CN106544317A - A kind of method that spermatid and excretion body are isolated and purified from pig semen - Google Patents
A kind of method that spermatid and excretion body are isolated and purified from pig semen Download PDFInfo
- Publication number
- CN106544317A CN106544317A CN201610975313.8A CN201610975313A CN106544317A CN 106544317 A CN106544317 A CN 106544317A CN 201610975313 A CN201610975313 A CN 201610975313A CN 106544317 A CN106544317 A CN 106544317A
- Authority
- CN
- China
- Prior art keywords
- spermatid
- exosome
- supernatant
- seminal fluid
- precipitation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000000582 semen Anatomy 0.000 title claims abstract description 97
- 238000000034 method Methods 0.000 title claims abstract description 29
- 230000029142 excretion Effects 0.000 title claims abstract description 17
- 210000001808 exosome Anatomy 0.000 claims abstract description 75
- 239000006228 supernatant Substances 0.000 claims abstract description 48
- 238000001556 precipitation Methods 0.000 claims abstract description 42
- 238000005119 centrifugation Methods 0.000 claims abstract description 29
- 210000002966 serum Anatomy 0.000 claims abstract description 29
- 108091070501 miRNA Proteins 0.000 claims abstract description 26
- 239000002679 microRNA Substances 0.000 claims abstract description 25
- 210000001082 somatic cell Anatomy 0.000 claims abstract description 20
- 238000010839 reverse transcription Methods 0.000 claims abstract description 18
- 210000004027 cell Anatomy 0.000 claims abstract description 16
- 108020004999 messenger RNA Proteins 0.000 claims abstract description 14
- 239000000725 suspension Substances 0.000 claims abstract description 13
- 239000013592 cell lysate Substances 0.000 claims abstract description 10
- 238000005199 ultracentrifugation Methods 0.000 claims abstract description 8
- 238000005336 cracking Methods 0.000 claims abstract description 7
- 238000001914 filtration Methods 0.000 claims abstract description 6
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 15
- 102000006382 Ribonucleases Human genes 0.000 claims description 14
- 108010083644 Ribonucleases Proteins 0.000 claims description 14
- 238000010790 dilution Methods 0.000 claims description 14
- 239000012895 dilution Substances 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 12
- 102000007327 Protamines Human genes 0.000 claims description 11
- 108010007568 Protamines Proteins 0.000 claims description 11
- 239000003085 diluting agent Substances 0.000 claims description 11
- 239000012634 fragment Substances 0.000 claims description 11
- 229940048914 protamine Drugs 0.000 claims description 11
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 238000013518 transcription Methods 0.000 claims description 9
- 230000035897 transcription Effects 0.000 claims description 9
- 239000012295 chemical reaction liquid Substances 0.000 claims description 8
- 238000011534 incubation Methods 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 210000001519 tissue Anatomy 0.000 claims description 7
- 108091092584 GDNA Proteins 0.000 claims description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 3
- 229930182566 Gentamicin Natural products 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 claims description 3
- 108091093189 Mir-375 Proteins 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 239000002299 complementary DNA Substances 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 108091091807 let-7a stem-loop Proteins 0.000 claims description 3
- 108091057746 let-7a-4 stem-loop Proteins 0.000 claims description 3
- 108091028376 let-7a-5 stem-loop Proteins 0.000 claims description 3
- 108091024393 let-7a-6 stem-loop Proteins 0.000 claims description 3
- 108091091174 let-7a-7 stem-loop Proteins 0.000 claims description 3
- 108091007423 let-7b Proteins 0.000 claims description 3
- 108091027034 miR-148a stem-loop Proteins 0.000 claims description 3
- 108091070404 miR-27b stem-loop Proteins 0.000 claims description 3
- 108091090583 miR-34c stem-loop Proteins 0.000 claims description 3
- 108091082133 miR-34c-1 stem-loop Proteins 0.000 claims description 3
- 108091076732 miR-99a stem-loop Proteins 0.000 claims description 3
- 108091064318 miR-99a-1 stem-loop Proteins 0.000 claims description 3
- 108091086202 miR-99a-2 stem-loop Proteins 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 3
- 239000000376 reactant Substances 0.000 claims description 3
- 239000011435 rock Substances 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- 239000001117 sulphuric acid Substances 0.000 claims description 3
- 235000011149 sulphuric acid Nutrition 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims 1
- 238000000926 separation method Methods 0.000 abstract description 2
- 239000012535 impurity Substances 0.000 abstract 1
- 238000004062 sedimentation Methods 0.000 abstract 1
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 230000004720 fertilization Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000007670 refining Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- LRYZPFWEZHSTHD-HEFFAWAOSA-O 2-[[(e,2s,3r)-2-formamido-3-hydroxyoctadec-4-enoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium Chemical class CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](NC=O)COP(O)(=O)OCC[N+](C)(C)C LRYZPFWEZHSTHD-HEFFAWAOSA-O 0.000 description 1
- 241000208340 Araliaceae Species 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 108091029474 Y RNA Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000004709 cell invasion Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000001085 differential centrifugation Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000035992 intercellular communication Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 230000021121 meiosis Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000029052 metamorphosis Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 230000008010 sperm capacitation Effects 0.000 description 1
- 210000004336 spermatogonium Anatomy 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0608—Germ cells
- C12N5/061—Sperm cells, spermatogonia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Reproductive Health (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides a kind of method that spermatid and excretion body are isolated and purified from pig semen, and step is：Collection boar semen dilutes in proportion, after filtering centrifugation precipitation and supernatant；After the cracking of precipitation somatic cell lysate, Jing centrifugal sedimentations spermatid obtains spermatid precipitation again；Retain supernatant after supernatant centrifugation, be continuously removed by filtration cell debriss therein, then exosome is precipitated by ultracentrifugation, add PBS solution suspension exosome.Total serum IgE in extracting spermatid and exosome suspensions, reverse transcription mRNA and miRNA, and cell and exosome purity are detected by quantitative mark mRNA and miRNA.The inventive method can improve the separation accuracy rate of pig semen spermatid and exosome, and the purity of the spermatid that is effectively ensured after separating and exosome, and operational approach is simple, impurity is few.
Description
Technical field
The invention belongs to cell biology, and in particular to one kind isolates and purifies spermatid and excretion from pig semen
The method of body (exosome).
Background technology
Normal semen is a kind of sticky liquid mixture, is made up of sperm and refining.
Sperm is the sexual cell of buck.In mammalian sperm generating process, primordial germ cells are budded into
Spermatogonium, then develop for spermatocyte, spermatocyte becomes circular spermatid through meiosis twice, and these are circular fine
Development by metamorphosis process of the born of the same parents through spermiogenesis tail (spermiogenesis), excludes most cells matter, and internal generation is a series of
Change, becomes sperm.The structure of ripe sperm (spermatozoon) can be divided into head, 3 part of neck and tail.People think always
Sperm has now found that it in the vital movement of after fertilization, plays more important on fertilization highly important impact of successfully have
Effect.The accuracy of the composition and its structure of sperm genome, after deep effect fertilization, embryo's generation and fetal birth
Survive.Therefore, for the research of spermatid has become more deep.
In the refining of mammal containing by high concentration cholesterol, sphingomyelins and complex structure protein etc. into being grouped into
Film vesicle (exosome), it is consistent with excretion body in morphology and molecular composition, so we are using term " outside seminal fluid
Secrete body " (seminal exosomes, SE).Correlational study shows seminal fluid excretion body except rich in proteins, lipid, also containing very
Many non-coding RNAs such as tRNA, miRNA, piwi-RNA, YRNA, rRNA and mRNA.Pig refining exosome can Inhibit sperm capacitation
The outflow of sperm top Membrane cholesterol and the mobility of film are shown, and the TYR phosphorylation of 14-kD polypeptide proteins disappears
Lose, and these activation for all possessing fertility with sperm are relevant.Exosomes is released to extracellular not just to list
The albumen discarded in the scavenger cell of pure ground, it also take part in intercellular communication.Which can transmit life as intercellular carrier
The physiology and pathological information of thing, take part in various different physiology and pathological process.The current research to exosomes is not only
In its function aspects, application --- the exploitation i.e. in terms of methods for diagnosis and treatment of reality is further related to.In view of exosomes contains
Have the bio informations such as substantial amounts of transport protein, mRNA, microRNA, can adjust immunoreation, angiogenesiss, cell propagation and
Tumor cell invasion, which can be originated as ideal biomarker, be expected to send out in the early diagnosiss of various diseases
The effect of waving.Therefore, the research for pig semen exosome has become more and more extensive.
In order to avoid polluting containing somatic cell in spermatid, the present invention adds somatic cell in the precipitation after seminal fluid centrifugation
Lysate, removes the somatic cell pollution in spermatid, can effectively improve the purity of spermatid.Seminal fluid exosome is extracted
Conventional method is ultracentrifugation method, and the method for ultraspeed mainly first removes cell and thin by the method for differential centrifugation
Born of the same parents' fragment, finally separates exosome using the method for ultracentrifugation.
The content of the invention
The present invention for above-mentioned problem make improvement, i.e. the technical problem to be solved in the present invention be to provide one kind from
The method that spermatid and excretion body (exosome) are isolated and purified in pig semen, the method can be efficiently separated, purify sperm
Cell and seminal fluid exosome, and detect the purity of spermatid and seminal fluid exosome.
For achieving the above object, the technical solution used in the present invention is：One kind isolates and purifies spermatid from pig semen
With the method for excretion body, step includes：
(1) the boar semen semen diluent for collecting is diluted in proportion, 40 μm of filters of Jing are filtered out in seminal fluid
Fragment of tissue and debris, then by centrifugation precipitation and supernatant；
(2) precipitation Jing centrifugations spermatid again after the cracking of somatic cell lysate, adds PBS solution cleaning, resuspended
Spermatid, then after being centrifuged, obtain spermatid precipitation；
(3) supernatant retains supernatant again Jing after centrifugation, continues through 0.45 μm and 0.22 μm of filter is filtered out
Cell debriss in exosome, then exosome is precipitated by ultracentrifugation, add PBS solution suspension exosome；
(4) extract the total serum IgE in spermatid and exosome suspensions with Trizol methods, carry out respectively mRNA and
MiRNA reverse transcriptions and related gene it is quantitative, and purity is detected.
Further：Step (1) concrete operations are as follows：
1) by the boar semen semen diluent for collecting by 1:1 dilution proportion；
2) seminal fluid after dilution is dissolved into 20min at room temperature；
3) again seminal fluid after dilution is placed in 37 DEG C of water-baths and is incubated 15min；
4) seminal fluid after incubation is mixed, the fragment of tissue and debris in seminal fluid is filtered out by 40 μm of filter；
5) by the seminal fluid refrigerated centrifuger after filtration, under the conditions of 4 DEG C, 1000 × g is centrifuged 10min, gently takes after centrifugation
Go out to shake and rock, separate supernatant and precipitation, supernatant and precipitation are taken in centrifuge tube respectively.
Further：Step 1) in, the semen dilution formula of liquid is D-glucose 27.5g/L, sodium citrate 6.9g/
Sticking L, sodium bicarbonate 1g/L, EDTA2.35g/L, citric acid 2.9g/L, dihydroxy first (base) aminomethane 5.065g/L, sulphuric acid mould more
Element (B) 0.0169g/L, gentamycin sulfate 0.15g/L and distilled water.
Further：Step (2) concrete operations are as follows：
1) to income centrifuge tube in precipitation in by volume be 8:1 adds somatic cell lysate, mixes, is placed on and incubates on ice
Educate 40min；
2) by the precipitation refrigerated centrifuger after incubation, under conditions of 4 DEG C, 600 × g is centrifuged 5min, and supernatant is abandoned after centrifugation
Retain precipitation；
3) PBS solution of its volume 25% is added to mix in precipitating, again with refrigerated centrifuger under conditions of 4 DEG C after mixing
600 × g is centrifuged 5min, abandons supernatant and retains precipitation, be repeated 2 times after centrifugation.
Further：Step 1) in, the somatic cell cracking formula of liquid is：SDS 0.001g/mL、Triton-100 5μ
L/mL and DEPC.
Further：Step (3) concrete operations are as follows：
1) supernatant 10000 × g centrifugations 45min under conditions of 4 DEG C, abandons precipitation and retains supernatant；
2) supernatant is continued through into 0.45 μm and 0.22 μm of filter is filtered；
3) by supernatant, in Ultracentrifuge, 130000 × g is centrifuged 90min；
4) supernatant is outwelled after being centrifuged, dissolve again suspension exosome with 100 μ L PBS solutions.
Further：Step (4) is concretely comprised the following steps：
1) extracting sperm total serum IgE and seminal fluid exosome total serum IgEs carries out reverse transcription；
2) the addition RNase Free dH in the inverse transcription reaction liquid for obtaining2O dilutes 4 times；
3) carried out with protamine and reference gene GAPDH quantitatively, detection protamine is in spermatid and seminal fluid
Expression in exosome, reaction system are II 5.0 μ L of SYBR premix Taq, 0.5 μ L of forward primer, 0.5 μ of downstream primer
L, cDNA 1.0 μ L, DEPC supply 10 μ L；
4) according to fluorescent quantitation result, respectively to protamine and reference gene in spermatid and seminal fluid exosome
The relative expression quantity of GAPDH is calculated, and when mean difference multiple is more than 40 times, spermatid is successfully separated；
5) extracting spermatid total serum IgE and seminal fluid exosome total serum IgEs carries out miRNA reverse transcriptions；
6) RNase Free dH are added in inverse transcription reaction liquid2O dilutes 5 times；
7) it is chosen at the high expression miRNA identified in seminal fluid exosome：let-7b、miR-148a、let-
7a, miR-375, miR-99a, miR-27b, miR-34c, and reference gene U6 carries out quantitatively, reaction system is：2×SYBR
5.0 μ L of mix, 0.5 3 ' Primer of μ L, mRQ of miRNA-specific primer, 0.5 μ L, cDNA1.0 μ L, DEPC supply 10
μ L systems；
8) according to fluorescent quantitation result, the phase to miRNA and reference gene U6 in spermatid and seminal fluid exosome respectively
Expression is calculated when mean difference multiple is more than 40 times, seminal fluid exosome is successfully separated.
Further：Step 1) in, the sperm total serum IgE and seminal fluid exosome total serum IgE reverse transcriptions include two steps,
Step one is：5 × gDNA Eraser buffer, 2.0 μ L, 1.0 μ L of gDNA Eraser, TotalRNA 1.0 μ L and RNase
Free dH26.0 μ L of O, reaction condition are 42 DEG C of 2min；Step 2 is：Prime Script RT Enzyme MixI 1.0μ
L、RT Primer Mix 1.0μL、5×Prime Script Buffer2 4.0μL、RNase Free dH2O4.0 μ L, step
One reactant liquor, 10.0 μ L, reaction condition are 37 DEG C of 15min, 85 DEG C of 5s.
The method have the benefit that：The present invention removes somatic dirt in spermatid using somatic cell lysate
Dye, effectively raises the purity of spermatid；Essence is filtered using cell sieve (continuing through 0.45 μm and 0.22 μm of filter)
Liquid exosome, somatic cell, spermatid and the cell debriss remained in removing seminal fluid exosome, effectively raises seminal fluid
The purity of exosome；Ultracentrifugation can carry out the separation of exosome to larger semen sample, and isolate
Exosome has higher biological activity, so as to not interfere with follow-up experiment.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described,
Obviously, described embodiment is only a part of embodiment of the invention, rather than the embodiment of whole.Based in the present invention
Embodiment, the every other embodiment obtained under the premise of creative work is not made by those of ordinary skill in the art, all
Belong to the scope of protection of the invention.
Embodiment 1
A kind of method that spermatid and excretion body are isolated and purified from pig semen, step include：
(1) the boar semen semen diluent for collecting is diluted in proportion, 40 μm of filters of Jing are filtered out in seminal fluid
Fragment of tissue and debris, then by centrifugation precipitation and supernatant, concretely comprise the following steps：
1) by the boar semen semen diluent for collecting by 1:1 dilution proportion；Semen dilution formula of liquid is dextrorotation
Glucose 27.5g/L, sodium citrate 6.9g/L, sodium bicarbonate 1g/L, EDTA2.35g/L, citric acid 2.9g/L, dihydroxy first (base)
Mycin (B) 0.0169g/L, gentamycin sulfate 0.15g/L and distilled water are sticked aminomethane 5.065g/L, sulphuric acid more.
2) seminal fluid after dilution is dissolved into 20min at room temperature；
3) again seminal fluid after dilution is placed in 37 DEG C of water-baths and is incubated 15min；
4) seminal fluid after incubation is mixed, the fragment of tissue and debris in seminal fluid is filtered out by 40 μm of filter；
5) by the seminal fluid refrigerated centrifuger after filtration, under the conditions of 4 DEG C, 1000 × g is centrifuged 10min, gently takes after centrifugation
Go out to shake and rock, separate supernatant and precipitation, supernatant and precipitation are taken in centrifuge tube respectively.
(2) precipitation Jing centrifugations spermatid again after the cracking of somatic cell lysate, adds PBS solution cleaning, resuspended
Spermatid, then spermatid precipitation after being centrifuged, is obtained, concretely comprise the following steps：
1) to income centrifuge tube in precipitation in by volume be 8:1 adds somatic cell lysate, mixes, is placed on and incubates on ice
Educate 40min；Somatic cell cracks formula of liquid：SDS 0.001g/mL, Triton-100 5 μ L/mL and DEPC.
2) by the precipitation refrigerated centrifuger after incubation, under conditions of 4 DEG C, 600 × g is centrifuged 5min, and supernatant is abandoned after centrifugation
Retain precipitation；
3) PBS solution of its volume 25% is added to mix in precipitating, again with refrigerated centrifuger under conditions of 4 DEG C after mixing
600 × g is centrifuged 5min, abandons supernatant and retains precipitation, be repeated 2 times after centrifugation.
(3) supernatant retains supernatant again Jing after centrifugation, continues through 0.45 μm and 0.22 μm of filter is filtered out
Cell debriss in exosome, then exosome is precipitated by ultracentrifugation, add PBS solution suspension exosome；
1) supernatant 10000 × g centrifugations 45min under conditions of 4 DEG C, abandons precipitation and retains supernatant；
2) supernatant is continued through into 0.45 μm and 0.22 μm of filter is filtered；
3) by supernatant, in Ultracentrifuge, 130000 × g is centrifuged 90min；
4) supernatant is outwelled after being centrifuged, dissolve again suspension exosome with 100 μ L PBS solutions.
(4) extract the total serum IgE in spermatid and exosome suspensions with Trizol methods, carry out respectively mRNA and
MiRNA reverse transcriptions and related gene it is quantitative, and purity is detected.
1) extracting sperm total serum IgE and seminal fluid exosome total serum IgEs carries out reverse transcription；Sperm total serum IgE and seminal fluid exosome are total
RNA reverse transcriptions include two steps, and step one is：5×gDNA Eraser buffer 2.0μL、gDNA Eraser 1.0μL、
1.0 μ L and RNase Free dH of TotalRNA26.0 μ L of O, reaction condition are 42 DEG C of 2min；Step 2 is：Prime
Script RT Enzyme MixI 1.0μL、RT Primer Mix 1.0μL、5×Prime Script Buffer2 4.0μ
L、RNase Free dH24.0 μ L of O, 10.0 μ L of step one reactant liquor, reaction condition are 37 DEG C of 15min, 85 DEG C of 5s.
2) the addition RNase Free dH in the inverse transcription reaction liquid for obtaining2O dilutes 4 times；
3) carried out with protamine and reference gene GAPDH quantitatively, detection protamine is in spermatid and seminal fluid
Expression in exosome, reaction system are II 5.0 μ L of SYBR premix Taq, 0.5 μ L of forward primer, 0.5 μ of downstream primer
L, cDNA 1.0 μ L, DEPC supply 10 μ L；
4) according to fluorescent quantitation result, respectively to protamine and reference gene in spermatid and seminal fluid exosome
The relative expression quantity of GAPDH is calculated, and when mean difference multiple is more than 40 times, spermatid is successfully separated；
5) extracting spermatid total serum IgE and seminal fluid exosome total serum IgEs carries out miRNA reverse transcriptions；
6) RNase Free dH are added in inverse transcription reaction liquid2O dilutes 5 times；
7) it is chosen at the high expression miRNA identified in seminal fluid exosome：let-7b、miR-148a、let-
7a, miR-375, miR-99a, miR-27b, miR-34c, and reference gene U6 carries out quantitatively, reaction system is：2×SYBR
5.0 μ L of mix, 0.5 3 ' Primer of μ L, mRQ of miRNA-specific primer, 0.5 μ L, cDNA1.0 μ L, DEPC supply 10
μ L systems；
8) according to fluorescent quantitation result, the phase to miRNA and reference gene U6 in spermatid and seminal fluid exosome respectively
Expression is calculated when mean difference multiple is more than 40 times, seminal fluid exosome is successfully separated.
Embodiment 2
(1) by 1:1 ratio adds semen diluent 20mL, mixes, and loads in 50mL centrifuge tubes.Semen diluent is being adopted
Preparation in 1 hour, its composition and compound method such as table 1 before collection seminal fluid,
1 semen diluent composition of table and preparation
(2) seminal fluid after dilution is dissolved into 20min at room temperature；
(3) 15min is incubated in being then placed in 37 DEG C of water-baths；
(4) seminal fluid after incubation is mixed, the fragment of tissue and debris in seminal fluid is filtered out by 40 μm of filter；
(5) by the seminal fluid refrigerated centrifuger after filtration under the conditions of 4 DEG C 1000 × g centrifugation 10min, separate supernatant and
Precipitation, supernatant is taken in a new centrifuge tube, and precipitation is retained in former centrifuge tube；
(6) 5mL somatic cell lysates are prepared：25 μ L Triton-100 and 0.005g SDS are added in 5mL DEPC water,
Mix.5mL somatic cell lysates are added in centrifuged pellet, is put on ice for being incubated 40min, it is dirty to remove somatic cell
Dye；
(7) by the precipitation refrigerated centrifuger after incubation, under conditions of 4 DEG C, 600 × g is centrifuged 5min, to remove after cracking
Somatic cell fragment, abandon after centrifugation supernatant retain precipitation；
(8) cleaned with PBS solution, resuspended spermatid, wherein after dilution, precipitation uses 10mL PBS obtained by seminal fluid per 40mL
Solution dissolves, and mixes, be repeated 2 times after adding PBS solution；
(9) after mixing, with refrigerated centrifuger, under conditions of 4 DEG C, 600 × g is centrifuged 5min again, to remove the somatic cell of residual
Fragment, abandons supernatant and retains precipitation after centrifugation；
(10) 1mL Trizol reagents are added in gained precipitation, carries out spermatid total serum IgE extracting；
(11) by the supernatant obtained in (5) step, under the conditions of 4 DEG C, 10000 × g is centrifuged 45min, to remove seminal fluid
Cell and fragment of tissue in exosome, after centrifugation is transferred to supernatant in one new centrifuge tube, discards precipitation；
(12) supernatant is continued through into 0.45 μm and 0.22 μm of filter filters out the cell debriss in exosome；
(13) supernatant is proceeded in Ultracentrifuge, 130000 × g centrifugations 90min in Ultracentrifuge；
(14) after ultracentrifugation, supernatant is outwelled, suspension exosome is dissolved again with 100 μ L PBS solutions；
(15) every 200 μ L exosome suspension samples add 0.8mL Trizol reagents, carry out seminal fluid exosome total serum IgEs
Extracting；
Embodiment 3
Spermatid total serum IgE and seminal fluid exosome total serum IgEs to extracting carries out mRNA and miRNA reverse transcriptions respectively
It is quantitative with related gene, and purity is detected.
(1) mRNA reverse transcriptions are carried out to sperm total serum IgE and seminal fluid exosome total serum IgEs respectively first, are divided into two steps,
Step one reaction system such as table 2,
2 mRNA reverse transcription steps of table, one system
Reaction condition is 42 DEG C of 2min；
Step 2 reaction system such as table 3,
3 mRNA reverse transcription steps of table, two system
Reaction condition is 37 DEG C of 15min → 85 DEG C 5s；
(2) the addition RNase Free dH in the inverse transcription reaction liquid for obtaining2O complements to 80 μ L, dilutes 4 times；
(3) 1 μ L diluents are taken to be added in the Real Time PCR reaction systems of next step, detection by quantitative is carried out.Reaction
System such as table 4, arranges sample (being repeated 3 times), a positive control and a negative control on every plate every time, and table 7 is fixed for mRNA
Amount Ct values compare,
4 Real Time PCR reaction systems (mRNA) of table
The quantitative Ct values of 7 mRNA of table compare
(4) according to quantitative result, in sperm in protamine expression and seminal fluid exosome protamine fold differences
For 40.4 and 526, show that spermatid purity is strictly very high.
(5) miRNA reverse transcriptions, reaction system such as table 5 are carried out again to spermatid total serum IgE and seminal fluid exosome total serum IgEs：
5 miRNA reverse transcription systems of table
Reaction condition is 37 DEG C of 60min → 85 DEG C 5min；
(6) the addition RNase Free dH in the inverse transcription reaction liquid for obtaining2O complements to 100 μ L, dilutes 5 times；
(7) 1 μ L diluents are taken to be added in the Real Time PCR reaction systems of next step, detection by quantitative is carried out.Reaction
System such as table 6, arranges sample (being repeated 3 times), a positive control and a negative control on every plate every time, and table 8 is fixed for miRNA
Amount CtValue compares：
6 Real Time PCR reaction systems (miRNA) of table
The quantitative C of 8 miRNA of tabletValue compares
(8) according to quantitative result, the fold differences of seminal fluid exosome and miRNA in spermatid are 108 and 209, and
Most of miRNA is only expressed in seminal fluid exosome, shows that seminal fluid exosome purity is strictly very high.
Those skilled in the art of the present technique are appreciated that unless otherwise defined all terms used herein (include technology art
Language and scientific terminology) with art of the present invention in those of ordinary skill general understanding identical meaning.Should also
It is understood by, those terms defined in such as general dictionary are should be understood that with the meaning in the context with prior art
The consistent meaning of justice, and unless defined as here, will not be with idealizing or excessively formal implication is explaining.
It should be noted last that：Above example is only to illustrative and not limiting technical scheme, although ginseng
The present invention is described in detail according to above-described embodiment, it will be apparent to an ordinarily skilled person in the art that：Still can be to this
Invention is modified or equivalent, any modification or partial replacement without departing from the spirit and scope of the present invention, and which is equal
Should cover in the middle of scope of the presently claimed invention.
Claims (8)
1. a kind of method that spermatid and excretion body are isolated and purified from pig semen, it is characterised in that step includes：
(1) the boar semen semen diluent for collecting is diluted in proportion, 40 μm of filters of Jing filter out the group in seminal fluid
Fragment and debris are knitted, then by centrifugation precipitation and supernatant；
(2) precipitation Jing centrifugations spermatid again after the cracking of somatic cell lysate, adds PBS solution cleaning, resuspended sperm
Cell, then after being centrifuged, obtain spermatid precipitation；
(3) supernatant retains supernatant again Jing after centrifugation, continues through 0.45 μm and 0.22 μm of filter is filtered out in exosome
Cell debriss, then by ultracentrifugation precipitate exosome, add PBS solution suspension exosome；
(4) total serum IgE in spermatid and exosome suspensions is extracted with Trizol methods, carry out mRNA respectively and miRNA is anti-
Transcription is quantitative with related gene, and purity is detected.
2. the method for isolating and purifying spermatid and excretion body according to claim 1 from pig semen, it is characterised in that step
Suddenly (1) concrete operations are as follows：
1) by the boar semen semen diluent for collecting by 1:1 dilution proportion；
2) seminal fluid after dilution is dissolved into 20min at room temperature；
3) again seminal fluid after dilution is placed in 37 DEG C of water-baths and is incubated 15min；
4) seminal fluid after incubation is mixed, the fragment of tissue and debris in seminal fluid is filtered out by 40 μm of filter；
5) by the seminal fluid refrigerated centrifuger after filtration, under the conditions of 4 DEG C, 1000 × g is centrifuged 10min, gently takes out not after centrifugation
Shake and rock, separate supernatant and precipitation, supernatant and precipitation are taken in centrifuge tube respectively.
3. the method for isolating and purifying spermatid and excretion body according to claim 2 from pig semen, it is characterised in that step
It is rapid 1) in, the semen dilution formula of liquid be D-glucose 27.5g/L, sodium citrate 6.9g/L, sodium bicarbonate 1g/L,
Glutinous mycin (B) 0.0169g/L more than EDTA2.35g/L, citric acid 2.9g/L, dihydroxy first (base) aminomethane 5.065g/L, sulphuric acid,
Gentamycin sulfate 0.15g/L and distilled water.
4. the method for isolating and purifying spermatid and excretion body according to claim 1 from pig semen, it is characterised in that：Step
Suddenly (2) concrete operations are as follows：
1) to income centrifuge tube in precipitation in by volume be 8:1 adds somatic cell lysate, mixes, is placed on and is incubated on ice
40min；
2) by the precipitation refrigerated centrifuger after incubation, under conditions of 4 DEG C, 600 × g is centrifuged 5min, and supernatant reservation is abandoned after centrifugation
Precipitation；
3) PBS solution of its volume 25% is added to mix in precipitating, again with refrigerated centrifuger 600 under conditions of 4 DEG C after mixing
× g is centrifuged 5min, abandons supernatant and retains precipitation, be repeated 2 times after centrifugation.
5. the method for isolating and purifying spermatid and excretion body according to claim 4 from pig semen, it is characterised in that：Step
It is rapid 1) in, somatic cell cracking formula of liquid is：SDS 0.001g/mL, Triton-100 5 μ L/mL and DEPC.
6. the method for isolating and purifying spermatid and excretion body according to claim 1 from pig semen, it is characterised in that step
Suddenly (3) concrete operations are as follows：
1) supernatant 10000 × g centrifugations 45min under conditions of 4 DEG C, abandons precipitation and retains supernatant；
2) supernatant is continued through into 0.45 μm and 0.22 μm of filter is filtered；
3) by supernatant, in Ultracentrifuge, 130000 × g is centrifuged 90min；
4) supernatant is outwelled after being centrifuged, dissolve again suspension exosome with 100 μ L PBS solutions.
7. the method for isolating and purifying spermatid and excretion body according to claim 1 from pig semen, it is characterised in that step
Suddenly (4) concretely comprise the following steps：
1) extracting sperm total serum IgE and seminal fluid exosome total serum IgEs carries out reverse transcription；
2) the addition RNase Free dH in the inverse transcription reaction liquid for obtaining2O dilutes 4 times；
3) carried out with protamine and reference gene GAPDH quantitatively, detection protamine is in spermatid and seminal fluid exosome
In expression, reaction system be II 5.0 μ L of SYBR premix Taq, 0.5 μ L of forward primer, downstream primer 0.5 μ L, cDNA
1.0 μ L, DEPC supply 10 μ L；
4) according to fluorescent quantitation result, respectively to protamine in spermatid and seminal fluid exosome and reference gene GAPDH
Relative expression quantity is calculated, and when mean difference multiple is more than 40 times, spermatid is successfully separated；
5) extracting spermatid total serum IgE and seminal fluid exosome total serum IgEs carries out miRNA reverse transcriptions；
6) RNase Free dH are added in inverse transcription reaction liquid2O dilutes 5 times；
7) it is chosen at the high expression miRNA identified in seminal fluid exosome：let-7b、miR-148a、let-7a、
MiR-375, miR-99a, miR-27b, miR-34c, and reference gene U6 carries out quantitatively, reaction system is：2×SYBR mix
5.0 μ L, 0.5 3 ' Primer of μ L, mRQ of miRNA-specific primer, 0.5 μ L, cDNA1.0 μ L, DEPC supply 10 μ L bodies
System；
8) according to fluorescent quantitation result, the relative table to miRNA and reference gene U6 in spermatid and seminal fluid exosome respectively
Calculated when mean difference multiple is more than 40 times up to amount, seminal fluid exosome is successfully separated.
8. the method for isolating and purifying spermatid and excretion body according to claim 7 from pig semen, it is characterised in that step
It is rapid 1) in, the sperm total serum IgE and seminal fluid exosome total serum IgE reverse transcriptions include two steps, and step one is：5×gDNA
2.0 μ L of Eraser buffer, 1.0 μ L of gDNA Eraser, TotalRNA 1.0 μ L and RNase Free dH26.0 μ L of O,
Reaction condition is 42 DEG C of 2min；Step 2 is：Prime Script RT Enzyme MixI 1.0μL、RT Primer Mix
1.0μL、5×Prime Script Buffer2 4.0μL、RNase Free dH24.0 μ L of O, 10.0 μ L of step one reactant liquor,
Reaction condition is 37 DEG C of 15min, 85 DEG C of 5s.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610975313.8A CN106544317B (en) | 2016-11-07 | 2016-11-07 | Method for separating and purifying spermatids and exosomes from pig semen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610975313.8A CN106544317B (en) | 2016-11-07 | 2016-11-07 | Method for separating and purifying spermatids and exosomes from pig semen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106544317A true CN106544317A (en) | 2017-03-29 |
| CN106544317B CN106544317B (en) | 2021-02-23 |
Family
ID=58394241
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610975313.8A Expired - Fee Related CN106544317B (en) | 2016-11-07 | 2016-11-07 | Method for separating and purifying spermatids and exosomes from pig semen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106544317B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107236822A (en) * | 2017-08-09 | 2017-10-10 | 四川农业大学 | It is a kind of to transfect miRNA into Boar spermatozoa and the method for assessing its transfection efficiency |
| CN107375234A (en) * | 2017-07-10 | 2017-11-24 | 武汉大学 | A kind of multifunctional carrier and preparation method and application based on cell source vesica in body fluid |
| CN108169199A (en) * | 2018-02-09 | 2018-06-15 | 大连理工大学 | A kind of method that excretion body fast quantification is carried out using ratio fluorescent |
| CN108414334A (en) * | 2018-02-23 | 2018-08-17 | 中山大学附属第医院 | Method for separating and purifying exosome from semen |
| CN108835031A (en) * | 2018-06-14 | 2018-11-20 | 华南农业大学 | A kind of method of the high reproductive performance herd boar of breeding |
| CN109468265A (en) * | 2018-11-06 | 2019-03-15 | 广州市创唯曦旺生物科技有限公司 | A method of extracting newborn excretion body |
| CN111876497A (en) * | 2020-08-31 | 2020-11-03 | 中国农业大学 | Method for identifying or assisting in identifying quality of animal semen |
| CN116676261A (en) * | 2023-04-19 | 2023-09-01 | 暨南大学附属第一医院（广州华侨医院） | Extraction method and application of human plasma exosome |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101543208A (en) * | 2009-03-31 | 2009-09-30 | 华南农业大学 | Powder for pig sperm dilution and room temperature preservation and preparation method and application thereof |
| CN105779586A (en) * | 2015-12-28 | 2016-07-20 | 四川农业大学 | Method for separating exosomes from animal plasma and for detecting purity |
| CN105934666A (en) * | 2013-08-21 | 2016-09-07 | 道格拉斯·T·卡雷尔 | Systems and methods for determining the effect of age-related changes in sperm epigenome on offspring phenotype |
-
2016
- 2016-11-07 CN CN201610975313.8A patent/CN106544317B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101543208A (en) * | 2009-03-31 | 2009-09-30 | 华南农业大学 | Powder for pig sperm dilution and room temperature preservation and preparation method and application thereof |
| CN105934666A (en) * | 2013-08-21 | 2016-09-07 | 道格拉斯·T·卡雷尔 | Systems and methods for determining the effect of age-related changes in sperm epigenome on offspring phenotype |
| CN105779586A (en) * | 2015-12-28 | 2016-07-20 | 四川农业大学 | Method for separating exosomes from animal plasma and for detecting purity |
Non-Patent Citations (3)
| Title |
|---|
| KADRI REKKER等: "Comparison of serum exosome isolation methods for microRNA profiling", 《CLINICAL BIOCHEMISTRY》 * |
| LUCIA VOJTECH等: "Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions", 《NUCLEIC ACIDS RESEARCH》 * |
| MADISON ET AL.: "Human semen contains exosomes with potent anti-HIV-1 activity", 《RETROVIROLOGY》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107375234A (en) * | 2017-07-10 | 2017-11-24 | 武汉大学 | A kind of multifunctional carrier and preparation method and application based on cell source vesica in body fluid |
| CN107375234B (en) * | 2017-07-10 | 2021-03-05 | 武汉大学 | Multifunctional carrier based on cell-derived vesicles in body fluid and preparation method and application thereof |
| CN107236822A (en) * | 2017-08-09 | 2017-10-10 | 四川农业大学 | It is a kind of to transfect miRNA into Boar spermatozoa and the method for assessing its transfection efficiency |
| CN108169199A (en) * | 2018-02-09 | 2018-06-15 | 大连理工大学 | A kind of method that excretion body fast quantification is carried out using ratio fluorescent |
| CN108169199B (en) * | 2018-02-09 | 2020-07-24 | 大连理工大学 | Method for quickly quantifying exosome by using fluorescence ratio |
| CN108414334A (en) * | 2018-02-23 | 2018-08-17 | 中山大学附属第医院 | Method for separating and purifying exosome from semen |
| CN108835031A (en) * | 2018-06-14 | 2018-11-20 | 华南农业大学 | A kind of method of the high reproductive performance herd boar of breeding |
| CN109468265A (en) * | 2018-11-06 | 2019-03-15 | 广州市创唯曦旺生物科技有限公司 | A method of extracting newborn excretion body |
| CN109468265B (en) * | 2018-11-06 | 2021-06-22 | 广州市创唯曦旺生物科技有限公司 | Method for extracting milk exosomes |
| CN111876497A (en) * | 2020-08-31 | 2020-11-03 | 中国农业大学 | Method for identifying or assisting in identifying quality of animal semen |
| CN111876497B (en) * | 2020-08-31 | 2021-08-31 | 中国农业大学 | Method for identifying or assisting in identifying quality of animal semen |
| CN116676261A (en) * | 2023-04-19 | 2023-09-01 | 暨南大学附属第一医院（广州华侨医院） | Extraction method and application of human plasma exosome |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106544317B (en) | 2021-02-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106544317A (en) | A kind of method that spermatid and excretion body are isolated and purified from pig semen | |
| CN104755628B (en) | The stabilisation of biological sample | |
| CN106574265A (en) | Method for isolating RNA with high yield | |
| EP2322613B2 (en) | Reagents and methods for isolation of purified RNA | |
| US20160122753A1 (en) | High-throughput rna-seq | |
| BR112012001077A2 (en) | nucleic acid extraction, methods to assess the quality of a nucleic acid extraction and biological sample, to obtain nucleic acid from a biological sample, to analyze microvesicle rna and to diagnose and monitor an individual, and, kit | |
| RU2017142155A (en) | METHOD FOR CARRYING OUT MULTI-ANALYSIS OF RARE CELLS EXTRACTED OR SEPARATED FROM BIOLOGICAL SAMPLES BY FILTRATION | |
| CN106244583A (en) | A kind of paramagnetic particle method method for extracting nucleic acid | |
| CN105112548B (en) | A kind of miRNA molecule mark miR-145 of quick detection sow embryo survival and its application | |
| CN108796086A (en) | A kind of circular rna circBCBM1 and its nondiagnostic fluorescence quantitative detecting method | |
| CN102286462A (en) | Method and kit for extracting free RNAs | |
| Milhoransa et al. | Micro RNA 146a-5p expression in Kidney transplant recipients with delayed graft function | |
| CN103289990A (en) | Rapid extraction method of blood deoxyribonucleic acid (DNA) | |
| Lizarraga et al. | miRNAs differentially expressed by next-generation sequencing in cord blood buffy coat samples of boys and girls | |
| CN104862301A (en) | Method for separating and enriching free fetus DNA from maternal blood plasma | |
| RU2556825C1 (en) | Method of extraction of exosomes from blood | |
| CN109402108A (en) | The extracting method of RNA in a kind of extracellular vesica | |
| CN114540297A (en) | Method for separating mesenchymal stem cell exosomes and analyzing miRNA | |
| CN102191238A (en) | Method for extracting small molecular RNA | |
| CN111876497B (en) | Method for identifying or assisting in identifying quality of animal semen | |
| CN112011624B (en) | Application of miR-155 as marker in identification or auxiliary identification of animal semen quality | |
| RU2678988C1 (en) | Method for isolating extracellular vesicles from biological fluids | |
| Monfared et al. | Determination and comparison miR 5a in the serum between women with GDM, non-pregnant type diabetes, healthy pregnant and control group | |
| KR102587121B1 (en) | Composition for isolating rna | |
| CN102127600B (en) | Method for detecting piwi-interacting ribonucleic acid (piRNA) in gastric juice |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210223 |