl groups on its surface and has the properties of hydrophilicity, easy usability, high porosity, high mechanical strength.
  • Cotton fiber, a natural material also contains hydroxyl groups on its surface.
  • Glass fiber is a kind of synthetic fiber and is formed of silica-based thin strands. Such fibrous material can be 100 pm - 500 pm thick. Examples of fibrous material include filter paper such as Flinders Technology Associates (FTA) cards®, Nobuto filter paper, Whatman® paper, and iBIot Filter Paper.
  • FDA Flinders Technology Associates
  • Absorbent material may also be in the form of e.g. polyethylene foam.
  • Absorbent material may also be in the form of polyurethane foam.
  • the invention also provides a solid support comprising the agent as defined in any of the embodiments herein.
  • the solid support is capable of binding to the agent defined herein, or otherwise containing the agent defined herein.
  • binding to the agent we include the meaning that the agent is immobilised onto a solid support such as an absorbent material. Immobilisation may be via a covalent or non-covalent interaction.
  • agent-coated supports can be achieved using an in situ polymerization method or by incubating material or surfaces with the inhibitory agent.
  • Those of ordinary skill in the art will appreciate that other means for directly or indirectly (through a linker) coupling of agents of this invention to solid supports are available in the art and can be employed in the practice of this invention.
  • the support may be coated with a moiety that binds non-covalently to the agent.
  • the agent can be adsorbed to the support either through the porous nature of the support or absorbent material, or through weak hydrophobic and/or polar interactions between the support and the agent.
  • the support may be coated with a pre-activated functional group to covalently immobilize the agent to the surface.
  • a pre-activated functional group to covalently immobilize the agent to the surface.
  • Any suitable system for covalent interactions may be used, including ELISA principles, and pre-activated surfaces to facilitate covalent bonds.
  • any suitable commercially available support that allows for covalent interactions can be used, such as those available from Corning®.
  • containing we include the meaning that the solid support has one or more pores within which the agents as defined herein can be retained.
  • biological samples e.g., whole blood, saliva, urine, tissue and cells and lysates thereof
  • Tubes, bottles and refrigerators are normally utilized to collect and store samples.
  • absorbent material has the advantages of low cost, porous structure, portability and ease of use. Thus, improved paper-based (and other absorbent material) sample storage and collection methods are required.
  • absorbent material pre-incubated also referred to as impregnated
  • the agents defined herein allow storage and collection of RIMA containing samples at room temperature and the eluted RNA is compatible with RNA sequencing.
  • the absorbent material can be stored at room temperature. After storage, the RNA can be eluted and analysed for RNA quality, cDNA yield by methods known in the art and as described herein.
  • the absorbent material is suitable for receiving a liquid solution comprising the agent.
  • the absorbent material is pre-incubated with a liquid solution comprising an effective amount of the agent, and may be dried to remove all or most of the liquid phase.
  • Effective amounts include lpg agent /mL water to lOOOmg agent /mL water.
  • the absorbent material is made of cotton fiber or wood pulp, or other cellulose-based materials.
  • the absorbent material is polyethylene foam and the agent is sodium alginate.
  • the absorbent material is polyethylene foam and the agent is PVSA.
  • the absorbent material is polyethylene foam and the agent is heparin.
  • the absorbent material is polyethylene foam and the agent is dextran sulfate. In an embodiment the absorbent material is polyurethane foam and the agent is sodium alginate.
  • the absorbent material is polyurethane foam and the agent is PVSA.
  • the absorbent material is polyurethane foam and the agent is heparin.
  • the absorbent material is polyurethane foam and the agent is dextran sulfate.
  • the device is in the form of a column.
  • such a column is in a support, such as in a tube.
  • the elusion procedure of such a column can include centrifugation.
  • kits refers to one or more suitably aliquoted compositions or reagents for use in the methods of the present disclosure, preferably together with a device as disclosed. Some components of the kits may be packaged either in aqueous or lyophilized form, while other may be dry or substantially dry.
  • the container means of the kits may include at least one vial, test tube, flask, bottle, syringe, or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third, or other additional container into which the additional components may be separately placed.
  • kits of the present disclosure also will typically include a means for containing the reagent containers in close confinement for commercial sale.
  • Such containers may include injection or blow moulded plastic containers into which the desired vials are retained, for example.
  • absorbent material includes but is not limited to cellulose paper, such as filter paper, alpha cellulose cotto paper (such as e.g. Whatman filter paper) wood pulp paper such as coffee filter (tree pulp) and foams such as e.g. polyethylene foam or polyurethane foam.
  • absorbent is intended that the material is able to absorb and retain a liquid such as an aqueous solution of an RNase inhibitor and/or other compounds, or a biological sample such as e.g. a lysate obtained from a tissue or cell culture or other sources.
  • Absorbent materials according to the present invention may absorb a liquid, undergo drying and be able to absorb a liquid or sample multiple times such as at least once. During drying it is contemplated that only the aqueous phase is removed from the absorbent material, whereas solutes and/or other components may retain absorbed in the material.
  • the material may, optionally subsequent to drying, be said to be impregnated with said one or more RNase inhibitors.
  • Solutes or components absorbed in the absorbent material may be subject to elution from the material. This includes elution of RNA, such as for use in reverse transcription. It is contemplated that such elution may also release RNase inhibitor from the absorbent material, rendering the eluted RNA under continued protection from RNase activity.
  • antimicrobial agents can be incorporated into absorbent materials, sample buffers, and lysis buffers. These agents may include compounds such as sodium azide, triclosan, sorbic acid, and benzoic acid, as well as metal-based compounds like silver nitrate, silver sulfadiazine, silver nanoparticles, and copper-based compounds such as copper sulfate and copper oxide. Additional antimicrobial agents and combinations may be used depending on specific needs and circumstances, including the biological sample source, sampling environment, and storage conditions.
  • Sodium alginate (NaC6H?O6)n, NaAIg, is the sodium salt of alginic acid, and is a carboxylated polysaccharine derived from algae with a repeating disaccharide block.
  • the structure of the repeating blocks are (1— >4)-linked 0-D-mannuronate (M) and o-L-guluronate (G) residues.
  • Heparin is a member of the glycosaminoglycan family of carbohydrates and consists of a variably sulfated repeating disaccharide unit.
  • the repeating unit consists of is glucosamine and uronic acid.
  • Dextran sulfate is a sulfated polymer consisting of (1— >6)-o-linked anhydroglucose molecules. It has a molecular weight of greater than 500,000 Daltons.
  • Fucoidan is a sulfated polysaccharine found in algae consisting predominantly fucose sugar molecules. It has a molecular weight ranging from approximately 50-1000 kiloDaltons.
  • the examples demonstrate that effective dry-storage devices for biological RIMA samples can be constructed by impregnating an absorbent material with sodium alginate (NaAIg), dextran sulfate (DexSulf), fucoidan, heparin, polyvinyl sulfonic acid (PVSA), vinyl sulfonic acid (VSA), and/or 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic acid (HEPES), onto which lysed samples can be placed and absorbed.
  • NaAIg sodium alginate
  • DexSulf dextran sulfate
  • fucoidan fucoidan
  • heparin polyvinyl sulfonic acid
  • PVSA polyvinyl sulfonic acid
  • VSA vinyl sulfonic acid
  • HEPES 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic acid
  • FIG. 1 Various chemical compounds preventing RNA degradation in RNA dry-storage devices after 1 day of storage.
  • the x-axis represents fragment size in base pairs and the y axis represents fluorescence units (FU) relating to detected cDNA abundance.
  • Tick marks on the y-axis correspond to increments of 500 FUs. Concentrations from left to right for each row denote decreases in the inhibitor by 10-fold.
  • FIG. 2 Various chemical compounds preventing RNA degradation in RNA dry-storage devices after 3 days of storage.
  • the x-axis represents fragment size in base pairs and the y axis represents fluorescence units (FU) relating to detected cDNA abundance.
  • Tick marks on the y-axis correspond to increments of 500 FUs. Concentrations from left to right for each row denote decreases in the inhibitor by 10-fold.
  • FIG. 3 Effect of elution time on RNA library yield.
  • Bioanalyzer traces from amplified cDNA libraries generated from the equivalent of 25 cells-worth of RNA eluted from lysed cells stored for 1 day on cellulose paper material and eluted (a) The eluted samples were incubated at room temperature for 2 minutes prior to Smart-seq2 cDNA preparation, (b) The eluted samples were incubated at room temperature for 5 minutes prior to Smart-seq2 cDNA preparation, (c) The eluted samples were incubated at room temperature for 30 minutes prior to Smart-seq2 cDNA preparation.
  • the x-axis represents fragment size in base pairs and the y axis represents fluorescence units (FU) relating to detected cDNA abundance.
  • FU fluorescence units
  • FIG. 4 Various chemical compounds preventing RNA degradation in RNA dry-storage devices after 1 day of dry storage and 6 days of elution. Bioanalyzer traces from amplified cDNA libraries generated from the equivalent of 25 cells-worth of RNA eluted from lysed cells stored dry for 1 day in cellulose paper material and then kept incubated in elution solution for an additional 6 days ("1+6").
  • Cellulose paper material was pre-treated with (a) NaAIg, (b) fucoidan, (c) heparin, (d) VSA, (e) PVSA, (f) HEPES, (g) DexSulf, (h) MES, (i) VPA, (j) PVPA, (k) chondroitin, (I) MOPS, or (m) TAPS at various concentrations stated in each panel, or (n) water.
  • the x-axis represents fragment size in base pairs and the y axis represents fluorescence units (FU) relating to detected cDNA abundance. Tick marks on the y-axis correspond to increments of 500 FUs. Concentrations from left to right for each row denote decreases in the inhibitor by 10-fold.
  • FIG. 5 Comparison of different materials used as sample carrier in RNA dry-storage. Bioanalyzer traces from amplified cDNA libraries generated from the equivalent of 25 cells-worth of RIMA eluted from lysed cells stored in various materials treated with various amounts of PVSA (3000 pg/mL, 300 pg/mL, 30 pg/mL) or water (left to right). The stated PVSA concentrations apply to all materials displayed columnwise. Samples were stored on (a) polyethylene foam, (b) o-cellulose sheet, (c) wood pulp filter, (d) cellulose sheet, or (e) PES filter for samples eluted after 1 day.
  • PVSA polyethylene foam
  • FIG. 6 Polyurethane foam and polyethylene foam are suitable sample carrier materials for RNA dry-storage.
  • Bioanalyzer traces from amplified cDNA libraries generated from the equivalent of 25 cells- worth of RIMA eluted from lysed cells stored in various materials treated with two concentrations of PVSA (300 pg/mL and 30 pg/mL, left to right). The stated PVSA concentrations apply to all materials displayed column-wise. Samples were stored on (a) polyethylene foam, (b) polyurethane foam, (c) wood pulp filter (d) cellulose sheet, or (e) PES filter for samples eluted after 3 days. For all panels, the x-axis represents fragment size in base pairs and the y axis represents fluorescence units (FU) relating to detected cDNA abundance. Tick marks on the y-axis correspond to increments of 500 FUs.
  • FU fluorescence units
  • FIG. 7 Combinatorial analysis of materials and chemical inhibitors for RNA preservation.
  • Bioanalyzer traces from amplified cDNA libraries generated from the equivalent of 25 cells-worth of RNA eluted from lysed cells stored in various materials pre-treated with the following inhibitors and concentrations: (a) 400 pg/ml NaAIg, (b) 10k pg/ml fucoidan, (c) 20 pg/ml heparin, (d) 200k pg/ml VSA, (e) 20k pg/ml HEPES, (f) ) 250 pg/ml DexSulf, and (g) water (no inhibitor).
  • Samples were stored on polyethylene foam, polyurethane foam, wood pulp filter, cellulose sheet, or PES filter (left to right) for samples eluted after 3 days.
  • the stated dry-storage matrix applies to all materials displayed column-wise.
  • the x-axis represents fragment size in base pairs and the y axis represents fluorescence units (FU) relating to detected cDNA abundance. Tick marks on the y-axis correspond to increments of 500 FUs.
  • FIG. 8 Long-term RNA preservation in polyurethane foam.
  • the stated PVSA concentrations apply to all materials displayed column-wise. Samples were stored on (a) polyurethane foam, (b) o-cellulose sheet, (c) wood pulp filter (d) cellulose sheet, or (e) PES filter for samples eluted after 11 months.
  • the x-axis represents fragment size in base pairs and the y axis represents fluorescence units (FU) relating to detected cDNA abundance. Tick marks on the y-axis correspond to increments of 500 FUs.
  • concentrations tested for these agents in our study were: 40000, 4000, 400, 40, 4 pg/mL for NaAIg; 10000, 1000, 100, 10, 1 pg/mL for fucoidan; 2000, 200, 20, 2, 0.2 pg/mL for heparin; 200000, 20000, 2000, 200, 20 pg/mL VSA; 30000, 3000, 300, 30, 3 pg/mL for PVSA; 20000, 2000, 200, 20, 2 pg/mL for HEPES; 2500, 250, 25, 2.5, 0.25 pg/mL for DexSulf; 40000, 4000, 400, 40, 4 pg/mL for MES; 200000, 20000, 2000, 200, 20 pg/mL for VPA; 100000, 10000, 1000, 100, 10 pg/mL for PVPA; 20000, 2000, 2000, 200, 20, 2 pg/mL for chondroitin; 100000, 10000, 1000, 100, 10
  • the impregnated absorbent material was then lifted out from the solution using stainless steel tweezers, moved into sterile 24-well polystyrene plates with ventilating lids (loose-fitting lids protecting samples from dust but allowing evaporation), and let dry at room temperature for 24 hours.
  • HEK293FT Human embryonic kidney cells were cultured and expanded in standard medium (DMEM/10% FBS) in 5% CO2 and 37°C. Aliquots of 2 million cells were collected in a 1.5 mL microcentrifuge tube, washed with lx PBS, pelleted by brief centrifugation upon which the PBS supernatant was discarded, and frozen in a -80°C deep freezer. For the following storage experiments, cell pellets were diluted in a cell lysis solution, consisting of 1000 pL 1% Triton-XlOO, and pipetted up and down to achieve a lysed cell suspension.
  • DMEM/10% FBS standard medium
  • the lysed cell suspension was vortexed for 30 seconds then centrifuged at 300 g for 3 minutes to pellet potential cell debris, such as incompletely lysed nuclei and other potentially remaining cell compartments. Consequently, the lysed cell suspension contained the input equivalent of 2000 lysed HEK293FT cells per pL.
  • sample devices The dried, pre-treated absorbent materials (sample devices) were spotted with 1.25 pL of lysed cell suspension corresponding to 2500 lysed cells (lysed supernatant after centrifugation, as described in the previous section), by pipetting the suspension onto the middle of the sample devices and letting the sample absorb and dry into the material.
  • the lysed-cell-spotted storage devices were then incubated/dried for 1 or 3 days at room temperature in 24-well polystyrene plates with ventilating lids (loose-fitting lids protecting samples from dust but allowing evaporation).
  • the cell-spotted absorbent materials were individually placed in the wells of a sterile 96-well polystyrene plate, and sample material was eluted by adding 100 pL of nuclease-free water followed by incubation at room temperature for 3 minutes.
  • the 96-well plate was sealed using plastic PCR sealing film, vortexed for 20 seconds, and the plates were finally centrifuged at 300 g for 1 minute.
  • the resulting eluates were immediately used as RIMA input to generate full- length cDNA libraries using the Smart-seq2 protocol (Picelli 2013, Picelli 2014), using 1 pL of eluate as input to the Smart-seq2 reaction.
  • This volume (1 pL) corresponded to an original input of 25 lysed cells, not taking potential losses into count, e.g., due to potential trapping of material inside the absorbent material.
  • RNA sample material absorbed and dried into the storage device material might require prolonged elusion time for maximum release into the elusion solution.
  • Smart-seq2 cDNA library generation was performed as follows: Smart-seq2 lysis buffer composition was 2.5mM dNTP/each and 2.5mM Smart seq2 oligo-dT (5'- AAGCAGTGGTATCAACGCAGAGTACT30VN-3') with 1 pL of eluant in a total buffer volume 4.5 pL. Samples in lysis buffer were incubated at 72°C for 3 min in a Bioer Life ECO thermocycler to hybridize the oligo-dT and placed on ice prior to first-strand synthesis.
  • the following reverse transcriptase reaction contained lx Superscript II buffer, 5mM DTT, IM betaine, 10 mM MgCL, 1 pM Smart-seq2 TSO (5'- AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3'), and 10 U Superscript II for a total reaction volume of 10 pL.
  • RT thermocycles were 42°C for 90 min, followed by 10 cycles of 42°C for 2 min and 50°C for 2 min.
  • the following cDNA amplification reaction contained lx KAPA HiFi HotStart Ready Mix and 80 nM ISPCR primers (5'- AAGCAGTGGTATCAACGCAGAGT-3'); total reaction volume 25 pL.
  • Thermocycles for Smart-seq2 cDNA amplification were 98°C for 3 min, followed by 18 cycles of 98°C for 20 sec, 67°C for 15 sec, and 72°C for 6 min, followed by a final incubation at 72°C for 5 min.
  • Amplified cDNA samples were bead purified (AMPure XP, Beckman) at a ratio of 0.8: 1 beads:cDNA (20 pL beads:25 pL cDNA for Smart-seq2) and inspected on a Bioanalyzer 2100 (High Sensitivity DNA kit, Agilent).
  • Input control libraries samples going into the storage devices were generated directly from the original lysed-cell supernatant (i.e., samples not kept in storage devices) diluted in nuclease-free water so that their input to the Smart- seq2 reactions corresponded to 25 lysed cells.
  • cDNA yield and quality were generated directly from the original lysed-cell supernatant (i.e., samples not kept in storage devices) diluted in nuclease-free water so that their input to the Smart- seq2 reactions corresponded to 25 lysed cells.
  • the resulting purified Smart-seq2 cDNA libraries were analyzed using Agilent Bioanalyzer 2100 High sensitivity DNA chips.
  • the shape of full-length cDNA traces is known in the field to reflect the quality and integrity of the underlying mRNA sample (Trombetta 2014), and an intact library (satisfactory library) is expected to have a peak around ⁇ 2kb, reflecting the median length of full-length mRNA transcripts in human cells.
  • Exact patterns of spikes in the cDNA traces can further be experiment- or cell-type specific, as abundant cell-type-specific or condition-specific RNA transcripts, for which one or a few transcripts account for a large proportion of total cellular transcripts lead to additional peaks on the Bioanalyzer trace.
  • RNA preservation being successfully achieved if satisfactory cDNA traces are achieved above a given concentration threshold, i.e., NaAIg (>400 pg/mL); fucoidan (>10000 pg/mL); heparin (> 2 pg/mL); VSA (>20000 pg/mL); PVSA (>30 pg/mL); HEPES (>2000 pg/mL); DexSulf (>250 pg/mL).
  • concentration threshold i.e., NaAIg (>400 pg/mL); fucoidan (>10000 pg/mL); heparin (> 2 pg/mL); VSA (>20000 pg/mL); PVSA (>30 pg/mL); HEPES (>2000 pg/mL); DexSulf (>250 pg/mL).
  • RNA stability was demonstrated up to day 3 of dry storage at room temperature and atmospheric conditions; it is expected that longer term stability also feasible.
  • elusion time affects RNA recovery from the dry-storage device, and that eluted RNA samples from the dry-storage devices produced intact full-length cDNA profiles even upon a 6-day elusion from the dry-storage device (incubation at room temperature).
  • RNA can be kept stably in dry storage devices for considerable amounts of time.
  • HEK human embryonic kidney
  • usefulness is contemplated with various other biological input materials, such as for example lysed tissues, blood, biopsies, solid or liquid tumors and extracts, saliva, culture cells.
  • Human, mammalian and other animal or plant RNA as well as microbial RNA could be sampled, stored, and detected from the device.
  • a product kit may be developed to include one of the identified chemical RNase inhibitors (e.g., NaAIg, fucoidan, heparin, VSA, PVSA, HEPES, DexSulf) also in the sample lysis buffer.
  • a filter capturing incompletely lysed cell debris may be included (e.g., mesh filter in spin-down column or compression syringe), as alternative to the cell debris removal by centrifugation.
  • separation or selection of sampled RNA based on physical or chemical RNA features e.g., RNA size, three-dimensional structure, or chemical modification
  • RIMA separation within the storage device e.g. by integrating a gradient of absorbency or pore size within the matrix.
  • This could be tailored to facilitate the selective passage of RNA molecules of specific sizes or configurations.
  • a gradated material beginning with a looser, larger-pored section that transitions to a denser, smaller-pored one— larger RNA fragments could be retained earlier in the matrix, while smaller fragments travel further, enabling size-based stratification directly within the device
  • by functionalizing different sections of the matrix with chemically distinct agents it may be possible to target and isolate RNA molecules with specific chemical modifications.
  • a section of the matrix could be functionalized to have an affinity for methylated RNA, thus capturing and segregating these molecules from the rest of the RNA population during the diffusion process.
  • the application of a controlled electric field across the device could induce directional movement of RNA molecules, enhancing separation based on their charge-to-mass ratio— a technique reminiscent of gel electrophoresis.
  • This method could be finely tuned to improve the precision of RNA separation by exploiting the minute differences in the electrical properties of RNA molecules.
  • the device could incorporate binding domains or molecules with shape-specific affinity. This could be particularly useful for capturing uniquely structured RNA, such as tRNA or rRNA, which could be bound and thus separated from mRNA.
  • incorporating microfluidic channels could lead to more advanced control over the flow and distribution of the sample, allowing for a more precise separation of RNA molecules based on their physical and chemical characteristics.
  • These channels could be lined with various absorbent materials, each section providing a different selection pressure or chemical environment.
  • integrating sensor technology within the device could offer real-time monitoring and characterization of the RIMA as it is separated, providing immediate analytical data.
  • Such a device would not only serve as a storage medium but also as a tool for RNA analysis, significantly streamlining the process from sample storage to RNA sequencing and identification.
  • RNA storage devices could increase the functionality of RNA storage devices, transforming them from mere preservation tools to comprehensive RNA processing and analysis platforms.
  • RNA-detection steps multiple other downstream RNA detection and analysis methods could be utilized in the RNA-detection steps, such as for example, but not limited to, RT-PCR, RT-LAMP (loop-mediated isothermal amplification), direct RNA-sequencing, RNA-FISH.
  • RT-PCR RT-PCR
  • RT-LAMP loop-mediated isothermal amplification
  • direct RNA-sequencing RNA-FISH.
  • cellulose filter paper (3 x 4 mm 2 ), alpha cellulose Whatman filter paper (4 x 4 mm 2 ), wood pulp coffee filter (tree pulp) (4 x 7 mm 2 ), polyethersulfone (PES) membrane filter (3 x 5 mm 2 ), polyethylene foam (4 x 3 x 3 mm 2 ).
  • PES polyethersulfone
  • the saturated materials were extracted using stainless steel tweezers and transferred to sterile 24-well polystyrene plates capped with ventilated lids— these loose-fitting covers shield the samples from contaminants while permitting evaporation.
  • the assemblies were then left to dry at ambient room temperature for a duration of 24 hours.
  • HEK293FT Human embryonic kidney cells were cultured and expanded in standard medium (DMEM/10% FBS) in 5% CO2 and 37°C. Aliquots of 2 million cells were collected in a 1.5 mL microcentrifuge tube, washed with lx PBS, pelleted by brief centrifugation upon which the PBS supernatant was discarded, and frozen in a -80°C deep freezer. For the following storage experiments, cell pellets were diluted in a cell lysis solution, consisting of 1000 pL 1% Triton-XlOO, and pipetted up and down to achieve a lysed cell suspension.
  • DMEM/10% FBS standard medium
  • the lysed cell suspension was vortexed for 30 seconds then centrifuged at 300 g for 3 minutes to pellet potential cell debris, such as incompletely lysed nuclei and other potentially remaining cell compartments. Consequently, the lysed cell suspension contained the input equivalent of 2000 lysed HEK293FT cells per pL.
  • sample devices The dried, pre-treated materials (sample devices) were spotted with 1.25 pL of lysed cell suspension corresponding to 2500 lysed cells by pipetting the suspension onto the middle of the sample devices and letting the sample absorb and dry into the material.
  • the lysed-cell- spotted storage devices were then incubated/dried for 1 or 6 days at room temperature in 24-well polystyrene plates with ventilating lids.
  • Smart-seq2 cDNA library generation was performed as follows: Smart-seq2 lysis buffer composition was 2.5mM dNTP/each and 2.5mM Smart seq2 oligo-dT (5'-AAGCAGTGGTATCAACGCAGAGTACT30VN-3') with 1 pL of eluant in a total buffer volume 4.5 pL.
  • RNA samples in lysis buffer were incubated at 72°C for 3 min in a Bioer Life ECO thermocycler to hybridize the oligo- dT and placed on ice prior to first-strand synthesis.
  • the following reverse transcriptase reaction contained lx Superscript II buffer, 5mM DTT, IM betaine, 10 mM MgCh, 1 pM Smart-seq2 TSO (5'- AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3'), and 10 U Superscript II for a total reaction volume of 10 pL.
  • RT thermocycles were 42°C for 90 min, followed by 10 cycles of 42°C for 2 min and 50°C for 2 min.
  • the following cDNA amplification reaction contained lx KAPA HiFi HotStart Ready Mix and 80 nM ISPCR primers (5'- AAGCAGTGGTATCAACGCAGAGT-3'); total reaction volume 25 pL.
  • Thermocycles for Smart-seq2 cDNA amplification were 98°C for 3 min, followed by 18 cycles of 98°C for 20 sec, 67°C for 15 sec, and 72°C for 6 min, followed by a final incubation at 72°C for 5 min.
  • Amplified cDNA samples were bead purified (AMPure XP, Beckman) at a ratio of 0.8: 1 beads:cDNA (20 pL beads:25 pL cDNA for Smart-seq2) and inspected on a Bioanalyzer 2100 (High Sensitivity DNA kit, Agilent). cDNA yield and quality
  • the resulting purified Smart-seq2 cDNA libraries were analyzed using Agilent Bioanalyzer 2100 High sensitivity DNA chips.
  • the shape of full-length cDNA traces is known in the field to reflect the quality and integrity of the underlying mRNA sample (Trombetta 2014), and an intact library (satisfactory library) is expected to have a peak around ⁇ 2kb, reflecting the median length of full-length mRNA transcripts in human cells.
  • Exact patterns of spikes in the cDNA traces can further be experiment- or cell-type specific, as abundant cell-type- specific or condition-specific RNA transcripts, for which one or a few transcripts account for a large proportion of total cellular transcripts lead to additional peaks on the Bioanalyzer trace.
  • RNA dry-storage devices Multiple agents of the invention can be combined to achieve a mix of RNase inhibiting agents that could be used to impregnate RNA drystorage devices.
  • RNase inhibiting agents that could be used to impregnate RNA drystorage devices.
  • combining two or more of the chemical agents each demonstrated to be functional as RNase-inhibiting agent in the RNA dry-storage devices presented in EXAMPLE 1 were useful in RNA storage devices.
  • An enhanced capability to preserve RNA was gained from combinatorial effects of the different chemical agents in cocktail, relative to a single RNase inhibitor alone.
  • RNAse A, RNase, B, RNase C which are structurally non-identical, due to differences in conformational and steric properties both of RNases and RNase- inhibiting agents.
  • the chemicals selected for combinatorial testing were that showed promise individually in EXAMPLE 1 : sodium alginate (NaAIg), fucoidan, heparin, vinyl sulfonic acid (VSA), polyvinyl sulfonic acid (PVSA), 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid (HEPES), and dextran sulfate (DexSulf).
  • NaAIg sodium alginate
  • VSA vinyl sulfonic acid
  • PVSA polyvinyl sulfonic acid
  • HPES 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid
  • DexSulf dextran sulfate
  • the saturated materials were extracted using stainless steel tweezers and transferred to sterile 48-well polystyrene plates capped with ventilated lids— these loose-fitting covers shield the samples from contaminants while permitting evaporation.
  • the assemblies were then left to dry at ambient room temperature for a duration of 24 hours.
  • RNA filter paper (3 x 4 mm 2 ), wood pulp coffee filter (tree pulp) (4 x 7 mm 2 ), polyethersulfone (PES) membrane filter (3 x 5 mm 2 ), polyethylene foam (4 x 3 x 3 mm 3 ), and polyurethane foam (4 x 4 x 4 mm 3 ).
  • RNA filter paper (3 x 4 mm 2 ), alpha-cellulose Whatman filter paper (4 x 4 mm 2 ), wood pulp coffee filter (tree pulp) (4 x 7 mm 2 ), polyethersulfone (PES) membrane filter (3 x 5 mm 2 ), polyurethane foam (4 x 4 x 4 mm 3 ).
  • PES polyethersulfone
  • the materials were extracted using stainless steel tweezers and transferred to sterile 24-well polystyrene plates capped with ventilated lids— these loose-fitting covers shield the samples from contaminants while permitting evaporation.
  • the assemblies were then left to dry at ambient room temperature for a duration of 24 hours.
  • the impregnated materials were used for long-term (11 months) storage tests of lysed cells as described in the following sections.
  • HEK293FT Human embryonic kidney cells were cultured and expanded in standard medium (DMEM/10% FBS) in 5% CO2 and 37°C. Aliquots of 2 million cells were collected in a 1.5 mL microcentrifuge tube, washed with lx PBS, pelleted by brief centrifugation upon which the PBS supernatant was discarded, and frozen in a -80°C deep freezer. For the following storage experiments, cell pellets were diluted in a cell lysis solution, consisting of 1000 pL 1% Triton-XlOO, and pipetted up and down to achieve a lysed cell suspension.
  • DMEM/10% FBS standard medium
  • the lysed cell suspension was vortexed for 30 seconds then centrifuged at 300 g for 3 minutes to pellet potential cell debris, such as incompletely lysed nuclei and other potentially remaining cell compartments. Consequently, the lysed cell suspension contained the input equivalent of 2000 lysed HEK293FT cells per pL.
  • sample devices The dried, pre-treated absorbent materials (sample devices) were spotted with 1.25 pL of lysed cell suspension corresponding to 2500 lysed cells (lysed supernatant after centrifugation, as described in the previous section), by pipetting the suspension onto the middle of the sample devices and letting the sample absorb and dry into the material.
  • the lysed-cell-spotted storage devices were then incubated/dried for 3 days (combinatorial) or 11 months (long term storage) at room temperature in 24-well polystyrene plates with ventilating lids (loose-fitting lids protecting samples from dust but allowing evaporation).
  • the cell-spotted absorbent materials were individually placed in the wells of a sterile 96-well polystyrene plate, and sample material was eluted by adding 100 pL of nuclease-free water followed by incubation at room temperature for 3 minutes.
  • the 96-well plate was sealed using plastic PCR. sealing film, vortexed for 20 seconds, and the plates were finally centrifuged at 300 g for 1 minute.
  • the resulting eluates were immediately used as RNA input to generate full-length cDNA libraries using the Smart - seq2 protocol (Picelli 2013, Picelli 2014), using 1 pL of eluate as input to the Smart-seq2 reaction.
  • Smart-seq2 cDNA library generation was performed as follows: Smart-seq2 lysis buffer composition was 2.5mM dNTP/each and 2.5mM Smart seq2 oligo- dT (5'-AAGCAGTGGTATCAACGCAGAGTACT30VN-3') with 1 pL of eluant in a total buffer volume 4.5 pL.
  • RNA samples in lysis buffer were incubated at 72°C for 3 min in a Bioer Life ECO thermocycler to hybridize the oligo-dT and placed on ice prior to first-strand synthesis.
  • the following reverse transcriptase reaction contained lx Superscript II buffer, 5mM DTT, IM betaine, 10 mM MgCI2, 1 pM Smart-seq2 TSO (5'- AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3'), and 10 U Superscript II for a total reaction volume of 10 pL.
  • RT thermocycles were 42°C for 90 min, followed by 10 cycles of 42°C for 2 min and 50°C for 2 min.
  • the following cDNA amplification reaction contained lx KAPA HiFi HotStart Ready Mix and 80 nM ISPCR primers (5'- AAGCAGTGGTATCAACGCAGAGT-3'); total reaction volume 25 pL.
  • Thermocycles for Smart-seq2 cDNA amplification were 98°C for 3 min, followed by 18 cycles of 98°C for 20 sec, 67°C for 15 sec, and 72°C for 6 min, followed by a final incubation at 72°C for 5 min.
  • Amplified cDNA samples were bead purified (AMPure XP, Beckman) at a ratio of 0.8: 1 beads:cDNA (20 pL beads:25 pL cDNA for Smart-seq2) and inspected on a Bioanalyzer 2100 (High Sensitivity DNA kit, Agilent). cDNA yield and quality
  • the resulting purified Smart-seq2 cDNA libraries were analyzed using Agilent Bioanalyzer 2100 High sensitivity DNA chips.
  • the shape of full-length cDNA traces is known in the field to reflect the quality and integrity of the underlying mRNA sample (Trombetta 2014), and an intact library (satisfactory library) is expected to have a peak around ⁇ 2kb, reflecting the median length of full-length mRNA transcripts in human cells.
  • Exact patterns of spikes in the cDNA traces can further be experiment- or cell-type specific, as abundant cell-type-specific or condition-specific RNA transcripts, for which one or a few transcripts account for a large proportion of total cellular transcripts lead to additional peaks on the Bioanalyzer trace.
  • Polyurethane foam is a suitable sample carrier material for RNA drystorage
  • RNA integrity of the samples we used microfluidic gel electrophoresis (Agilent Bioanalyzer high-sensitivity DNA chips) and inspected the full-length Smart-seq2 cDNA libraries generated from eluates.
  • polyethylene foam, wood pulp, and cellulose pre-treated with 300 pg/mL PVSA yielded high-quality cDNA libraries with the characteristic high- molecular-weight peak around 2 kbp, indicating successful RNA preservation throughout dry storage in these conditions (Fig. 6, left panels).
  • samples stored in polyethersulfone did not yield cDNA (Fig. 6e).
  • Example 1- 2 We combinatorically characterized how different absorbent materials performed together with various chemical compounds serving as RNase inhibitors. For this testing, we selected six of the top-performing chemical RNase inhibitors based on our previous experimental data (Example 1- 2), specifically: sodium alginate (NaAIg) (400 pg/mL), fucoidan (10,000 pg/mL), heparin (20 pg/mL), vinyl sulfonic acid (VSA) (200,000 pg/mL), 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid (HEPES) (20,000 pg/mL), and dextran sulfate (250 pg/mL), or water.
  • NaAIg sodium alginate
  • fucoidan 10,000 pg/mL
  • heparin 20 pg/mL
  • VSA vinyl sulfonic acid
  • HEPES 4-(2-hydroxyethyl)-l-piperazine-ethanes
  • a storage device for a biological sample comprising an absorbent material impregnated with a solution of at least one chemical RNase inhibitor wherein the at least one RNase inhibitor or RNase inhibitors is/are selected from the group comprising sodium alginate (NaAIg), heparin, fucoidan, vinyl sulfonic acid (VSA), polyvinyl sulfonic acid (PVSA), 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic acid (HEPES), and dextran sulfate (DexSulf).
  • NaAIg sodium alginate
  • VSA vinyl sulfonic acid
  • PVSA polyvinyl sulfonic acid
  • HEPES 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic acid
  • DexSulf dextran sulfate
  • absorbent material is selected from the group comprising cellulose paper, alpha cotton cellulose paper, wood pulp paper, polyurethane foam, and polyethylene foam.
  • a kit comprising a storage device according to any one of the preceding items and a container comprising a lysis buffer.
  • a method of preserving and eluting RNA from a biological sample comprising the steps of a. Providing a storage device or a kit according to any one of the preceding items b. Providing to said storage device a biological sample comprising RNA c. Optionally allowing the storage device comprising said biological sample to dry d. Eluting RNA from said storage device comprising said biological sample
  • a storage device for a biological sample comprising an absorbent material impregnated with a solution of at least one chemical RNase inhibitor wherein the at least one RNase inhibitor or RNase inhibitors is/are selected from the group consisting of sodium alginate (NaAIg), fucoidan, vinyl sulfonic acid (VSA), and 4-(2- hydroxyethyl)-l-piperazine-ethanesulfonic acid (HEPES).
  • NaAIg sodium alginate
  • VSA vinyl sulfonic acid
  • HEPES 4-(2- hydroxyethyl)-l-piperazine-ethanesulfonic acid
  • a kit comprising a storage device according to any one of the preceding items and a container comprising a lysis buffer.
  • a method of preserving and eluting RNA from a biological sample comprising the steps of a. Providing a storage device or a kit according to any one of the preceding items b. Providing to said storage device a biological sample comprising RNA c. Optionally allowing the storage device comprising said biological sample to dry d. Eluting RNA from said storage device comprising said biological sample

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Abstract

L'invention concerne des dispositifs et des procédés de stockage de matériel biologique contenant de l'ARN, la stabilité de l'ARN étant augmentée.
PCT/EP2024/081517 2023-11-08 2024-11-07 Stockage à sec Pending WO2025099161A2 (fr)

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