METHOD AND KIT FOR THE PURIFICATION OF FUNCTIONAL RISC-ASSOCIATED SMALL RNAS
20220220467 · 2022-07-14
Inventors
Cpc classification
C12Q2527/125
CHEMISTRY; METALLURGY
C12N15/101
CHEMISTRY; METALLURGY
C12N15/101
CHEMISTRY; METALLURGY
International classification
Abstract
The invention relates to methods and kits for the purification of functional RISC-associated small RNAs in organisms, organs, tissues, cells or biological fluids.
Claims
1. Method for the purification of RISC-associated sRNAs, comprising the following steps: a) providing a native sample derived from a biological specimen; b) lysing the sample using a native lysis buffer; c) selectively removing non RISC-associated nucleic acids from the lysate; and d) collecting RISCs comprising RISC-associated sRNAs.
2. The method of claim 1, wherein in step c), non-RISC associated nucleic acids are removed from the lysate by loading the lysate onto a column comprising a resin allowing the fixation of nucleic acids.
3. The method of claim 1, wherein in step d) RISCs are collected by applying an elution buffer to the column.
4. The method of claim 2, wherein the resin is an anion exchange resin.
5. The method of claim 1, wherein in step b) the lysate is clarified by centrifugation or by filtration.
6. The method of claim 2, wherein the column is a 96 well plate or a microfluidic chip.
7. The method of claim 1, wherein the biological specimen is from a human.
8. The method of claim 1, wherein the biological specimen is a RISC-containing sample generated by in vitro, in cellulo or in vivo RISC production.
9. The method of claim 1, further comprising: e) removing the protein content from the collected RISCs using phenol/chloroform/isoamyl-alcohol extraction; or f) removing the protein content from the collected RISCs using proteinase K treatment.
10. A method of diagnosing a disease whose diagnosis or prognosis is characterized by the presence of particular sRNAs, comprising performing the method of claim 1 and analyzing the RISC-associated sRNAs to diagnose the disease.
11. The method of claim 10, wherein the disease is a cancer, a metabolic disorder, an hereditary condition or an infectious disease.
12. A kit for the purification of RISC-associated sRNAs, comprising: a native lysis buffer; an elution buffer; and a column comprising an anion exchange resin stored in storage buffer.
13. The kit of claim 12, wherein the lysis buffer comprises 20 mM HEPES-KOH (pH 7.9), 10 to 20% (v/v) glycerol, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT, 100 mM CH.sub.3CO.sub.2K and 0.1% Triton X-100, with a measured conductivity from 7.5 to 8.5 mS/cm.sup.2.
14. The kit of claim 12, wherein the elution buffer comprises 20 mM HEPES-KOH (pH 7.9), 10 to 20% (v/v) glycerol, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT and a CH.sub.3CO.sub.2K concentration comprised between 400 and 800 mM, to reach a measured conductivity comprised between 30 and 50 mS/cm.sup.2.
15. The kit of claim 12, wherein the storage buffer comprises 20 mM HEPES-KOH (pH 7.9), 10 to 20% (v/v) glycerol, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT, 100 mM CH.sub.3CO.sub.2K, with a measured conductivity from 7.5 to 8.5 mS/cm.sup.2, and 2 mM NaN.sub.3.
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
[0071] In one aspect, the present invention relates to methods for the purification of RISC-associated sRNAs, comprising the following steps: [0072] a) providing a native sample derived from a biological specimen containing RISC-associated sRNAs; [0073] b) lysing the sample using a native lysis buffer; [0074] c) selectively removing non RISC-associated nucleic acids from the lysate; and [0075] d) collecting RISCs comprising RISC-associated sRNAs.
[0076] The method according to the invention is called TraPR, standing for Transkingdom rapid and affordable Purification of RISCs.
[0077] Herein, the term “sRNAs” relates to small RNA molecules with a length of 18 to 40 nucleotides. The term is intended to cover microRNA (miRNA), Piwi-interacting RNA (piRNA), small interfering RNA (siRNA), scan RNA (scnRNA). In particular, the term refers to any sRNA molecule functionally associated with an Argonaute (AGO)-family protein as part of a RISC which may be engaged in gene regulation. The terms “sRNA”, “sRNA molecule” and “sRNAs” are used interchangeably.
[0078] The term “RISC-associated sRNAs” herein refers to sRNAs that are functionally incorporated into a RISC, e.g. via association with or loading onto an AGO-family protein. The term is not intended to cover free RNAs that do not interact with a RISC and/or are not loaded onto a RISC at the time of purification. RISC-associated sRNAs are the RNA type most interesting since their interaction with and loading onto an AGO-family proteins is a prerequisite for them to convey mRNA regulation. It is therefore highly preferable to isolate the RISC-loaded sRNA rather than the general pool of sRNAs present in a sample of interest. In other words, the invention is directed to a method of isolating only those sRNAs that are loaded onto a RISC at the time of purification i.e. RISC-loaded sRNAs.
[0079] The term “Argonaute (AGO)-family protein” herein refers to members of the Argonaute protein family which form the core components of any RISC acting at the RNA or DNA level. AGO proteins are evolutionary conserved among eukaryotes and can be separated into AGO and PIWI sub-families. All AGO/PIWI proteins comprise three key domains: PIWI, PAZ and Mid, and bind different classes of sRNAs which guide them to their specific targets through nucleotide sequence complementarity (base pairing). AGO-bound sRNAs might be functionally inert or might promote mRNA cleavage, enhanced mRNA decay and/or translation inhibition or, alternatively, chromatin compaction and/or altered transcription or, alternatively, physical genome modification/editing. While the complete suite of RISC components is yet to be fully elucidated, AGO proteins have been confirmed as invariable key elements of such complexes. Consequently, the terms “AGO-associated”, “AGO-bound”, “AGO-loaded”, “RISC-associated”, “RISC-bound” and “RISC-loaded” are herein used interchangeably.
[0080] The terms “associated” or “loaded” herein refers to non-covalent binding of a sRNA molecule to an AGO-family protein.
[0081] Like IP-based or AGO-PAPP methods, the principle of the invention is based on the notion that the most useful sRNA information will be contained within functional RISCs, i.e., those sRNAs potentially engaged in gene regulation by guiding an AGO. Isolating AGO-bound sRNAs also concomitantly offers the advantage of significantly eliminating other nucleic acids unrelated to sRNAs. However, unlike IP or AGO-PAPP, the method according to the invention does not isolate RISC-associated sRNAs based on immuno-enrichment or AGO-AGO-hook affinity, but instead by exploiting conserved biochemical properties exhibited by all known AGO/PIWI proteins loaded with sRNA cargoes, including, chiefly, a isoelectric point comprised between 9.3 and 9.8 under physiological conditions.
[0082] By contrast, under such conditions, all free nucleic acids are heavily negatively charged. Therefore, it is possible to selectively remove non RISC-associated nucleic acids from a native lysate. For example, the lysate may be exposed to a positively charged resin, so that all contaminating, free nucleic acids (RNA, DNA) remain bound to it and are thus removed from the lysate. Once the resin is washed, mild salt concentrations can be applied to collect RISC-associated sRNAs. Thus, the isolation principle of the method according to the invention is based upon retention as opposed to enrichment (see
[0083] The invention is directed to the purification of RISC-associated sRNAs from a sample, in particular to the purification of RISC-loaded sRNAs. The sample may be derived from a biological specimen such as cells, biological fluids, biopsies of a tissue, or organ of an animal, fungus, protozoan or plant. In one embodiment, the biological specimen is a whole organ of an animal, fungus, protozoan or plant. Likewise, the sample may be derived from a biological specimen of cell culture of animal, fungus, protozoan or plant cells. In this aspect, the sample may also comprise the supernatant from a cell culture specimen. The specimen may also consist of one or several whole organisms.
[0084] In a preferred embodiment, the sample is a biological specimen from a mammal, in particular a specimen derived from a human subject or a human patient. The specimen may be a whole-blood sample, a serum sample, a plasma sample, a cerebro-spinal fluid sample, a saliva sample, a lachrymal fluid sample, a urine sample, a stool sample, a lymph sample, a milk sample, a seminal fluid sample, an ascites or an amniotic fluid sample.
[0085] In another embodiment, the animal from which the specimen is derived is any animal of veterinary interest, including, but not restricted to, zoo animals, pets, cattle, poultry and fish. In another embodiment, the animal is a nematode or yeast.
[0086] In yet another embodiment, the specimen is a biological fluid from any plant, e.g. xylem or phloem.
[0087] Biological specimen may be fresh or frozen-stored as freezing does not modify the biochemical properties required for the application of the method according to the invention.
[0088] Biological specimens used to obtain the native samples according to the invention may be treated in order to facilitate purification of sRNAs prior to use as samples in the method of the invention. For example, the specimen may be washed with standard buffers (PBS for cell culture, M9 buffer for nematodes or sterile physiological water for biopsies derived from animals). Once the washing buffer is removed, dry pellets may be flash frozen in liquid nitrogen or dry ice and may be used as samples according to the invention.
[0089] Collected cells may be separated using suitable standard procedures. The skilled person knows how to select a suitable procedure for separating different cells in a specimen. Suitable methods may be Ficoll-Plaque® (GE Healthcare, 17-1440-02), Lymphoprep™ (STEMCELL Technologies, 07801) or fluorescence activated cell sorting (FACS). After sorting, cells may be pelleted by centrifugation. Once the washing buffer is removed from the pelleted cells, dry pellets may be flash frozen in liquid nitrogen or dry ice and may be used as samples according to the invention.
[0090] According to the invention, a biological fluid may be collected and subsequently flash frozen in liquid nitrogen or dry ice. Collected samples may be stored frozen, preferably at −80° C. The skilled person is aware that freezing/defrosting cycles should be minimized to preserve the quality of the material. In a preferred embodiment, aliquots of specimens are prepared prior to freezing, e.g. 2.5 million cells, 10 Drosophila ovary pairs, 20 mg of plant/animal material, 50 to 100 μL whole nematode or fungi pellets, 150 μL bio fluid.
[0091] In one embodiment of the invention, the biological specimen is a RISC-containing sample generated by in vitro, in cellulo or in vivo RISC production.
[0092] As used herein, the term “lysis” refers to destabilization, using detergents, of the cytoplasmic membranes, vesicles, organelles and nuclear envelopes of the sample, in order to access to their proteins content. The term “native lysis” refers to a lysis performed using detergents with low stringency at optimized concentration in order to retain the protein-protein interactions (protein complexes), RNA-protein interaction (ribonucleoprotein) and their enzymatic activity.
[0093] The skilled person is aware that the different buffers used for lysis and elution in any purification method should be compatible with each other. Consequently, the different buffers used in some embodiments of the invention are based on the same basic buffer and differ from each other only by the addition of specific compounds or adjustment of other properties, such as preserving agents, detergents or salt concentration necessary for the desired purpose (i.e., column storage, lysis, elution).
[0094] The buffers used in these embodiments are optimized to (i) solubilize RISCs while preserving the non-covalent interaction between sRNAs and AGO proteins, (ii) favor retention of all other nucleic acids on the positively charged matrix and (iii) allow AGO-bound sRNA separation based on differences in isoelectric point. These combined biochemical properties are obtained using specific salt concentrations which were surprisingly found by the inventors. In a preferred embodiment, potassium acetate (CH.sub.3CO.sub.2K) is used as the salt.
[0095] The specific salt concentrations in the different buffers used in the embodiments of the invention are monitored by conductivity measurements. The term “conductivity” herein refers to the ability of an electrolyte solution to conduct electricity. Conductivity measurement is a fast, inexpensive and reliable way to measure the ionic content (salt concentration) of a solution routinely used in industrial processes. The international unit for conductivity is Siemens per meter (S/m). The CH.sub.3CO.sub.2K concentration of the buffers used in these embodiments are adjusted by monitoring the buffer conductivity until specific values are reached.
[0096] In a preferred embodiment, the basic buffer consists of 20 mM HEPES-KOH adjusted to pH=7.9 to allow better pH stability over a range of temperatures; 10 to 20% (v/v) glycerol to preserve AGO-sRNA interactions; reducing agents such as 1 mM dithiothreitol (DTT) to prevent oxidization; and 0.2 mM EDTA to chelate divalent cations before addition of excess Mg.sup.2+ with 1.5 mM MgCl2.
[0097] In a preferred embodiment, the basic buffer is complemented with 2 mM NaN.sub.3 as a preserving agent and 100 mM CH.sub.3CO.sub.2K final to obtain the storage buffer (measured conductivity comprised between 7.5 and 8.5 mS/cm.sup.2). This buffer ensures optimal resin storage within the column.
[0098] In a preferred embodiment, the basic buffer is complemented with 0.1% (v/v) TRITON-X100 as zwitterionic detergent to solubilize the RISC content in basic buffer at 100 mM CH.sub.3CO.sub.2K final used for lysis (measured conductivity comprised between 7.5 and 8.5 mS/cm.sup.2).
[0099] In a preferred embodiment, buffers used in the invention are filtered at 0.22 μm and degassed. The buffers may further be validated by conductivity measurement and/or benchmarked sRNAs isolation. The buffers may further comprise additional, commercially available compounds suitable for protein and RNA stabilization.
[0100] According to the invention, the samples may be solid or liquid. Solid samples are mechanically transformed into powder using standard procedures adapted to the respective biological model, then homogenized in lysis buffer. Liquid samples are mixed with lysis buffer at a ratio of 1:1 (v/v). Lysis disrupts cell wall, membranes and the nuclear envelope, leading to subsequent RISCs solubilization. The skilled person is well aware of the properties of various lysing agents and of how to select an appropriate amount of lysing agent. In addition, the skilled person knows how to measure the effect of a lysing agent onto a given biochemical interaction, a given resin, using given salts and at a given pH.
[0101] Since the method of the invention aims at obtaining native state RISCs, the lysis buffer may not contain chaotropic agents such as guanidium salts or urea at concentrations that would dissociate the sRNA from the complex. In a preferred embodiment, the lysis buffer contains between 0.05% (v/v) and 0.2% (v/v) Triton X-100, preferably between 0.1% (v/v) and 0.2% (v/v) Triton X-100, most preferably 0.1% (v/v) Triton-X-100. The lysis buffer may additionally contain a zwitterionic detergent, such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and may be supplemented with an RNase inhibitor and/or a protease inhibitor.
[0102] The lysis buffer comprises or consists of 20 mM HEPES-KOH (pH 7.9), 10 to 20% (v:v) glycerol, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT and 100 mM CH.sub.3CO.sub.2K and 0.1% Triton X-100, with a measured conductivity from 7.5 to 8.5 mS/cm.sup.2. In a preferred embodiment, the lysis buffer have a measured conductivity of 8 mS/cm.sup.2.
[0103] In one embodiment, the lysate thus obtained can be directly used for subsequent steps, thereby facilitating handling of the sample.
[0104] In another embodiment, the native lysate is clarified from insoluble material once RISCs are solubilized in lysis buffer according to the invention. Such clarifying can be carried out through e.g. sedimentation, filtration or by a short centrifugation. Herein, the terms “spin” and “centrifugation” are used interchangeably.
[0105] In one embodiment, a centrifugation at 10 000×g, 4° C., 5 minutes is applied to the sample in order to remove insoluble material, such as, e.g., membrane debris or plant cell walls. The supernatant is transferred into a fresh tube, and constitutes the clarified lysate ready for purification of functional sRNAs according to the invention.
[0106] In another embodiment, the native lysate is clarified by filtration. For example, filtration is carried out by applying the lysate to a filtration column that retains insoluble material such as membrane debris or plant cell walls, but lets the solubilized RISCs pass through. Filtration can be carried out using centrifugation or by applying a vacuum to the lysate loaded filtration column. In a preferred embodiment the lysate is loaded onto a centrifugation column with a 0.45 μm cellulose acetate filter such as Costar Spin-X (Corning, Cat-No: CLS8162) and centrifugated for 5000×g, 4° C. for 2 minutes.
[0107] In yet another embodiment, the filtration membrane is included on top of the anion exchange resin in the column containing the resin, providing a first filtration compartment where the native lysate is added and filtered and a second compartment containing the anion exchange resin, thereby seamlessly integrating the clarifying filtration and the loading of the anion exchange resin. In a preferred embodiment the first filtration compartment is provided as a removable insert.
[0108] Clarifying the lysate offers the advantage that cell debris that could clog the column and otherwise hinder enrichment of the RISC-associated sRNAs are removed.
[0109] According to one embodiment of the invention, RISC-associated sRNAs are eluted from the column when an elution buffer is added to the column. The elution buffer differs from the lysis buffer by the absence of a detergent and by a higher salt concentration, validated by conductivity measurement. The elution buffer comprises or consists of 20 mM HEPES-KOH (pH 7.9), 10 to 20% (v/v) glycerol, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT and a CH.sub.3CO.sub.2K concentration comprised between 400 and 800 mM, to reach a measured conductivity comprised between 30 and 50 mS/cm.sup.2. In a preferred embodiment, the elution buffer have a measured conductivity of 40 mS/cm.sup.2.
[0110] The column according to one embodiment of the invention comprises a column body and an resin. The column body may have any suitable shape and volume. The volume of the column body depends on the desired application and may be between 50 μl and 50 L, preferably between 100 μl and 1000 μl, particularly preferably between 200 μl and 800 μl. In one embodiment, the column body may have a volume of 50 μl, 100 μl, 150 μl, 200 μl, 250 μl, 300 μl, 350 μl, 400 μl, 450 μl, 500 μl, 550 μl, 600 μl, 650 μl, 700 μl, 750 μl, 800 μl, 900 μl, 1000 μl. In a preferred embodiment, the volume of the column body is 1000 μl. In another preferred embodiment, the volume of the column body is 200 μl. A low volume of the column body allows working with minimal amounts of material. According to the invention, the column body is suitable for centrifuging the clarified sample and elution buffer in a 2 mL microcentrifuge tube in order to streamline the procedure. Typically, the column body is made of a 1000 μL polypropylene tube, but any other material showing similar properties may be used. In a preferred embodiment, Microspin™ columns (GE healthcare, GE27-35-650, REF 27356501) are used as column bodies.
[0111] In one embodiment, the column body is a 96 well plate. This allows using the method according to the invention for automated high throughput analysis. In another embodiment, the column is a microfluidic chip that also allows the automatization of the purification procedure.
[0112] According to one embodiment of the invention, the column body comprises an anion exchange resin stored, preferably in a storage buffer, i.e. the column body is packed with said resin. Any anion exchange resin may be used for the invention, as long as the system preserves the non-covalent interaction between sRNAs and AGO proteins, while allowing separation of all other AGO-free nucleic acids on the positively charged matrix to proceed with the separation. The skilled person knows how to choose a suitable anion exchange resin to allow for sRNA purification using different buffers, different salts and at different pH values. In a preferred embodiment, Q Sepharose HP resin (GE healthcare GE17-5072-01), is used as anion exchange resin in the method of the invention.
[0113] Before packing the anion exchange resin into the column body, the anion exchange resin may be equilibrated. In a preferred embodiment, the anion exchange resin is equilibrated in equilibration buffer comprising 20 mM HEPES-KOH (pH 7.9), 10 to 20% (v/v) glycerol, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT and 100 mM CH.sub.3CO.sub.2K, with a measured conductivity of 8 mS.Math.cm.sup.2. This influences the anion exchange resin's separation properties. According to the invention, the anion exchange resin may be washed 1, 2, 3, 4 or 5 times prior to packing the column, then re-suspended in a suitable volume of storage buffer and packed into the column body. In a preferred embodiment, the complete volume of the column body is packed with the anion exchange resin in order to maximize the column's separation property for a given elution volume. Typically, this consists in 8004 of storage buffer/resin in ratio of 3:5 in GE Microspin™ columns.
[0114] In another embodiment, the column body is only partially packed with the anion exchange resin in order to lower costs, considering that a lower column capacity can suffice for further sRNA analysis since the amount of sample may be decreased in proportion to the amount of resin. In another embodiment, the column body size and the volume of packed resin is decreased in order to allow sRNA purification from minute amount of samples, (e.g., 5000 Arabidopsis embryonic cells or up to one Arabidopsis flower bud).
[0115] Columns and buffers used in the invention are prepared in batches in advance, with each batch being validated by conductivity measurement and molecular assays, and subsequently stored at 4° C. The storage buffer, according to the invention, may additionally comprise compounds preventing microbial contamination, e.g., sodium azide (NaN.sub.3).
[0116] In a preferred embodiment, the elution step of the method of the invention is repeated for a second time. This ensures that all RISC-associated sRNAs are washed off the column, resulting in a higher yield of RISC-associated sRNAs. In another embodiment of the invention, the elution step may be performed by applying a gradient of increasing salt concentration.
[0117] The method according to the invention enables robust and consistent purification of AGO-associated sRNAs in the most complex organisms, the most recalcitrant tissues and/or from the most limiting amounts of starting biological material.
[0118] The kit according to the invention is conditioned, shipped and operational at room temperature, and overcomes the main caveats of other state-of-the-art methods by providing a highly simplified, universal and single-step anion-exchange purification procedure for RISC-associated sRNAs.
[0119] The method according to the invention can be run within 15 minutes on the bench with bare minimal laboratory requirements, thus greatly reducing work time and costs.
[0120] The method according to the invention is fully suitable for sRNA isolation from notoriously difficult-to-handle tissues including starchy plant storage roots or Heparin/EDTA-treated mammalian blood samples.
[0121] RISC-associated sRNAs isolated according to the invention are not affected by harsh conditions in the sample leading to global RNA degradation in the sample. In addition, RISC-associated sRNAs isolated according to the invention are immediately suitable for northern analysis, quantitative RT-PCR and microarray analysis, as well as deep-seq using any in-house or commercial cloning protocols, and all state-of-the-art sequencing platforms. By restricting the analysis to AGO-bound sRNAs, the invention allows a higher multiplexing of the sRNAs libraries prior to deep-seq, thereby significantly reducing costs of downstream analyses.
[0122] RISC-associated sRNAs isolated according to the invention are also particularly resilient to degradation and may be frozen after isolation.
[0123] The method according to the invention typically achieves >95% enrichment of the desired sRNA species over contaminating/degraded RNAs, thus providing unprecedented quality of the isolated sRNA.
[0124] The method according to the invention allows sRNA deep-seq with a yield, purity and quality at least on par with that achieved by gold standard size selection on gel, both in plants and animals. The method of the invention does not exhibit sequencing biases based on endo-siRNA and miRNA correlation analyses in either organisms.
[0125] In a preferred embodiment, the method of the invention is combined with the NEBnext® smallRNA library Prep kit (NEB, Ipswich, Mass., Catalog #E7330), allowing truly direct sRNA cloning bypassing altogether the step of post-PCR size selection of the library.
[0126] The RISC-associated sRNA fraction obtained when performing the method of the invention may be adapted to a downstream silicate-based separation procedure using commercially available columns to bypass the need for RNA precipitation altogether, thus allowing direct access to purified sRNAs in less than 30 minutes. Following RISCs purification, in one embodiment of the invention, a de-protenization step using phenol/chloroform/isoamylic alcohol is performed, followed by isopropanol-based precipitation to allow sRNA recovery, in order to remove the protein content from the collected RISCs. This step, however, requires long incubation at low temperature followed by centrifugation at high speed which is technically challenging and time consuming.
[0127] Although nucleic precipitation is a cost effective and high output standard procedure, commercially available silicate columns can be used to directly isolate sRNAs after de-protenization. In one embodiment of the invention, the method entails fixation of sRNAs after removal of the protein content to a column matrix in the presence of alcohol, thereby relying on hydrophobic interaction. The sRNAs may then be washed from impurities and finally eluted in water. In a preferred embodiment, ZYMO microspin IC columns (Zymo Research, Freiburg, Germany, REF C1004-50) are used according to the manufacturer's instruction. The inventors have surprisingly found that the sRNA output versus elution volume are optimal compared to similar commercially available systems, thus allowing for subsequent sequencing.
[0128] The inventions also relates to a kit for the purification of AGO-associated sRNA, comprising [0129] a) a lysis buffer; [0130] b) an elution buffer; and [0131] c) a column comprising an anion exchange resin.
[0132] The kit according to the invention may comprise buffers as described above.
[0133] The present invention may be used for the diagnosis of diseases or subtypes of diseases associated with the presence of particular sRNAs. For example, in human, increased levels of the Let-7 miRNA are nearly always positively correlated with lung cancer, while miR-21 levels are usually increased in glioblastoma and breast cancer. miR-15a/16a is frequently absent or strongly reduced in B-cells leukemia while miR-155 is increased in B-lymphoma and breast cancer. miRNA profiling may also be used to refine the state or the complex composition of tumors in biopsies, and may also help determining the tissues from which a tumor might originate during metastasis or in case of undifferentiated tumors. All these miRNA-based readouts hold great promises in terms of profiling cancers and their origins, effectively granting much more personalized and targeted disease treatment. The potential use of miRNAs as biomarkers is not restricted to cancer and may apply to any condition in which cellular homeostasis is perturbed by say, a metabolic or genetic dysfunction, or an infection. For instance, elevated miR-122 in hepatocytes is usually a sign of liver dysfunction and/or hepatitis virus infection. Examples of the few circulating miRNAs already being used as biomarkers include miR-14 in the plasma, whose abundance has become a major criterion for the unambiguous and non-invasive diagnostic of prostate cancer; high plasma levels of miR-141 are associated with poor prognosis in colorectal cancer. Several other circulating, plasma miRNAs have been correlated with cardiovascular diseases, organ dysfunctions (kidney/miR-215, liver/miR-122, pancreas/miR-375) or even complicated gestation due to trophoblast/placental defects.
[0134] Consequently, the invention also relates to a method to identify sRNAs as biomarkers of a pathological state, to diagnose such pathological state and ultimately to provide information on its possible evolution (prognosis). Given that the method of the invention yields highly pure AGO-associated sRNAs even from notoriously recalcitrant specimen, such as plasma, it can be used for identification of circulatory biomarkers. This implies access to plasma samples from a cohort of healthy individuals comparable in gender and age to a cohort of patients. The method according to the invention may be used to purify plasma-borne AGO-associated sRNAs to be then subjected to deep-seq. The resulting datasets, corresponding to circulating sRNA populations of each individual may subsequently be analyzed in order to determine significant differences in term of identity or abundance (up- or down-regulation) of a particular set of sRNAs in patients versus healthy individuals. The relevance of such markers for diagnosis/prognosis may be then validated on an independent set of patients. Once a set of sRNAs identified as biomarkers is validated, the method of the invention may be coupled to targeted quantification via qRT-PCR in patients. The inventors, in collaboration with clinicians, have already obtained preliminary results demonstrating how the method according to the invention can be used reliably to identify patients afflicted by a rare auto-inflammatory disease.
Embodiments
[0135] (1) A method for the purification of RISC-associated sRNAs, comprising the following steps: [0136] a) providing a native sample derived from a biological specimen; [0137] b) lysing the sample using a native lysis buffer; [0138] c) selectively removing non RISC-associated nucleic acids from the lysate; and [0139] d) collecting RISCs comprising RISC-associated sRNAs.
[0140] (2) A method according to embodiment (1), wherein in step c), non-RISC associated nucleic acids are removed from the lysate by loading the lysate onto a column comprising a resin allowing the fixation of nucleic acids.
[0141] (3) A method according to embodiment (1) or (2), wherein the lysis buffer comprises or consists of 20 mM HEPES-KOH (pH 7.9), 10 to 20% (v:v) glycerol, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT and 100 mM CH.sub.3CO.sub.2K and 0.1% Triton X-100, with a measured conductivity from 7.5 to 8.5 mS/cm.sup.2.
[0142] (4) A method according to embodiment (1) or (2), wherein in step d) RISCs are collected by applying an elution buffer to the column.
[0143] (5) A method according to embodiment (3), wherein the elution buffer comprises or consists of 20 mM HEPES-KOH (pH 7.9), 10 to 20% (v/v) glycerol, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT and a CH.sub.3CO.sub.2K concentration comprised between 400 and 800 mM, to reach a measured conductivity comprised between 30 and 50 mS/cm.sup.2.
[0144] (6) A method according to any of the embodiments, wherein the resin is an anion exchange resin.
[0145] (7) A method according to any of the embodiments, wherein in step b) the lysate is clarified by a short centrifugation or by filtration.
[0146] (8) A method according to any of the embodiments, wherein the column is a 96 well plate or a microfluidic chip.
[0147] (9) A method according to any of the embodiments, wherein the specimen is from a human.
[0148] (10) A method according to any of the embodiments, wherein the biological specimen is a RISC-containing sample generated by in vitro, in cellulo or in vivo RISC production.
[0149] (11) A method according to any of the embodiments, further comprising: [0150] e) removing the protein content from the collected RISCs using phenol/chloroform/isoamyl-alcohol extraction; or [0151] f) removing the protein content from the collected RISCs using proteinase K treatment.
[0152] (12) A method according to any of the embodiments for use in the diagnosis of a disease whose diagnosis or prognosis is characterized by the presence of particular sRNAs.
[0153] (13) A method for use according embodiment (10), wherein the disease is a cancer, a metabolic disorder, an hereditary condition or an infectious disease.
[0154] (14) A kit for the purification of RISC-associated sRNAs, comprising: [0155] a native lysis buffer; [0156] an elution buffer; and [0157] a column comprising an anion exchange resin stored in storage buffer.
[0158] (15) A kit according to embodiment (12), wherein the lysis buffer comprises 20 mM HEPES-KOH (pH 7.9), 10 to 20% (v/v) glycerol, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT, 100 mM CH.sub.3CO.sub.2K and 0.1% Triton X-100, with a measured conductivity from 7.5 to 8.5 mS/cm.sup.2.
[0159] (16) A kit according to embodiments (12) or (13), wherein the elution buffer comprises 20 mM HEPES-KOH (pH 7.9), 10 to 20% (v/v) glycerol, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT and a CH.sub.3CO.sub.2K concentration comprised between 400 and 800 mM, to reach a measured conductivity comprised between 30 and 50 mS/cm.sup.2.
[0160] (17) A kit according to embodiments (12) to (14), wherein the storage buffer comprises 20 mM HEPES-KOH (pH 7.9), 10 to 20% (v/v) glycerol, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT, 100 mM CH.sub.3CO.sub.2K, with a measured conductivity from 7.5 to 8.5 mS/cm.sup.2, and 2 mM NaN.sub.3.
EXAMPLES
Example 1
[0161] Principle of the Method According to the Invention
[0162] The scheme presented in
[0163] Detailed Procedure for the Use of Mini-TraPR Kit
[0164] As depicted in the scheme presented in
[0165] Buffers
[0166] Resin equilibration buffer (20 mM HEPES-KOH pH=7.9, 10% (v/v) glycerol, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT, conductivity adjusted at 8 mS.Math.cm.sup.−2 with potassium acetate at an indicative concentration of ˜0.1M))
[0167] Resine storage buffer (20 mM HEPES-KOH pH=7.9, 10% (v/v) glycerol, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT, 0.2 mM sodium azide, conductivity adjusted at 8 mS.Math.cm.sup.−2 with potassium acetate (KoAc) at an indicative concentration of ˜0.1M)
[0168] TraPR Lysis buffer (20 mM HEPES-KOH pH=7.9, 10% (v/v) glycerol, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT, 0.1% (v/v) Triton X100, conductivity adjusted at 8 mS.Math.cm.sup.−2 with KoAc at an indicative concentration of ˜0.1M)
[0169] TraPR Elution buffer (20 mM HEPES-KOH pH=7.9, 10% (v/v) glycerol, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT, conductivity adjusted at 40 mS.Math.cm.sup.−2 with KoAc at an indicative concentration of ˜0.44M)
[0170] TraPR High Salt buffer (20 mM HEPES-KOH pH=7.9, 10% (v/v) glycerol, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT, conductivity of the HS buffer is adjusted at 110 mS.Math.cm.sup.−2 with KoAc at the indicative concentration of ˜4M)
[0171] 1. Sample Lysis and Clarification
[0172] Lyse flash frozen samples in 400 μL TraPR Lysis buffer (nitrogen precooled mortar, dounce, or others method);
[0173] Transfer lysate into a fresh DNase, RNAse free 1.5 mL microcentrifuge tube;
[0174] Clarify lysate is by centrifugation at 10 000×g, 5 minutes, 4° C.;
[0175] Transfer 300 μL of clarified lysate into a fresh DNase-, RNAse-free 2 mL microcentrifuge tube;
[0176] 2. Mini TraPR Kit Column Preparation
[0177] Re-suspend the resin in the column by vortex few second;
[0178] Loosen the cap one quarter turn and twist off the bottom closure (do not trash the bottom closure);
[0179] Place the column in a 2 mL microcentifuge tube;
[0180] Centrifuge 15 seconds on bench microcentrifuge;
[0181] Discard the collection tube, and place the column on fresh 2 mL microcentrifuge tube labeled “TraPR E fraction”
[0182] 3. Sample Application
[0183] Close the column with the bottom closure;
[0184] Open the column cap, apply 300 μL of sample to the top center of the resin;
[0185] Close column cap and mix sample and resin by inverting vigorously;
[0186] Remove the bottom closure, and place the column into previous 2 mL microcentrifuge tube labeled “TraPR E fraction”
[0187] 4. Elution of RISC Loaded SmallRNAs
[0188] Centrifuge 15 seconds on bench microcentrifuge, save flowthrough;
[0189] Open the column cap, apply 300 μL of TraPR Elution Buffer;
[0190] Close the column and place into previous 2 mL microcentrifuge tube labeled “TraPR E fraction”;
[0191] Centrifuge 15 seconds on bench microcentrifuge;
[0192] Repeat step steps 15 to 17 once, collect eluate in the same 2 mL microcentrifuge tube labeled “TraPR E fraction”;
[0193] Close the “TraPR E fraction” collection tube (900 μL) at store on ice for immediate use, or store at −80° C. This fraction contains the RISCs proteins (AGOs) loaded with their cognate sRNAs.
[0194] 5. RNA Extraction (Precipitation)
[0195] Add 500 μL acidic PCI to the fraction collected;
[0196] Vortex vigorously;
[0197] Centrifuge 5 min, full speed, 4° C.;
[0198] Collect the aqueous phase in new 1.5 mL microcentrifuge tube;
[0199] Add 10% of NaAcetate 3M, pH 5.2 and 1 μL Glycogen, homogenize;
[0200] Add 120% cold isopropanol, homogenize;
[0201] Incubate at least 30 min at −20° C.;
[0202] Centrifuge 30 min, full speed, 4° C.;
[0203] Discard liquid phase;
[0204] Add 400 μL cold 80% ethanol;
[0205] Centrifuge 5 min, full speed, 4° C.;
[0206] Repeat steps 29 to 30 two more time;
[0207] Remove all ethanol;
[0208] Add adapted volume of water (or LMNb loading buffer 1×) to the pellet;
[0209] Resuspend by pipetting up and down (can be heated few minutes at 60° C.);
[0210] RNA can be stored at −80° C., or immediately used.
[0211] 6. RNA Extraction (Zymo Ic Microspin Silicate Column)
[0212] Add 500 μL acidic PCI to the fractions collected;
[0213] Vortex vigorously;
[0214] Centrifuge 5 min, full speed, 4° C.;
[0215] Collect the aqueous phase in 2 new 1.5 mL microcentrifuge tubes (split the collected phase in 2 equal volumes) ;
[0216] Add 2 volumes of RNA MAX buffer in each tubes and mix well;
[0217] Transfer 800 μL of the mixture in Ic column, placed into a collection tube;
[0218] Centrifuge 30 sec., 12 000 g, discard the flow through;
[0219] Repeat steps 41 and 42 until all the mixture is passed through the column;
[0220] Add 400 μRNA Prep buffer to the column;
[0221] Centrifuge 1 min., 12 000 g, discard the flow through;
[0222] Add 800 μL RNA Prep buffer to the column;
[0223] Centrifuge 30 sec., 12 000 g, discard the flow through;
[0224] Repeat steps 46 and 47 with 400 μL RNA Prep buffer;
[0225] Centrifuge 2 min, 12 000 g the column to completely remove the buffer;
[0226] Transfer the column on a new 1.5 mL microcentrifuge tube;
[0227] Add 6 to 15 μL of water pre warmed at 60° C. on the resin;
[0228] Incubate at room temperature for 1 minute;
[0229] Centrifuge 1 min., 10 000 g to elute RNA;
[0230] Recovered RNA can be used immediately, or stored at −80° C.
Example 2
[0231] Arabidopsis AGOs and Their Associated sRNAs Co-Purify in the RISCs-Enriched E Fraction According to the Method of the Invention
[0232] The Arabidopsis thaliana genome encodes 10 paralogous AGO genes of which 9 are expressed as proteins classified into 3 major phylogenetic clades, as depicted in
[0233] To extend this analysis to the entire Arabidopsis AGO protein family (nine members), protein analysis by immunoblot was conducted using antibodies to detect 7 endogenous AGO proteins following RISC purification form Arabidopsis, using an elution buffer adjusted to 40 mS/cm.sup.2 according to the method of the invention (
[0234] Deep-seq analyses shows that total plant sRNAs are constituted of two major species of diagnostic, discrete length: highly abundant, 24-nt heterochromatic siRNAs derived from transposons and repeats are loaded into AGO4-clade AGOs whereas abundant 21-nt sRNAs are composed of mostly miRNAs loaded into AGO1-clade AGOs and of sRNAs loaded into AGO2. To test the purity of sRNAs purified according to the method of the invention, all fractions (I, E and HS) were subjected to 5′-end labelling with polynucleotide kinase (PNK).
[0235] The results presented in
[0236] The Method of the Invention Defines a Universal RISCs Purification Procedure in a Broad Range of Organisms
[0237] Given that AGO proteins and their interaction with sRNAs to form RISCs is a highly conserved feature in all kingdoms of life, the purification procedure according to the method of the invention was tested in a range of organisms. The schematic phylogenetic tree presented in
[0238] Ciliates can be studied at synchronized stages representative of either the vegetative stage (WT) accumulating 23-nt siRNAs or of the sexual stage (T0) accumulating 25-nt long scnRNA uniquely involved in DNA elimination. No commercial antibody is available against ciliate AGO/PIWI proteins. Nonetheless, 5′-end labelling by PNK following purification according to the method of the invention shows a specific and strong enrichment, in the E fraction of cognate 25-nt scnRNA species in sexual stage cells. These are otherwise barely detectable, if at all, in the I and HS displaying instead strong labelling of longer RNAs.
[0239] Similar analyses were performed in two key staple crops using 5′-end labeling of sRNAs purified from lysates of rice leaves and cassava storage roots according to the method of the invention. Rice, unlike Arabidopsis, has a waxy leaf cuticle not favorable to RNA extraction. The rice genome is also much more replete in transposons and repeats such that the 24-nt siRNAs are disproportionately abundant. The results of 5′-end labelling after RISCs purification according to the method of the invention is in full agreement with this notion, showing a strong 24-nt sRNA and more moderate 21-nt sRNA enrichment in the E compared to the I and HS fractions. A second analysis involved lysate of cassava storage roots, which, due to their extremely high starch content, constitute an even more challenging tissue for RNA extraction. The cassava used for the analysis is a farmer-preferred genotype grown in Africa. As already observed in Arabidopsis and rice, 5′-end labelling following RISCs purification according to the method of the invention showed a strong enrichment in 21-nt and 24-nt sRNA species and a near-absent background in the E, but not in the I or HS fraction. Importantly, and unlike in Arabidopsis, no antibody is currently available against cassava AGO proteins, precluding RISC isolation via immunoprecipitation. These results demonstrate that the purification according to the method of the invention allows fast and robust isolation of RISCs from crops notoriously recalcitrant to RNA extraction and for which no AGO antibody is available.
[0240] Similar analysis were conducted in a variety of fungal and metazoan samples ranging from unicellular yeast to more complex organisms such as whole C. elegans or mouse whole organs. In fission yeast, heterochromatic siRNAs derived from pericentromeric repeats constitute the largest, if not unique, bulk of sRNAs. Their size is less well defined than in other organisms, but still around 23-nt. The 5′-end labelling by PNK, following RISC purification according to the method of the invention, shows a very strong enrichment of 23-nt siRNAs in the E compared to I and HS fractions displaying instead labelling of longer RNAs. This result is remarkable because S. Pombe's heterochromatic siRNAs are typically undetectable by northern analysis, even using sRNA species-specific radiolabeled probes.
[0241] Worms possess a complex sRNA machinery involving more than 25 proteins in the AGO/PIWI family for which only few commercial antibodies are available and reliable. As seen with all other examples, 5′-end radiolabeling of RNA reveals a strong enrichment of sRNAs in the E but not I or HS fractions following purification according to the method of the invention. Also as seen with all other organisms, the E fraction is markedly devoid of background labelling unlike the I and HS in which mostly long RNA contaminants or breakdown products are labelled.
[0242] RISCs purification according to the method of the invention have been tested mouse adult brain. 5′-end radiolabeling reveals an enrichment of sRNAs centered on 22-nt (the cognate size of mammalian Dicer products) with, again, low background in the E, unlike in the I and HS fractions in which mostly long RNA contaminants or breakdown products are labelled. To generalize the notion that the method according to the invention enables universal isolation of RISCs including that of metazoan-specific piRNAs, similar experiments were conducted on lysates from mouse testis, a tissue where piRNAs are highly expressed. As seen with all other examples, 5′-end radiolabeling reveals an enrichment of sRNAs centered on 30-nt (the cognate size of mouse piRNAs) with, again, low background in the E, unlike the I and HS fractions in which mostly long RNA contaminants or breakdown products are labelled.
[0243] We conclude from all these analyses that the purification according to the method of the invention can be used for fast and robust isolation of RISCs across all kingdoms of life including from tissues notoriously recalcitrant to RNA extraction. The RISCs co-purify with their cognate cargoes, be they siRNAs, miRNAs, piRNAs, or scnRNAs defining the full range of all currently known silencing small RNAs.
Example 3
[0244] RISC-Associated sRNAs Purified by the Method of the Invention are Directly Amenable to Silicate-Based Extraction, Bypassing Precipitation Step
[0245] As described above, the method according to the invention allows access to RISCs-associated sRNAs. Following sample lysis, the E fraction can be generated in 15 minutes, from which sRNAs are usually extracted directly although this fraction might be also stored at −80° C. (the AGO/PIWI-bound sRNA are particularly resilient to degradation).
[0246] sRNA are commonly extracted from RISCs with phenol followed by alcohol precipitation which takes a minimum of 90 minutes to a full day in total (
[0247]
[0248] The coupling of Zymo™ micro columns decreases (30 minutes) the time required to access RISC-associated sRNAs according to the method of the invention (
[0249] RISC-Associated sRNAs Purified by the Method of the Invention are Highly Suitable for miRNA Detection Via RT-qPCR in Various Biological Systems
[0250] Deep-seq remains a gold standard to identify and quantify, at the whole-genome scale and without a priori, the sRNA populations within a given biological sample. Despite its increasing affordability, (economically and technically) the systematic use of Deep-seq to investigate biological processes or for mere diagnosis is still prohibitive for most research laboratories, notwithstanding the expertise required for large sRNA data curation/analysis. In most cases, Deep-seq is used as a downstream procedure for identifying robust sRNA candidates linked to a particular process, cellular state or pathology. Once such candidates are validated, the preferred downstream method relies upon targeted RT-qPCR-based quantification of these sRNA candidates as opposed to genome-wide sRNA sequencing. RT-qPCR allows accurate quantification of multiple sRNA sequences on a large number of samples, at a modest cost.
[0251] The main limiting aspect of RT-qPCR-based sRNA quantification is the reverse-transcription (RT) step, where specific sRNA sequences are reverse-transcribed into cDNA to enable the downstream PCR amplification. The complexity of the RNA preparation including the potential low abundance of the sRNA sequence of interest might indeed compromise the RT efficiency, thereby negatively impacting the quality and robustness of quantification. Given that the purification according to the method of the invention dramatically enriches RISC-associated sRNAs in the E fraction, its suitability for miRNA quantification was tested using an in-house loop-based RT-qPCR procedure on sRNAs purified from Arabidopsis inflorescences. With the RISC-associated sRNAs purification according to the method of the invention, the miRNAs tested (miR159, miR171) were enriched in the E compared to HS fraction. The HS fraction was, by contrast, enriched in the Arabidopsis small nucleolar RNA snoRNA85, which is not loaded into any AGO (FIG. 4C). In a second example involving a sRNA-rich mammalian tissue such as the mouse liver, a similar pattern was observed with the enrichment of two mammalian miRNAs in the E fraction and of snoRNA202 in the HS fraction, respectively (
Example 4
[0252] RISC-Associated sRNAs Purified by the Method of the Invention are Directly Amenable to Deep-Seq in a Range of Organisms
[0253] As shown using radiolabeling of RNAs in
[0254] Analyses in Arabidopsis were conducted in three technical triplicates from the same batch of inflorescences. We compared the results of deep-seq from sRNA isolated via of three independent gel size selection events, three independent TRIzol-based extraction of total RNA, or three independent batches of RISC-associated sRNAs purified according to the method of the invention without size selection on gel (
[0255] Comparative deep-seq analyses were also conducted on Drosophila ovaries in which extraction and cloning of sRNAs provides a high-level benchmark in terms of difficulty and tediousness. In this complex tissue, three sRNAs classes are found: 22-nt miRNAs and 21-nt siRNAs are respectively loaded into AGO1 and AGO2. By contrast, the metazoan-specific piRNAs, 23-to-29-nt in length, are loaded into PIWI proteins. piRNA sequencing has been very much optimized in the fly, due to the highly abundant 2S rRNA (30-nt) in the same size range. The current, laborious, procedure entails first to gel-select, in a very precise manner, sRNAs with a length comprised between 18 and 29 nucleotides. In a second step, the purified sRNAs are ribo-depleted using commercial kits, then oxidized in order to remove RNA not harboring a 3′ methyl group. Metazoan siRNAs and piRNAs harbor this modification and are thus protected from oxidization unlike miRNAs or the 2S rRNA. A major caveat is that, after oxidization, the sample is depleted not only from the main contaminant (2S), but also from the information encoded by miRNAs, which is highly valuable nonetheless.
[0256] In a second set of experiments, a deep-seq analysis was conducted on RNA from Drosophila ovaries. Libraries were generated in biological duplicates using an optimized in-house cloning procedure developed in the Brennecke laboratory (IMBA, Vienna), in which gel selected sRNAs are ribodepleted, then oxidized. This golden standard was compared to direct cloning of RISC-associated sRNAs purified according to the method of the invention without any ribodepletion and oxydization (
[0257] A clustering analysis was conducted on the miRNA content of the three library types. The heatmap in
[0258] One recurring and legitimate question raised by the use of the method of the invention concerns the qualitative and quantitative correlation of sRNA content yielded by the various procedures. In other words, by selecting exclusively functional i.e. RISC-associated sRNAs, does the method according to the invention induce an underrepresentation or a loss of certain sRNA species isolated via other methods? To address potential biases, a correlation analysis was conducted on the sRNA content of all Drosophila libraries (
[0259] Taking ribodepleted sRNA libraries as reference, a good correlation (>0.99) is observed when comparing the miRNA- and TE-mapping reads (piRNAs and siRNAs) between the ribodepleted and RISC-associated sRNAs libraries (
Example 5
[0260] The Method of the Invention Isolates Native RISCs and Improves the Quality of Immunoprecipitation
[0261] In Arabidopsis, AGO1- and AGO2-clade AGOs preferentially associate with 21-nt long sRNA species starting with a 5′ Uracil (U) whereas the AGO4 clade associates preferentially with 24-nt species starting with a 5′ Adenine (A). To confirm that the native RISC purification enabled by the method of the invention is applicable to the complete suite of Arabidopsis AGO proteins, a 5′ nucleotide analysis was conducted using the sRNA sequencing libraries presented in
[0262] The method according to the invention isolates functionally active pools, i.e. AGO-loaded, sRNAs and, as such, infers the purification of native RISCs. To confirm the native state of isolated RISCs, Arabidopsis AGO1 immunoprecipitation (IP) experiments were conducted in parallel from total lysates or RISCs-enriched E fractions isolated according to the method of the invention. The experiment was conducted in inflorescences from a transgenic Arabidopsis line expressing Flag-tagged AGO1 under its endogenous promoter, which is detected with a commercial anti-Flag antibody. The protein blot presented in
Example 6
[0263] RISC-Associated sRNAs Purified by the Method of the Invention are Highly Resilient to Degradation
[0264] RNAs are unstable molecules sensitive to degradation at any step of their preparation and handling, from sample collection to long-term storage. Although their loading into AGO proteins makes the regulatory sRNAs more stable than other RNA species, the degradation products of long RNA will strongly contaminate sRNA libraries prepared from samples of suboptimal quality via total RNA or sRNA gel-size selection. Since the method of the invention isolates RISCs in which sRNAs are bound to their cognate AGO effectors, its use was anticipated to strongly select against longer RNA degradation products found in suboptimal quality samples, thereby potentially enabling high quality sRNA deep-seq libraries to be prepared even from highly degraded RNA preparations. To test this idea, the non-clarified lysate from a mouse liver was treated with RNase T1 and incubated at room temperature for 30 minutes before being subjected to sRNA purification according to the method of the invention. Deep-seq libraries were prepared in biological triplicates, from input (total RNA) and RISC-associated sRNAs, using intact or RNAse-treated samples.
[0265] Prior to deep-seq, low molecular weight RNA blot analysis was conducted (
[0266] This notion was indeed confirmed upon inspection of the sequencing results from the sRNA libraries prepared with the standard Truseq (Illumina) procedure. The annotation profile in
[0267] To test potential biases of the method according to the invention when it is applied to degraded samples, a correlation analysis was conducted with the sRNAs sequenced from the various libraries. The results presented in
[0268] This result is highly relevant for work conducted with unstable samples, samples collected at various time intervals and/or stored under various conditions including some not preventing RNA degradation. The method of the invention is therefore uniquely suited to the study of large cohorts of patient-derived biopsies or biological fluids, which are prone to degradation and collected sometimes over many years (e.g. >10 years). This would normally strongly limit robust comparisons of sRNA cohorts contained in the samples via deep-seq, but the method according to the invention enables normalization of sRNA libraries through their RISCs contents.
Example 7
[0269] The Method According to the Invention Enables Highly Reproducible and Robust sRNA Isolation From Mammalian Plasma
[0270] The complexity, RNA-degradation proneness and very low sRNA content of mammalian plasma has so far drastically impeded the robust exploration of sRNA biomarkers in this, and other body fluids, in clinical research. The same impediment applies to the use of RT-qPCR to reliably detect already identified circulating biomarkers for diagnosis/prognosis. We thus tested the performance of the method according to the invention with mouse plasma to assess if the procedure could remedy these major, often unsurmountable, burdens. Plasma samples were collected from four individual mice. Each sample was subjected to total RNA extraction from 150 μL of plasma, or RISCs-associated sRNA purification according to the method of the invention, from the same volume. For all conditions, the RNA was cloned following the smallRNA library preparation kit produced by Lexogen.
[0271]
[0272] Exploration of sRNAs typifying a tissue- or cell-type-specific state or pathological condition, is usually achieved via deep-seq. Once robust sRNA markers of this state/condition are identified, they can then be used in a targeted manner as quantitative indicators of said state/condition. The results presented in
[0273] During the validation of the method according to the invention, as a robust asset to produce high quality sequencing libraries of functional sRNA in complex samples (Arabidopsis and Drosophila), different cloning strategies were applied. A standardized TRUseq (Illumina) procedure was applied for plant samples (
[0274] We aimed at confirming this hypothesis by comparing yet another commercial cloning kit (produced by Lexogen) with the Illumina TRUseq. The results presented in
[0275] We conclude that the method of the invention is amenable to high-quality sRNAs preparation suitable for RT-qPCR quantification and deep-seq analysis including, chiefly, of miRNAs. The method according to the invention therefore opens great prospects for improved diagnosis/prognosis in terms of reproducibility and depth, offering the guarantee of consistent and robust detection of qualitative and quantitative variations in complex in a multitude of samples including mammalian plasma.