Microrna is separated from biofluid
阅读说明：本技术 从生物流体中分离微小rna (Microrna is separated from biofluid ) 是由 C.克里德 于 2014-11-25 设计创作，主要内容包括：本公开提供用于从生物流体中分离miRNA的方法和试剂盒。具体地说,所述方法包括使生物流体与表面活性剂和抗miRNA结合蛋白试剂接触,其中所述表面活性剂使生物流体组分解离并且所述抗miRNA结合蛋白试剂与和miRNA缔合的miRNA结合蛋白相互作用从而形成免疫沉淀miRNA复合物；并且将miRNA从所述免疫沉淀miRNA复合物中释放。进一步提供可与循环miRNA缔合的miRNA结合蛋白的实例。(The disclosure provides the method and kit for separating miRNA from biofluid.Specifically, the method includes contacting biofluid with surfactant and anti-miRNA binding protein reagent, wherein the surfactant make biofluid component dissociate and the anti-miRNA binding protein reagent with and miRNA association miRNA binding protein interactions to form immunoprecipitation miRNA compound；And miRNA is discharged from the immunoprecipitation miRNA compound.Further providing for can be with the protein-bonded example of miRNA of circulation miRNA association.)
1. method of the one kind for separating Microrna (miRNA) from biofluid, the method includes (a) to make the biology Fluid is contacted with surfactant and anti-miRNA binding protein reagent, wherein the surfactant decomposes biofluid group From and the anti-miRNA binding protein reagent with and miRNA association miRNA binding protein interactions to formed be immunized Precipitate miRNA compound；(b) miRNA is discharged from the immunoprecipitation miRNA compound.
2. the method as described in claim 1, wherein the biofluid includes vesica miRNA and non-vesica miRNA.
3. the method as described in claim 1 or claim 2, wherein the biofluid is blood plasma or serum.
4. method according to any one of claims 1 to 3, wherein the glass or plastic containers.
5. method according to any one of claims 1 to 4, wherein the biofluid simultaneously with the surfactant and The anti-miRNA binding protein reagent contact.
6. the method as described in any one of claims 1 to 5, wherein by the biofluid, the surfactant and institute Anti- miRNA binding protein reagent is stated to be incubated for about 30 minutes.
7. such as method described in any one of claims 1 to 6, wherein being incubated at room temperature the biofluid, the surface Activating agent and the anti-miRNA binding protein reagent.
8. the method as described in any one of claims 1 to 7, wherein the anti-miRNA binding protein reagent includes being attached to The anti-Argonaut antibody of solid carrier.
9. method according to claim 8, wherein the solid carrier is magnetic beads.
10. method as claimed in any one of claims 1-9 wherein, wherein step (b) includes keeping the immunoprecipitation miRNA multiple Object is closed to contact with proteolytic enzyme.
11. method as claimed in claim 10, wherein the proteolytic enzyme is Proteinase K.
12. the method as described in claim 10 or claim 11, wherein the immunoprecipitation miRNA compound and albumen water Enzyme is solved to contact about 10 minutes.
13. the method as described in any one of claim 10 to 12, wherein the immunoprecipitation miRNA compound is at room temperature It is contacted with proteolytic enzyme.
14. the method as described in any one of claims 1 to 13, wherein discharged from the immunoprecipitation miRNA compound The miRNA is free of other types of RNA molecule.
15. a kind of for separating the kit of Microrna from biofluid, the kit includes surfactant and resists MiRNA binding protein reagent.
16. kit as claimed in claim 15, wherein the glass or plastic containers.
17. the kit as described in claim 15 or claim 16, further includes Proteinase K.
18. the kit as described in any one of claim 15 to 17, wherein the anti-miRNA binding protein reagent includes attached It is connected to the anti-Argonaut antibody of solid carrier.
19. kit as claimed in claim 18, wherein the solid carrier is magnetic beads.
Technical field
This disclosure relates to the method for separating Microrna from biofluid.
Background
Microrna (miRNA) is small non-coding RNA, by matching Thermodynamic parameters via particular bases come to specific MRNA (mRNA) target carries out post-transcriptional control and influences gene regulatory network.MiRNA has been demonstrated to deposit with cell-free form It is in human biological fluids.These are cell-free miRNA can for non-vesica, by the protein in miRNA- protein complex Lai In conjunction with and protect, be closed in film combination vesica such as allochthon or microvesicle or two kinds of situations exist simultaneously.Exist in view of miRNA Critical function effect in disease, this group of nucleic acid molecules contain candidate, and the candidate is used to diagnose and prognosis disease, and In the subject before various patients and performance disease, in readily available biological sample, such as serum and blood plasma, urine or The response for treatment is monitored in saliva.The method of current separation miRNA is for the relative abundance in cell and tissue MiRNA, it is more complex and be related to toxic chemical using being not easy to automate or expand large-scale adsorption column, or may be specific Ground separates vesica or non-vesica miRNA.In addition, the miRNA to attract attention in terms of diagnosis or prognosis is often with the presence of low abundance In biofluid, so that challenging detect to it using current separation method.Therefore, it is necessary to for separating life Wholly or largely miRNA in logistics body it is simple, effective, can automate and can scale method.
Summary of the invention
An aspect of this disclosure provides the method that Microrna (miRNA) is separated from biofluid.The method includes Contact biofluid with surfactant and anti-miRNA binding protein reagent, wherein surfactant makes biofluid component Dissociate and anti-miRNA binding protein reagent with and miRNA association miRNA binding protein interactions to form immune sink Shallow lake miRNA compound.The method also includes discharging miRNA from immunoprecipitation miRNA compound.
Another aspect of the disclosure forgives the kit for separating Microrna from biofluid.Kit includes Surfactant, anti-miRNA binding protein reagent and miRNA discharge reagent.
The other aspects of the disclosure and repetition are in following more detail.
Brief description
Fig. 1 offer will use TriBD or RNA immune precipitation (RIP) separates miRNA from 0.2ml blood plasma Two charts being compared.Tri is shown to employ by the scale of the miRNA of RIP separationThe miRNA of BD separation Fold difference.(A) describe let-7a-5p, 23a-3p and 191-5p miRNA level, and (B) show 142-3p and The level of 451a miRNA.
Fig. 2, which is provided, to be shown using Ago-RIP or Qiagen Column kit (Q1, Q2) miRNA's separated from blood plasma Three horizontal charts.Show let-7a, 23a and 142miRNA (A), 191 miRNA (B) and 451a miRNA (C) to wash De- miR copy number/μ l is the amount of the separation miRNA of unit.
Fig. 3 offer, which shows to use, utilizes biotinylation (b-Ago2 or b-Ago) or non-biotinylated (Ago2) antibody and chain The three horizontal charts for the miRNA that the RIP that mould avidin or a-protein bead carry out is separated from blood plasma.It illustrates The amount for separating miRNA as unit of eluting miR copy number/μ l to indicate of let-7a (A), 23a (B) and 191 (C) miRNA. Used antibody (clone in round parentheses) indicates in x-axis.
Fig. 4 provides the two horizontal charts shown using the miRNA that exothermic Ago-RIP is separated from blood plasma is accompanied by.Q generation Table Qiagen column purification；RIP-Q represents the subsequent Qiagen column purification of immune precipitation；BRIP-Q represents anti-using biotinylation The subsequent Qiagen column purification of the immune precipitation of body.(A) describe using Qiagen column purification, the RIP that is combined with column purification and Accompany by the level of the synthesis cel-miR-39-3p tracking calibration object of the RIP of proteolytic enzyme K release.The level of tracking calibration object It is expressed as tracking the percent of total of the synthesis cel-miR-39-3p of calibration during the separation.(B) describe pure using Qiagen column The level for the let7amiRNA that the slave blood plasma of the RIP and the RIP with proteolytic enzyme K release that change, combine with column purification separate. The level of let7a recycles the copy number of the let7a in sample with 1 μ l to indicate.
Fig. 5 provides what displaying was separated using the Ago-RIP for accompanying by proteolytic enzyme K release at different temperatures from blood plasma Two horizontal charts of miRNA.(A) describe by the level of the proteolytic enzyme K let7a miRNA discharged (in each temperature Under left side stick), and the level (the right side stick at each temperature) being held on bead.The horizontal of let7a miRNA indicates For using the miRNeasy serum/plasma kit of Qiagen come the let7a miRNA's in the isolated identical blood plasma of 0.2ml Percent of total.(B) describe the level (the left side stick at each temperature) by the proteolytic enzyme K miR451a miRNA discharged, With the level (the right side stick at each temperature) being maintained on bead.The level of miR451a miRNA is expressed as using The miRNeasy serum/plasma kit of Qiagen is come total percentage of the miR451a miRNA in the isolated identical blood plasma of 0.2ml Than.
Fig. 6 provides displaying and commercially available miRNA separation method, or use is used to accompany by proteolytic enzyme in standard test tube Two horizontal charts of let7a (A) or miR451a (B) miRNA that the separation of the RIP of K release is separated from blood plasma.E1 and E2 is represented to be separated using miRCury RNA separating kit-biofluid miRNA from Exiqon.Q1 and Q2 representative makes It is separated with the miRNA of the miRNeasy serum/plasma kit from Qiagen.RIP-std-Q represent immune precipitation with Qiagen column purification afterwards.The level of miRNA is expressed as the total copy number recycled from 0.2ml blood plasma.
Fig. 7, which is provided, to be shown using commercially available miRNA separation method, or uses the Ago- for accompanying by proteolytic enzyme K release The two horizontal charts for the let7a miRNA that the separation (RIP1-4) of RIP is separated from blood plasma.(A) show test 1 and (B) test 2 is shown.E1 and E2, which is represented, uses miRCury RNA separating kit-biofluid miRNA from Exiqon Separation.Q1 and Q2 is represented to be separated using the miRNA of the miRNeasy serum/plasma kit from Qiagen.The level of let7a It is expressed as total copy number of the let7a recycled from 0.2ml blood plasma.
Fig. 8, which is provided, to be shown presence or absence of proteolytic enzyme and RNase inhibitor, and existing or There is no in the pretreated situation of detergent, the miRNA separated from blood plasma is discharged using the subsequent proteolytic enzyme K of Ago-RIP Two horizontal charts.+ pre ,+inh；Igepal and inhibitor are added to blood plasma and are incubated for~30 minutes, is then added Add to Ago2- bead.+ pre ,-inh；Igepal is added to blood plasma in the absence of inhibitors and is incubated for~30 points Clock is then added to Ago2- bead.- pre ,+inh；Igepal and inhibitor and Ago2- bead are added to blood plasma simultaneously.- Pre ,-inh；Igepal is added to blood plasma with Ago2- bead in the absence of inhibitors simultaneously.(A) describe recycling The copy number of let7amiRNA, and (B) describes the copy number of the miR451a miRNA of recycling.
Fig. 9 provides the freedom (the left side stick of each miRNA) and capsule for showing the percentage as total IGEPAL processing miRNA Steep the horizontal chart of (the right side stick of each miRNA) instruction miRNA.
Figure 10, which is provided, to be shown using the subsequent proteolytic enzyme K release (RIP) of Ago-RIP from 0.2 or 0.4ml blood plasma, or Use the water for the miRNA that miRCury RNA separating kit-biofluid (E) from Exiqon is separated from 0.2ml blood plasma Three flat charts.(A) describe the copy number of the let7a miRNA of recycling, (, B) and describe copying for the miR191 miRNA recycled Shellfish number, and (C) describes the copy number of the miR451a miRNA of recycling.
Figure 11 provides the level for showing the let7a separated from blood plasma using the subsequent proteolytic enzyme K release of Ago-RIP Chart.RIP incubation is to last 5,15,30 or 60 minutes (5 ', 15 ', 30 ', 60 ') at room temperature.5, those of 15 or 30 minutes It is incubated for and is all washed 5 times before Proteinase K release.Incubation in those of 60 minutes wash 5,4,3,2 or 1 times (5w, 4w, 3w, 2w,1w).Show the gross production rate of the let7a recycled from 0.2ml blood plasma.
Figure 12 A shows the water of the let7amiRNA separated from blood plasma using Ago-RIP or base for post miRNA separating kit It is flat.Three different experiments (Exp) are provided.S1 and S2 represents the separation for using Ago-RIP；E1 and E2 is represented using from Exiqon The separation of miRCury RNA separating kit-biofluid；And Q1 and Q2, which is represented, uses the miRNeasy from Qiagen The separation of serum/plasma kit.
Figure 12 B provide the RNU6 small nuclear rna separated from blood plasma using Ago-RIP or base for post miRNA separating kit and The level of SNORD48 little nucleolar RNA.Three different experiments are provided.S1 and S2 represents the separation for using Ago-RIP；E1 and E2 generation Table uses miRCury RNA separating kit-biofluid separation from Exiqon；And Q1 and Q2 represents use and comes from The separation of the miRNeasy serum/plasma kit of Qiagen.
Figure 12 C show the GAPDH mRNA separated from blood plasma using Ago-RIP or base for post miRNA separating kit, The level of RN18S rRNA and RN28S rRNA.Three different experiments are provided.S1 and S2 is represented using Ago-RIP's Separation；E1 and E2, which is represented, uses miRCury RNA separating kit-biofluid separation from Exiqon；And Q1 and Q2 represents the separation using the miRNeasy serum/plasma kit from Qiagen.
Figure 13 provides displaying and uses anti-Ago1 antibody, anti-Ago2 antibody or anti-Ago1 and anti-Ago2 antibody via Ago-RIP The specified miRNA that is separated from blood plasma of combination horizontal chart.
Detailed description of the invention
Have been found that the method efficiently and quickly of separation circulation miRNA.As shown in example, disclosed method can be same When separation vesica association and non-vesica association circulation miRNA.Advantageously, disclosed method and kit allow quickly and special Surely pure miRNA preparation is separated without the pollution of other type RNA.In addition, method disclosed herein and kit allow with High yield separates miRNA from dilute extra-cellular fluid.In addition, method of the invention can scale, allow from increasing volume MiRNA is separated in extracellular biological fluid.
The level of miRNA is associated with disease, including cancer, cardiovascular disease, and in many other diseases and development In the process, including schizophrenia, Alzheimer's, immunocyte development and adaptability and congenital immunity adjusting, Stem cell maintains and versatility, nervous system development, endocrine disease include diabetes, the development of pancreas, crisp folding X syndrome, skin Skin wound healing, cell cycle progression, transplanting tissue rejection, anoxic, skeletal muscle differentiation.In addition, miRNA is also by expressing viral, And the target gene of those miRNA is identified.Therefore, disclosed method and kit may be used in readily available Biological sample, such as blood, serum or blood plasma prepare miRNA for use in diagnosing the illness or the measurement of disease condition.
I. method
The disclosure covers the method that Microrna (miRNA) is separated from biofluid.The method includes making biofluid It is contacted with surfactant and anti-miRNA binding protein reagent.The biofluid component that surfactant makes dissociates and anti-miRNA Binding protein reagent with and miRNA association miRNA binding protein interactions to form immunoprecipitation miRNA compound. The method also includes discharging miRNA from immunoprecipitation miRNA compound.
Method disclosed herein specifically separates miRNA.As being described in detail in following example 12, other types of tiny RNA is (such as Small nuclear rna or little nucleolar RNA) it is not separated by disclosed method, and larger RNA molecule (such as mRNA or ribosomes RNA it) is not separated by disclosed method.
(a) biofluid
Disclosed method includes that cell outer circulation miRNA is being separated from the biologicfluid sample that subject obtains.Such as Term as used herein " subject " refers to human or animal.Subject can be embryo, childhood or adult.Subject can be male Or female.Suitable animal includes vertebrate such as mammal, birds, reptile, amphibian and fish.It is suitble to feed The example of newborn animal may include but be not limited to rodent, companion animals, domestic animal and primate.Rodent it is unrestricted Example includes mouse, rat, hamster, gerbil jird and cavy.Suitable companion animals include but is not limited to cat, dog, rabbit, hedgehog and snow Ermine.The non-limiting example of domestic animal includes horse, goat, sheep, pig, ox, camel and alpaca.Suitable primate includes but unlimited In capuchin monkey, chimpanzee, mongoose lemur, macaque, marmoset, thin,tough silk hair monkey, Ateles, Squirrel monkey and black guenon.Birds it is unrestricted Example includes chicken, turkey, duck and goose.Exemplary subject is people.
Term " biofluid " can refer to all biofluids and excreta separated from any given subject.Biology stream The non-limiting example of body may include blood and its part, serum, blood plasma, urine, excreta, sperm (semen), sperm Secretion (examining amber liquid), pleural effusion, tears, saliva before (seminal fluid), seminal plasma, prostatic fluid, ejaculation, Phlegm, sweat, biopsy, ascites, cerebrospinal fluid, amniotic fluid, lymph, marrow, cervical secretions, vaginal fluid, endometrial secretions, stomach Intestinal secretion object, bronchial secretion, mammal gland secretion, ovarian cyst secretion, tissue fluid, tumour aspirate and tissue fluid Sample.In some embodiments, biofluid is serum.In other embodiments, biofluid is blood plasma.
The method for obtaining blood plasma or blood serum sample from subject is well known in the art.For example, existing or not depositing In the case where conduit, venipuncture can be used for collecting blood sample to prepare serum.Blood plasma and blood are prepared from blood sample Clear method is known in the art.In general, blood sample be sufficiently large to supply sufficient amount blood plasma or serum for It is as further discussed below to handle.Blood plasma or blood serum sample can immediately treat after a sample is collected.Alternatively, blood plasma or serum Sample can freeze to handle later.
Biologicfluid sample can be obtained and collecting sample recently from subject.Alternatively, biologicfluid sample can from Preceding collection and the sample acquisition stored.For example, sample can be from the blood for storing and saving when biofluid is blood plasma or serum It is obtained in the set of sample.In some embodiments, sample is obtained by collecting sample recently.In other embodiments In, sample from collecting and the sample stored obtains in the past.
In some embodiments, biologicfluid sample is undiluted.In other embodiments, biologicfluid sample is dividing It is diluted from before miRNA.Dilution may depend on the class that a number of factors includes but is not limited to miRNA, biofluid in sample Type, subject, the disease condition of subject, measurement for measuring miRNA type, and in the measurement for measuring miRNA Used in reagent.In one embodiment, biologicfluid sample is by addition original sample volume about 1/2 to original sample The diluent of volume within the scope of about 50,000 times of product volume dilutes.Diluent can be that miRNA is not interfered to separate or be used for Any fluid of other methods of subsequent processing steps.The non-limiting example of suitable diluents includes deionized water, distilled water, salt Aqueous solution, Ringer's solution, phosphoric acid buffers saline solution, TRIS buffered saline solution, normal saline solution citric acid and HEPES buffering Salt water.
(b) surfactant
Biofluid is contacted with surfactant (or being referred to as " surfactant " or " detergent ").As made herein With term " surfactant " can be used for describing to make may include any examination for recycling the biofluid component dissociation of miRNA Agent.May include recycle miRNA biofluid component non-limiting example include extracellular vesica such as lipoprotein, allochthon, Microvesicle, ectosome, apoptosis main body and other extracellular vesicas.
If those skilled in the art recognize, any surfactant that biofluid component can be made to dissociate can be used for this In disclosed method, as long as surfactant does not interfere the formation of the immunoprecipitation miRNA compound of the disclosure.For example, surface Activating agent can be anionic surfactant, cationic surfactant, zwitterionic surfactant, non-ionic surface active Agent or combinations thereof.The characteristic of surfactant of the invention may and will change, depending on may include in biofluid Recycle the characteristic of the biofluid component of miRNA, anti-miRNA binding protein reagent and separation miRNA.
In some embodiments, surfactant is anionic surfactant.Suitable anionic surfactants packet It includes but is not limited to dodecylbenzene sulfanilic acid；Octanol polyethers ammonium sulfate；Cumenesulfonic acid ammonium；Dihydroxy stearic acid ammonium；Dodecylbenzene sulfonic acid Ammonium；Laureth ammonium sulfate；- 12 ammonium sulfate of laureth；- 30 ammonium sulfate of laureth；Cocoyl sarcosine ammonium；Month Osmanthus base ammonium sulfate；Lauryl sulfosuccinic acid ammonium；Ammonium lignosulphonate；Myristyl alcohol polyethers ammonium sulfate；Naphthalene sulfonic acids ammonium；Nonylbenzene alcohol - 20 ammonium sulfate of ether；- 30 ammonium sulfate of nonoxinol；- 4 ammonium sulfate of nonoxinol；- 6 ammonium sulfate of nonoxinol；Nonoxynol-9 sulphur Sour ammonium；Oily ammonium sulfate；Perfluorooctanoic acid；Ammonium stearate；Ammonium xylene sulfonate；Butyl napsylate；Butyl phosphoric acid ester；Dodecane Benzene sulfonic acid calcium；Stearyl lactate；Tetrapropylene benzene sulfoacid calcium；- 9 carboxylic acid of octanol polyethers；Cetyl phosphate；Cumenesulfonic acid； DEA- cetyl phosphate；DEA- dodecylbenzene sulphonic acid ester；DEA- lauryl sulfate ester；- 4 phosphate of decyl alcohol polyethers；Lauryl Sulfosuccinic acid diammonium；Stearyl sulfosuccinamic acid diammonium；Sulfosuccinic acid diamyl sodium；Sulfosuccinic acid dicyclohexyl Sodium；Sulfosuccinic acid dihexyl sodium；Sulfosuccinic acid diisobutyl sodium；- 7 citrate of two laureths；Poly dimethyl silicon Oxygen alkanol；- 4 phosphoric acid of two nonoxinols；Sulfosuccinic acid dioctyl ammonium；Sulfosuccinic acid dioctyl sodium；Cetearyl alcohol sulfo group amber Amber amide acid disodium；Cocamidopropyl MEA- disodium sulfosuccinate；Cocamidopropyl PEG-3 disodium sulfosuccinate；Decyl alcohol - 6 disodium sulfosuccinate of polyethers；Decyl diphenyloxide disulfonate；Dodecyloxy propylsultaine succinamide acid disodium；It is different Decyl disodium sulfosuccinate；- 5 disodium sulfosuccinate of lanolin alcohol polyethers；Lauramide DEA- disodium sulfosuccinate；Month Osmanthus amide MEA- disodium sulfosuccinate；Laureth disodium sulfosuccinate；Lauryl disodium sulfosuccinate；Nutmeg Amide MEA- disodium sulfosuccinate；Oleoyl amido MEA- disodium sulfosuccinate；Oleoyl amido PEG-2 sulfosuccinic acid two Sodium；- 3 disodium sulfosuccinate of oleth；PEG-4 cocamidopropyl MIPA disodium sulfosuccinate；Ricinoleamide base MEA- disodium sulfosuccinate；Stearyl sulfosuccinamic acid disodium；Endecatylene amide groups MEA- disodium sulfosuccinate； The double tritriacontyl sodium of sulfosuccinic acid；Dodecenyl succinic anhydride；Dodecyl diphenyl oxide disulfonic acid；Dodecyl diphenyl oxide two Sulfonic acid；Dodecyl benzene sulfonic acid；Glyceryl dioleate SE；Glyceryl distearate SE；Glyceryl monoricinolein SE；Glycerol Base stearate citrate；Glyceryl monostearate SE；Glycol stearate SE；Phosphoric acid hexyl ester；P isopropylbenzoic acid base ester；It is different Propylamine dodecylbenzenesulfonate；- 2 phosphate of isosteareth；Different -3 phosphate of trideceth；Different tridecanol is poly- - 6 phosphate of ether；- 1 phosphate of laureth；- 12 carboxylic acid of laureth；- 3 phosphate of laureth；Laruyl alcohol is poly- - 4 phosphate of ether；- 6 phosphate of laureth；- 7 citrate of laureth；- 9 phosphate of laureth；Lauryl Phosphate；Lauryl sulfate lithium；Laureth magnesium sulfate；PEG-3 coconut oleoyl amine magnesium sulfate；MEA- laureth phosphoric acid Ester；MEA- lauryl sulfate ester；MIPA- laureth sulfuric ester；MIPA- lauryl sulfate ester；Myristoy sarcosine； Naphthalene-formaldehyde sulphonic acid ester；- 10 phosphate of nonoxinol；- 12 phosphate of nonoxinol；- 3 phosphate of nonoxinol；Nonoxinol -4 Phosphate；- 4 sulfuric ester of nonoxinol；- 6 phosphate of nonoxinol；- 7 phosphate of nonoxinol；- 8 phosphate of nonoxinol；Nonyl - 9 phosphate of menthylphenoxypolyethoxy ethanol；- 10 phosphate of nonyl nonoxinol；- 15 phosphate of nonyl nonoxinol；- 7 phosphoric acid of nonyl nonoxinol Ester；- 10 carboxylic acid of oleth；- 10 phosphate of oleth；- 3 carboxylic acid of oleth；- 4 phosphate of oleth；Oleyl alcohol is poly- - 5 phosphate of ether；- 6 carboxylic acid of oleth；- 7 phosphate of oleth；PEG-2 dilaurate SE；PEG-2 dioleate SE； PEG-2 distearate SE；PEG-2 laurate SE；PEG-2 oleate SE；PEG-2 stearate SE；PEG-9 stearmide Carboxylic acid；Cetyl phosphate potassium；- 4 potassium phosphate of decyl alcohol polyethers；Dodecylbenzene potassium sulfonate；- 2 potassium phosphate of isosteareth；Lauroyl Sarcosine potassium；Lauryl potassium sulfate；Potassium oleate；Oily potassium sulfate；Perfluorooctanoic acid potassium salt；Castor oil potassium sulfate；PPG-2 laurate SE；PPG-2 oleate SE；PPG-2 stearate SE；- 10 phosphate of PPG-5- cetanol；Glycol laurate SE；The third two Alcohol oleate SE；Propylene glycol monoricinolein SE；Propylene glycol stearate SE；Gantrez AN-119；2- ethylhexyl phosphoric acid sodium；2- Ethylhexyl sulfate；Paraffin sodium sulfonate；Allyloxy hydroxypropyl sulfonate；Behenyl acyl lactylate；Butoxyethoxy second Sour sodium；Sodium butylnaphthalenesulfonate；Butyl oleate sodium sulphate；Butyl oleate sodium sulfonate；Butyl phosphoric acid sodium；Caproyl lactylate sodium；Decoyl Base sodium sulfonate；Cetyl sulfate；Sodium taurocholate；Cumene sodium sulfonate；Decyl alcohol polyethers sodium sulphate；Decyl diphenyloxide sodium sulfonate；Decyl Sodium sulphate；NaTDC；Nekal；Docosyl benzene sulfonic acid sodium salt；Diisooctyl sodium sulfosuccinate；Two is different Naphthalene sulfonate；- 7 sodium citrate of two laureths；Dinonyl sodium sulfosuccinate；Dodecyl diphenyl oxide disulfonic acid Sodium；Disodium 4-dodecyl-2,4 '-oxydibenzenesulfonate；Neopelex；Glyceryl trioleate sodium sulphate；Palmityl biphenyl two Sodium sulfonate；Palmityl diphenyl oxide disulfonate；Hexyl diphenyl sodium disulfonate；Different sodium thiosulfate；Isodecyl sodium sulphate；It is different pungent Base sodium sulphate；Sodium stearoyl lactylate；Different -15 sodium sulphate of trideceth；Sodium lactate；Lauramide DEA- sulfosuccinic acid Sodium；Laureth sodium phosphate；Laureth sodium sulphate；Laureth sodium sulfosuccinate；- 10 phosphoric acid of laureth Sodium；- 11 carboxylic acid sodium of laureth；- 12 sodium sulphate of laureth；- 13 sodium acetate of laureth；Laureth -13 Carboxylic acid sodium；- 3 carboxylic acid sodium of laureth；- 4 carboxylic acid sodium of laureth；- 4 sodium phosphate of laureth；Laureth -6 Carboxylic acid sodium；- 7 carboxylic acid sodium of laureth；- 7 sodium sulphate of laureth；- 8 sodium sulphate of laureth；Lauroyl glutamate Sodium；Sodium lauroyl lactylate；Sodium lauroyl lactylate；Lauroyl methyl alanine sodium；Sodium lauroyl sarcosine；Lauryl phosphoric acid Sodium；NaLS；Lauryl sulfoacetate sodium；Sodium lignate；Sodium lignin sulfonate；Methallylsulfonic acid sodium；First Base laurel sodium acyl taurine；Methyl myristoyl sodium taurocholate；Methyl oleoyl taurate；Methyl palmitoyl taurine Sodium；Methyl tristearin sodium acyl taurine；Methyl naphthalene sulfonic acid sodium；M- nitrobenzene sodium sulfonate；Myristyl alcohol polyethers sodium sulphate；Myristoyl Base sodium glutamate；Myristoy sarcosine sodium；Myristyl sulfate；Nonoxinol sodium sulphate；- 10 sodium sulphate of nonoxinol； - 10 sodium sulfosuccinate of nonoxinol；- 15 sodium sulphate of nonoxinol；- 4 sodium sulphate of nonoxinol；- 5 sodium sulphate of nonoxinol； - 6 sodium phosphate of nonoxinol；- 6 sodium sulphate of nonoxinol；- 8 sodium sulphate of nonoxinol；Nonoxynol-9 sodium phosphate；Nonoxinol- 9 sodium sulphate；- 2 ethane sulfonic acid sodium of Octoxinol；- 3 sodium sulphate of Octoxinol；Sodium octyl sulfate；Octylphenoxy ethoxyethyl group Sodium sulfonate；Oily sodium sulphate；- 7 sodium phosphate of oleth；Oleyl alcohol sodium phosphate；Oleyl alcohol sodium sulphate；Oleyl alcohol sulfosuccinamic acid sodium； Palmitoyl sarcosine sodium；Phenylbenzimidazole sulfonic acid sodium；Propyl oleate sodium sulphate；Stearoyl lactate；Stearyl sulfosuccinamic acid Sodium；Trideceth sodium sulphate；- 3 carboxylic acid sodium of trideceth；- 6 carboxylic acid sodium of trideceth；Trideceth- 7 carboxylic acid sodiums；Tridecyl sulfate；Tridecyl benzene sulfonate；Sodium xylene sulfonate；Hamposyl S；TEA- lauroyl Glutamate；TEA- lauryl sulfate ester；Four sodium of dicarboxyl ethylstearyl sulfosuccinamic acid；TIPA- laureth sulphur Acid esters；- 4 phosphate of three ceteareths；- 5 phosphate of three ceteths；- 2 phosphate of trideceth；13 - 3 phosphate of alkanol polyethers；- 5 phosphate of trideceth；Tridecyl phosphate esters；With -4 phosphate of Trilaureth； And trioctyl phosphate.
In other embodiments, surfactant is cationic surfactant.Suitable cation surfactant Example includes but is not limited to alkyl trimethyl ammonium bromide；Benzalkonium chloride；Benzalkonium chloride；Benzyl dimethyl cetyl chloride ammonium； Benzyldimethyltetradecyla-mmonium chloride；Benzododecinium ammonium bromide；Benzyl trimethyl tetrachloro ammonium iodate；Cetyl Trimethylammonium bromide (CTAB)；Dimethyldioctadecylammonium base ammonium bromide；Dodecyl ethyl ditallowdimethyl ammonium bromide；Dodecyl three Methyl bromide ammonium；Dodecyl trimethyl ammonium bromide；Dodecyl trimethyl ammonium chloride；Ethylhexadecyl dimethyl bromination Ammonium；Girard reagent T；Cetyl trimethylammonium bromide；Cetyl trimethylammonium bromide；N, N ', N '-polyoxyethylene (10)-N- tallow -1,3- diaminopropanes；Thonzonium bromide；With trimethyl (tetradecyl) ammonium bromide.
In other embodiments, surfactant is zwitterionic surfactant.Suitable amphoteric ion surface-active Agent includes but is not limited to 3- [(3- gallbladder amido propyl) dimethylammonio] -2- hydroxyl -1- propane sulfonic acid (CHAPSO)；3- [(3- gallbladder Amido propyl) dimethylammonio] -1- propane sulfonic acid (CHAPS)；3- (4- heptyl) phenyl -3- hydroxypropyl) dimethylammonio third Alkyl sulfonic acid (C7BzO)；3- (N, N- dimethyl octyl ammonium) propane sulfonic acid inner salt (SB3-8)；3- (decyldimethylammonio) third Alkyl sulfonic acid inner salt (SB3-10；Caprylyl sulfobetaine)；3- (dodecyl dimethyl ammonium) propane sulfonic acid inner salt (SB3- 12)；3- (N, N- dimethyl tetradecyl base ammonium) propane sulfonic acid (SB3-14)；3- (N, N- Dimethylpalmitylammonio) propane sulphur Sour (SB3-16)；3- (N, N- dimethyl stearyl ammonium) propane sulfonic acid (SB3-18)；3- [N, N- dimethyl (3- nutmeg Acylaminopropyl) ammonium] propane sulfonic acid (ASB-14).Depending on embodiment, other suitable amphoteric ion detergents include: Acylated lecithin；Apricot amido propyl betaine；Babassu oil amido propyl betaine；Behenyl base glycine betaine；Double 2- ethoxys Tallow glycine；C12-14 alkyl dimethyl betaine；Canolamidopropyl glycine betaine；Octanoic acid/caprylyl amide Propyl betaine；Caprylamide propyl betaine；Cetyl betaine；Cocoamidopropyl betaine；Cocoamidopropyl dimethylamine Base hydroxy amino propyl Hydrolyzed Collagen；N- [3- cocamidopropyl)-propyl]-N, N- dimethyl betaine, sylvite；Cocounut oil Amido propyl hydroxyl sulfo betaine；Cocamidopropyl propyl amide sulfobetaines；Cocounut oil aminobutyric acid；Cocaminopropionic acid；Cocounut oil Acyl both sexes base dipropionic acid；Coco betaine；Cocodimonium -3- azochlorosulfonate propyl lycine；Cocounut oil imino-diacetic glycine； Cocounut oil dipropionic acid；Cocounut oil/oleamidopropyl betaine；Cocoyl amide DEA；DEA- cocounut oil acyl both sexes base Dipropionic acid；Dihydroxy ethyl tallow glycine；Dimethyl silicone polymer propyl PG- glycine betaine；N, N- dimethyl-N-lauric acid-acyl Amine propyl-N- (3- sulfopropyl)-ammonium glycine betaine；N, N- dimethyl-N-myristyl-N- (3- sulfopropyl)-ammonium glycine betaine；N, N- Dimethyl-N-palmityl-N- (3- sulfopropyl)-ammonium glycine betaine；N, N- dimethyl-N-stearoyl aminocarbonyl propyl-N- (3- sulphur third Base)-ammonium glycine betaine；N, N- dimethyl-N-stearyl-N- (3- sulfopropyl)-ammonium glycine betaine；N, N- dimethyl-N-tallow-N- (3- sulfopropyl)-ammonium glycine betaine；Caprinoyl both sexes base diethyl acid disodium；Caprinoyl both sexes base disodium beclomethasone；Decoyl both sexes base diethyl Acid disodium；Decoyl both sexes base disodium beclomethasone；Cocounut oil both sexes base diethyl acid disodium；Cocounut oil both sexes base disodium beclomethasone；Different tristearin Both sexes base disodium beclomethasone；- 5 carboxyl both sexes base diethyl acid disodium of laureth；Laurel iminodipropionate；Lauroyl Both sexes base diethyl acid disodium；Lauroyl both sexes base disodium beclomethasone；Octyl b- iminodipropionate；Oleoyl both sexes base diethyl Acid disodium；Oleoyl both sexes base disodium beclomethasone；- 7 carboxyl both sexes base diethyl acid disodium of PPG-2- isodecanol polyethers；Soybean oleoyl two Property base diethyl acid disodium；Stearoyl both sexes base diethyl acid disodium；Carvedilol oleoyl both sexes base disodium beclomethasone；Tallow acyl both sexes base two Acetic acid disodium；Tallow acylimino disodium beclomethasone；Wheat germ oleoyl both sexes base diethyl acid disodium；N, N- distearyl acyl group-N- first Base-N- (3- sulfopropyl)-ammonium glycine betaine；Erucyl amide hydroxysultaine；Ethylhexyl dipropionic acid；Ethyl methylol Oleyl alcohol oxazoline；Ethyl PEG-15 cocoa amine sulfuric ester；Hydrolecithin；Aminosal；Isostearoyl amine propyl betaine； Lauroylamidopropyl betaine；Dodecanamide propyl dimethyl betaine；Lauramide propionic acid；Laurel both sexes base dipropionic acid；Month Osmanthus acyl lysine；Lauryl betaine；Lauryl hydroxyl sulfo betaine；Lauryl sulfobetaines；Sub- oleamide propyl sweet tea Dish alkali；Lysolecithin；Butterfat matter amido propyl betaine；Myristamide propyl betaine；Octyl dipropionic acid；Octyl imido Base dipropionic acid；Oleamidopropyl betaine；Oleyl alcohol glycine betaine；4,4 (5H)-oxazole dimethanols, 2- (17 alkenyl)-；Palm Amido propyl betaine；Palm amine oxide；Ricinoleic acid amide propyl betaine；Ricinoleic acid amide propyl betaine/ IPDI copolymer；Sesame amido propyl betaine；C12-15 alkoxy propyl dipropionic acid sodium；Caprinoyl both sexes guanidine-acetic acid Sodium；Decoyl both sexes guanidine-acetic acid sodium；Decoyl both sexes base hydroxypropyl azochlorosulfonate acid sodium；Decoyl both sexes base sodium propionate；The poly- propylamine of carboxymethyl tallow Sodium；Cocoyl alanine sodium；Cocoyl both sexes guanidine-acetic acid sodium；Cocoyl both sexes base hydroxypropyl azochlorosulfonate acid sodium；Cocoyl both sexes base Propyl sodium；Two carboxyethyl cocoyl ethyl imidazoline sodium；Hydrogenated tallow dimethylglycine sodium；Different tristearin both sexes base third Sour sodium；Laurel dipropionic acid sodium；Sodium Lauroamphoacetate；Oleoyl both sexes base hydroxypropyl azochlorosulfonate acid sodium；Oleoyl both sexes base Sodium propionate；Stearic both sexes guanidine-acetic acid sodium；Carvedilol oleoyl both sexes base sodium propionate；Soybean oil amido propyl betaine；Stearyl beet Alkali；Tallow amidoethyl hydroxysultaine；The poly- carboxyl propionic acid of tallow both sexes base；Lauroyl both sexes base PG- acetic acid trisodium Phosphoric acid chloride；Endecatylene amido propyl betaine；With wheat-germ oil amido propyl betaine.
In other embodiments, surfactant is preferably nonionic surfactant.Suitable nonionic surface is living The example of property agent includes but is not limited to polyoxyethylene (10) cetyl etherPolyoxyethylene (20) cetyl etherPolyoxyethyleneglycododecyl dodecyl etherPolyoxyethylene (9) p-t- octyl phenol (NONIDETTMP-40)；Polyoxyethylene (4-5) p-t- octyl phenol (TRITONTMX-45)；Polyoxyethylene (7-8) p-t- octyl Phenol (TRITONTMX-114)；Polyoxyethylene (9-10) p-t- octyl phenol (TRITONTMX-100)；Polyoxyethylene (9-10) Nonyl phenol (TRITONTMN-101)；Polyoxyethylene (20) D-sorbite monolauratePolyoxyethylene (20) sorbitan monopalmitatePolyoxyethylene (20) sorbitol monooleateDimethyl decyl phosphine oxide (APO-10)；Dimethyl dodecyl phosphine oxide (APO-12)；Hexamethylene Base-n- ethyl-β-D-Maltose glycosides；Cyclohexyl-n- hexyl-β-D-Maltose glycosides；Cyclohexyl-n-methyl-beta-maltose glycosides；n- Capryl sucrose；N- decyl-β-D- glucopyranoside；N- decyl-β-pyrans maltoside；The thio malt of n- decyl-β-D- Glucosides；N- dodecanoyl sucrose；Ten ethylene glycol monododecyl ethers；N- caprinoyl-N- methyl glucose osamine；N- decyl α-D- pyrans Glucoside；Decyl β-D- pyrans maltoside；N- dodecanoyl-N- methyl glucose amide；N- dodecyl α-D-Maltose Glycosides；N- dodecyl β-D-Maltose glycosides；Heptane -1,2,3- triol；Seven ethylene glycol list last of the ten Heavenly stems ethers；Seven ethylene glycol list dodecyls Ether；Seven ethylene glycol list myristyl ethers；N- palmityl β-D-Maltose glycosides；Six ethylene glycol monododecyl ethers；Six ethylene glycol lists Cetyl ether；Six ethylene glycol list octadecyl ethers；Six ethylene glycol list myristyl ethers；Methyl -6-O- (N- heptyl carbamyl Base)-α-D- glucopyranoside；Nine ethylene glycol monododecyl ethers；N- nonanoyl-N- methyl glucose osamine；N- nonanoyl-N- methyl Gucosamine；Eight ethylene glycol list last of the ten Heavenly stems ethers；Eight ethylene glycol monododecyl ethers；Eight ethylene glycol list cetyl ethers；Eight ethylene glycol lists Octadecyl ether；Eight ethylene glycol list myristyl ethers；Octyl-beta-glucosidase；Octyl-β-glucosinolate；Octyl-β-D- Glucopyranoside；Octyl-β-D-1- thioglucopyranoside；Five ethylene glycol list last of the ten Heavenly stems ether；Five ethylene glycol monododecyl ether； Five ethylene glycol list cetyl ether；Five ethylene glycol monohexyl ether；Five ethylene glycol list octadecyl ether；Five ethylene glycol list octyl ether；Poly- second Diglycidyl ether；Polyglycol ether；10 tridecyl ether of polyoxyethylene；Polyoxyethylene (100) stearate；Polyoxy second Alkene (20) isocetyl ether；Polyoxyethylene (20) oleyl alcohol ether；Polyoxyethylene (40) stearate；Polyoxyethylene (50) stearic acid Salt；Polyoxyethylene (8) stearate；Polyoxyethylene is bis- (Imidazolylcarbonyl)；Polyoxyethylene (25) propylene glycol stearate；It comes from The saponin of Quillaia saponaria bark；Tetradecyl-β-D-Maltose glycosides；Tetraethylene glycol list last of the ten Heavenly stems ether；Tetraethylene glycol monododecyl ether；Tetrem Glycol list myristyl ether；Triethylene glycol list last of the ten Heavenly stems ether；Triethylene glycol monododecyl ether；Triethylene glycol list cetyl ether；Three Ethylene glycol monooctyl ether；Triethylene glycol list myristyl ether；Tyloxapol；N- hendecyl β-D- glucopyranoside, (octyl benzene Oxygen) polyethoxy ethanol (CA-630)；Polyoxyethylene (5) nonyl phenylate (CO-520)；And polyoxy Ethylene (150) dinonyl phenylate (DM-970).In one embodiment, surfactant is polyoxyethylene (5) Nonyl phenylate (CO-520).In another embodiment, surfactant is polyoxyethylene (150) dinonyl Phenylate (DM-970).In one embodiment, surfactant is preferably (octyl benzene oxygen) polyethoxy second Alcohol (CA-630)。
If those skilled in the art recognize, the amount for being added to the surfactant of biofluid may and will become Change, depending in biofluid may include recycle miRNA biofluid component characteristic.In some embodiments, raw The ultimate density of surfactant in logistics body can be in about 0.001 to about 10% range.In one embodiment, surface The concentration of activating agent can be in about 0.001 to about 0.01% range.In another embodiment, the concentration of surfactant can In about 0.01% to about 0.1% range.In another embodiment, the concentration of surfactant can be about 0.1% to about In 1% range.In another embodiment, the concentration of surfactant can be in about 1 to about 5% range.In other reality It applies in scheme, the concentration of surfactant can be in about 5% to about 10% range.
(c) anti-miRNA binding protein reagent
Biofluid is contacted with anti-miRNA binding protein reagent.Anti- miRNA binding protein reagent can be that can combine and follow The protein-bonded any reagent of miRNA of ring miRNA association.It can be bound directly with the miRNA binding protein of circulation miRNA association MiRNA, or can associate indirectly with the RNA- protein complex including miRNA.It can be tied with the miRNA of circulation miRNA association The non-limiting example of hop protein matter may include Argonaut, Dicer, human immunodeficiency virus's (HIV) trans-activation response RNA knot Hop protein (TRBP), the protein activation agent (PACT) of interferon inducible protein kinases, SMN compound, crisp folding X feeblemindedness egg White matter (FMRP), assumes DNA helicase at the protein (Tudor-SN) containing Tudor staphylococcal nuclease structural domain MOV10, and protein TNRC6B or the RISC compound containing RNA Recognition unit other components or can be with RISC compound The substance instantaneously or permanently to associate.
In some embodiments, biofluid is preferably contacted with anti-Argonaut reagent.Argonaut protein Non-limiting example may include Ago1, Ago2, Ago3 and Ago4.In one embodiment, biofluid connects with anti-Ago1 reagent Touching.In another embodiment, biofluid is contacted with anti-Ago2 reagent.In another embodiment, biofluid with Anti- Ago3 reagent contact.In another embodiment, biofluid is contacted with anti-Ago4 reagent.In another embodiment In, biofluid is preferably contacted with that can combine the reagent of more than one Argonaut protein.For example, biofluid It can be contacted with anti-Ago1 and anti-Ago2 reagent.In still another embodiment, biofluid preferably with can combine The reagent of Ago1, Ago2, Ago3 and Ago4 contact.
Anti- miRNA binding protein reagent can be epitope binding agent.Depending on target molecule, the non-limit of appropriate epitope bonding agent Example processed includes the agent selected from the group being made up of: aptamer, antibody, antibody fragment, double chain DNA sequence, modification of nucleic acids, nucleic acid Analogies, ligand, ligand fragment, receptor, receptor fragments, polypeptide, peptide, coenzyme, the auxiliary adjustment factor, allosteric molecules and ion.
In some embodiments, epitope binding agent is antibody.The non-limiting example of workable antibody includes polyclonal Antibody, ascites, Fab segment, Fab ' segment, monoclonal antibody, single-chain antibody, single domain antibody, humanized antibody and contain Other segments of the epitope binding site of antibody.
In some embodiments, biofluid is contacted with anti-Argonaut antibody.In one embodiment, biology stream Body is contacted with anti-Ago1 antibody.In another embodiment, biofluid is contacted with anti-Ago2 antibody.In another embodiment party In case, biofluid is contacted with anti-Ago3 antibody.In another embodiment, biofluid is contacted with anti-Ago4 antibody.In In another embodiment, biofluid is anti-with two kinds of anti-Ago selected from anti-Ago1, anti-Ago2, anti-Ago3 or anti-Ago4 antibody Body contact.For example, biofluid is contacted with anti-Ago1 and anti-Ago2 antibody.In another embodiment, biofluid It is contacted with three kinds of anti-Ago antibody selected from anti-Ago1, anti-Ago2, anti-Ago3 or anti-Ago4.In another embodiment, biological Fluid is contacted with all four anti-Ago antibody.In another embodiment, biofluid and more than one can be identified The antibody of Argonaut protein contacts.This kind of antibody can recognize a kind of, two kinds, three kinds or four kinds of Argonaut protein.In In one embodiment, biofluid connects with the anti-Argonaut antibody that can identify all four people Argonaut protein Touching.
Biofluid contacts to form immunoprecipitation miRNA compound with the anti-miRNA binding protein reagent of the disclosure.Cause This, when biofluid and fixed anti-miRNA binding protein reagent contact, anti-miRNA binding protein reagent is typically attached to solid Body carrier is to form immunoprecipitation miRNA compound.Solid carrier can be that can be modified to contain and be suitable for anti-miRNA combination The material in discrete individual sites of protein reagent attachment or association.The non-limiting example of solid carrier material includes glass, modification Or functional glass, plastics include the copolymer of acrylic resin, polystyrene and styrene and other materials, polypropylene, gather Ethylene, polybutene, polyurethanes or TeflonJ, nylon, nitrocellulose, polysaccharide, resin, silica or be based on two The material of silica includes silicon and modified silicon, carbon, metal, unorganic glass and plastics.The size and shape of solid carrier is alterable Without departing from the scope of the invention.Solid carrier can be plane, and solid carrier can be hole, that is, 364 orifice plates, or alternatively, solid carries Body can be bead or slide plate.In some embodiments, solid carrier is multiple-plate hole.In other embodiments, solid Carrier is the inner surface of pipette tips.In other embodiments, solid carrier is preferably bead.In some embodiments In, solid carrier is preferably magnetic beads.
Anti- miRNA binding protein reagent can be attached to solid carrier in many ways, as those skilled in the art recognize. The two can be then attached by anti-miRNA binding protein reagent and solid carrier with chemical functional group's derivatization.For example, Solid carrier can be with the chemical functional group of including but not limited to amino, carboxyl, oxo group or thiol group come derivatization.It uses These functional groups, functional group can be used directly to be attached for anti-miRNA binding protein reagent, or is attached indirectly using connector.Or Person, anti-miRNA binding protein reagent non-covalent can also be attached to solid carrier.For example, the anti-miRNA of biotinylation can be prepared Binding protein reagent causes to be attached in combination with to the solid carrier being covalently coated with streptavidin.It will resist Other methods that miRNA binding protein reagent is attached to solid carrier are well known in the art, and can be tested as announced Room handbook such as " Current Protocols in Molecular Biology " Ausubel et al. John Wiley&Sons, New York, 2003 or " Molecular Cloning:A Laboratory Manual " Sambrook&Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, the 3rd edition, described in 2001.In some embodiments In, the anti-miRNA binding protein reagent of biotinylation is prepared, in combination with the bead being extremely covalently coated with streptavidin Solid carrier causes to be attached.As described in following part I (d), in disclosed method, anti-miRNA binding protein is tried Agent is attached to solid carrier, then contacts biofluid.Alternatively, the biofluid of the disclosure can be simultaneously in conjunction with anti-miRNA Protein reagent and solid carrier contact, wherein anti-miRNA binding protein reagent is attached to solid carrier.The biofluid of the disclosure It can also be contacted before contacting biofluid with solid carrier with anti-miRNA binding protein reagent, wherein anti-miRNA combination egg White reagent is attached to solid carrier.If those skilled in the art recognize, the amount and concentration of anti-miRNA binding protein reagent may And it will change, depending in the characteristic, the volume of used biofluid, biofluid of anti-miRNA binding protein reagent MiRNA concentration and miRNA binding protein and other factors, and can experimentally determine.When anti-miRNA binding protein When reagent is antibody purification, about 0.5 to about 10 μ g antibody is can be used in every 0.2m1 blood plasma or blood serum sample.
(d) it contacts biofluid and separates miRNA
In disclosed method, biofluid is contacted with surfactant and anti-miRNA binding protein reagent.Such as ability Field technique personnel recognize that biofluid can be contacted with various other reagents without departing from the scope of the invention.For example, biofluid The formation to block disulfide bond and the inhibitory RNA enzymatic activity during miRNA is separated can be contacted with mercaptan-reducing agent.It closes Suitable mercaptan-reducing agent includes dithiothreitol (DTT) (DTT), 2 mercapto ethanol, 2-MEA and three (carboxyethyl) phosphines (TCEP).It is raw Logistics body can also be contacted with defoaming agent.The example of defoaming agent include defoaming agent 204 and defoaming agent O-30, defoaming agent A, defoaming agent B, Defoaming agent C, defoaming agent Y-30 and Sag 471.Biofluid can also be contacted with RNA and protein degradation inhibitor to save MiRNA and miRNA- protein complex.
In some embodiments, buffer can be used for remaining suitable for separating the pH of miRNA.As non-limiting example, Buffer may include but be not limited to trishydroxymethylaminomethane acetic acid esters, EDTA, three (methylol) aminomethanes, glycine and lemon Lemon acid.
In some embodiments, disclosed method includes making biofluid and surfactant fluid to dissociate biology Fluid components contact biofluid with anti-miRNA binding protein reagent to form immunoprecipitation miRNA compound.In In other embodiments, biofluid is contacted with surfactant and anti-miRNA binding protein reagent simultaneously.
In some embodiments, the undiluted sample of biofluid and surfactant and anti-miRNA binding protein try Agent contact.In other embodiments, biofluid is dilute before contacting with surfactant and anti-miRNA binding protein reagent It releases.The dilution of biofluid can be as described by above section I (a).
Contact between biofluid, surfactant and anti-miRNA binding protein reagent generally comprise incubation period with Allow to be formed immunoprecipitation miRNA compound.Biofluid can contact simultaneously with surfactant and anti-miRNA binding protein reagent And it is incubated for about 1,5,10,15,30,45,60,90,120,240 or 480 minute or longer.In some embodiments, biology stream Body contacts with surfactant and anti-miRNA binding protein reagent and is incubated for about 1,2,3,4,5,6,7,8,9,10,11,12, 13,14 or about 15 minutes.In other embodiments, biofluid connects with surfactant and anti-miRNA binding protein reagent It touches and is incubated for about 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or about 30 Minute.In other embodiments, biofluid is contacted with surfactant and anti-miRNA binding protein reagent and is incubated for about 20,25,30,35,40,45,50,55,60,65,70,75,80,85 or about 90 minutes.In other embodiments, biofluid It contacts and is incubated for about 90,120,240 or 480 minutes or longer with surfactant and anti-miRNA binding protein reagent.One In a embodiment, biofluid preferably contacts with surfactant and anti-miRNA binding protein reagent and be incubated for about 20, 25,30,35,40,45,50,55 or about 60 minutes.
Biofluid can about 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20, 21, at 22,23,24,25,26,27,28,29 or about 30 DEG C or higher temperature with surfactant and anti-miRNA binding protein Reagent contact.In some embodiments, biofluid about 0,1,2,3,4,5 or about 6 DEG C at a temperature of and surfactant It is contacted with anti-miRNA binding protein reagent.In other embodiments, biofluid about 5,6,7,8,9,10,11,12,13, It is contacted at a temperature of 14 or about 15 DEG C with surfactant and anti-miRNA binding protein reagent.In other embodiments, biological Fluid about 11,12,13,14,15,16,17,18,19,20,21,22,23,24 or about 25 DEG C at a temperature of and surfactant It is contacted with anti-miRNA binding protein reagent.In other embodiments, biofluid about 20,21,22,23,24,25,26, 27, it is contacted with surfactant and anti-miRNA binding protein reagent at a temperature of 28,29 or about 30 DEG C.
Typically, biofluid is contacted with surfactant and anti-miRNA binding protein reagent under stiring.In addition, In It is formed after compound, biofluid, which can be removed generally, precipitates miRNA compound with separating immune, and by immunoprecipitation The washing of miRNA compound.
(e) miRNA is discharged
According to disclosed method, discharge miRNA from immunoprecipitation miRNA compound.It is released from protein complex The method for putting nucleic acid such as miRNA is well known in the art and may include proteolytic enzyme digest, and makes nucleic acid-protein Protein denaturation in matter compound.In some embodiments, miRNA is multiple from immunoprecipitation miRNA by protein denaturation Close object release.For example, miRNA can by combine immunoprecipitation miRNA compound with guanidinium thiocyanate-phenol-chloroformic solution come It is discharged from immunoprecipitation miRNA compound.Then, release miRNA can be purified by precipitating or using adsorpting column chromatogram.
In other embodiments, miRNA preferably passes through proteolytic enzyme digest and releases from immunoprecipitation miRNA compound It puts.Term " proteolytic enzyme (protease) ", " protease (proteinase) " and " peptase " is used interchangeably herein And refer to the enzyme group of the hydrolysis of catalysis covalent peptide bonds.Proteolytic enzyme is well known in the art and may include acidity Proteolytic enzyme and serine protein hydrolase.In some embodiments, can be used for discharging miRNA in disclosed method Proteolytic enzyme be bace protein.In one embodiment, can be used for discharging miRNA in disclosed method Bace protein be pepsin.
In other embodiments, the proteolytic enzyme that can be used for discharging miRNA in disclosed method is acid egg White hydrolase.Six clusters of identified serine protein hydrolase, two of them maximum cluster is chymotrypsin-like With subtilopeptidase A sample cluster.Many novel subtilases are known.The some novel subtilases studied extensively include from The novel subtilases that various bacillus obtain, including subtilopeptidase A DY, subtilopeptidase A Carlsberg, Subtilopeptidase A BPN ' (also known as nagarse), the rod-shaped mycoproteinase of alkalinity and from woods Bai Shi Candida albicans The Proteinase K of acquisition, and the thermophilic protease obtained from Thermoactinomyces.In certain embodiments of the invention, egg White enzyme K is preferably proteolytic enzyme.However, other proteolytic enzymes, such as withered grass can also be used in certain embodiments Mycoproteinase.Therefore, proteolytic enzyme can be cause at least partly decomposition of the protein in immunoprecipitation miRNA compound with So that any one of many proteolytic enzymes of release miRNA.It in some embodiments, can in disclosed method Proteolytic enzyme for discharging miRNA is preferably proteolytic enzyme K.
In itself, release that miRNA from immunoprecipitation miRNA compound by contacting compound with proteolytic enzyme It puts.If those skilled in the art recognize, it may and will change for discharging the amount of proteolytic enzyme of miRNA, depend on Temperature during proteolytic enzyme, the abundance of immunoprecipitation miRNA compound, proteolytic enzyme digest, delaying for digestion Fliud flushing condition and digestion duration and other factors.In general, immunoprecipitation miRNA compound can be with the enzyme of about 0.3 unit The enzymatic activity of activity to about 30 units contacts.In certain embodiments, with the albumen water of immunoprecipitation miRNA complex contacts The amount for solving enzyme can be in about 0.3 to about 1 unit, about 1 to about 3 unit, about 3 units to about 10 units, or about 10 units to about 30 lists In the range of position.
In some embodiments, as described in Example 1, proteolytic enzyme digest is used at room temperature.As made herein With term " room temperature " is used to describe about 10 DEG C to about 30 DEG C of temperature.
Immunoprecipitation miRNA compound can be incubated with about 0.5 with proteolytic enzyme, 1,2,3,4,5,6,7,8,9,10, 11,12,13,14,15,16,17,18,1920,25 or about 30 minutes or longer.In some embodiments, immunoprecipitation MiRNA compound and proteolytic enzyme are incubated with about 0.5,1,2,3,4 or about 5 minute.In other embodiments, it is heavy to be immunized Shallow lake miRNA compound and proteolytic enzyme are incubated with about 5,6,7,8,9,10,11,12,13,14 or about 15 minutes.Other In embodiment, immunoprecipitation miRNA compound and proteolytic enzyme are incubated with about 15,16,17,18,1920,25 or about 30 Minute is longer.
Release miRNA is suitably adapted for using in the case where not being further purified for downstream.Alternatively, release miRNA can It is further purified and is used for downstream.Nucleic acid purification method, such as adsorpting column chromatogram or filtering technique are known in the art , for example, according to laboratory manual such as " Current Protocols in Molecular Biology " Ausubel is announced Et al. John Wiley&Sons, New York, 2003 or " Molecular Cloning:A Laboratory Manual " Sambrook&Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, the 3rd edition, 2001 institutes The method of description.
The downstream for discharging miRNA, which uses, can change.Discharge the non-limiting using including quantitative real-time PCR, micro- battle array of miRNA Column analysis, sequencing, restrictive fragment length polymerphism (RFLP) analysis, single nucleotide polymorphisms (SNP) analysis, microsatellite point Analysis, Short tandem repeatSTR (STR) is analyzed and comparative genome hybridization (CGH).
II. kit
The present invention further provides kits comprising surfactant, anti-miRNA binding protein reagent, and can be used for Other reagents of disclosed method.In some embodiments, it provides for the kit from biofluid separation miRNA, The kit includes anti-miRNA binding protein reagent and surface action reagent.Anti- miRNA binding protein reagent and surface-active Agent can be as described by above-mentioned part (I).In some embodiments, the anti-miRNA binding protein reagent in kit is attachment To the anti-Ago antibody of solid carrier.In certain embodiments, solid carrier can be bead, magnetic beads or multiple-plate hole. In other embodiments, solid carrier can be the inner surface of pipette tips.In some embodiments, the table in kit Face agent is IGEPAL.Kit can further include the component from immunoprecipitation miRNA compound release miRNA.In some realities It applies in scheme, kit includes for the proteolytic enzyme from immunoprecipitation miRNA compound release miRNA, for example, albumen water Solve enzyme K.
Definition
Unless otherwise defined, otherwise all technical and scientific terms used herein have fields of the present invention In the meaning that is generally understood of those of ordinary skill.It is provided for those skilled in the art in the present invention below with reference to document Many terms general definition: Singleton et al. Dictionary of Microbi010gy and M0lecular Biology (second edition .1994)；The Cambridge Dictionary ofScience and Technology(Walker Ed., 1988)；The Glossary of Genetics, the 5th edition, R.Rieger et al. (eds.), Springer Verlag (1991)；And Hale&Marham, The Harper Collins Dictionary ofBiology (1991).Such as this paper institute With unless otherwise specified, following term has the meaning for assigning it.
When introducing the element of the disclosure or one or more preferred aspect, article "/kind (a) ", "/kind (an) ", " (the) " and " (said) " means that there are one or more elements.The terms "include", "comprise" " having " is intended to inclusive, and means that other element may be present in addition to listed element.
As used herein, " Microrna " or " miRNA " refers to 5 to 40 nucleotide that can be detected in biological sample The small non-coding RNA sequence of length.Some miRNA are obtained from hairpin precursor, and the precursor is for example handled by enzyme DICER At mature substance, for example, about 18-25 nucleotide, preferably 21-23 nucleotide.Microrna variant is for example in different animals It is common between species.In addition, the variation of the 5 ' of miRNA and 3 ' ends is common, and can be by leading to during maturing It crosses caused by the inaccurate cracking of enzyme such as DICER.These variants, which show in miRNA sequence, does not weaken function or detection A series of acceptable variations of the ability of miRNA.Another seed type variant is to add nontemplated nucleotide extremely after Dicer is handled 3 ' the ends of miRNA (these nucleotide are non-templates, because it mismatches human genome).Most common variant is that have addition To the additional A of 3 ' ends or the miRNA sequence of U.
As used herein, term " biofluid " or " body fluid " are used interchangeably and refer to the stream separated from subject Body.
Term " biofluid ", " biologicfluid sample " or " biological sample " is used interchangeably and means from any given All biofluids and excreta of subject's separation.In situation of the invention, this kind of sample include but is not limited to blood and Its part, serum, blood plasma, urine, excreta, sperm (semen), sperm (seminal fluid), seminal plasma, prostatic fluid, Secretion (examining amber liquid), pleural effusion, tears, saliva, phlegm, sweat, biopsy, ascites, cerebrospinal fluid, amniotic fluid, lymph before ejaculation, Marrow, cervical secretions, vaginal fluid, endometrial secretions, gastrointestinal secretion object, bronchial secretion, mammal gland secretion, Ovarian cyst secretion and tissue fluid sample.
" separation " polynucleotides are by identification and at least one dirty with usually associating with it in its natural origin Contaminate the nucleic acid molecules of object separation.Isolated nucleic acid molecules be in when it is found in nature other than form or environment.Cause This, isolated nucleic acid molecules are different from the specific nucleic acid molecule when it is present in n cell.
Because various changes can made to above-mentioned animal, cell and method without departing from the scope of the invention, All items contained in above description and in embodiments given below all should be interpreted that illustrative rather than have limitation Property meaning.
Embodiment
Comprising following embodiment to illustrate the disclosure.Those skilled in the art are understood that be disclosed in the following embodiments Technology represent the inventors have found that the disclosure in practice in play preferable technology.But in view of the disclosure, ability Field technique personnel recognize can carry out many variations in the disclosure, and still obtain it is same or like as a result, without Away from the spirit and scope of the present invention, therefore, all items shown be interpreted as it is illustrative, without limited significance.