CN106434633A - Quick osseous tissue RNA extraction kit - Google Patents
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- CN106434633A CN106434633A CN201610938574.2A CN201610938574A CN106434633A CN 106434633 A CN106434633 A CN 106434633A CN 201610938574 A CN201610938574 A CN 201610938574A CN 106434633 A CN106434633 A CN 106434633A
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Abstract
The invention discloses a quick osseous tissue RNA extraction kit. The kit comprises at least a genomic DNA removal column, at least one RNA absorptive column, lysis buffer used cooperatively with the genomic DNA removal column and elution buffer used cooperatively with the RNA absorptive column. The kit has the advantages that the exclusive phenol/chloroform-free lysis buffer is used and a plurality of ingredients are added to remove proteoglycan. A particular buffer system and a bimodal pore silica gel purification system are utilized to solve the problem of remaining DNA, thus osseous tissue RNA with high purity and good integrity can be obtained. The particular genomic DNA removal column can effectively remove remaining genomic DNA (gDNA), thus the extracted RNA can be directly used in reverse transcription PCR and real-time PCR without DNase digestion. Extraction of single samples can be completed in 35 minutes generally, and RNA degradation due to long-time extraction, RNase contamination and other causes is avoided.
Description
Technical field
The present invention relates to RNA extracts, it is related to field of nucleic acid purification, is exactly specifically osseous tissue RNA rapid extraction reagent
Box.
Background technology
The research of molecular biology is concentrated mainly on nucleic acid and protein, and the molecular biology research of the overwhelming majority is required for
Obtain the nucleic acid of purification first, therefore nucleic acid purification is one of foundation stone of molecular biology, and world market is very huge, and the world is each
State all updates method for extraction and purification, with reach faster, purer obtain nucleic acid.Nucleic acid purification is generally divided into RNA
Extraction purification and DNA extraction purification two big class.
The RNA of main flow extracts mainly two kinds of solution in the world, and a kind of is Chomczynski invention in 1987
Guanidinium isothiocyanate/acid phenol/chloroform one-step method extracted total RNA (being commonly called as TRIzol method), this is first of RNA extraction purification technology
Milestone.Its Purification: Principles is：Under conditions of guanidinium isothiocyanate/acid phenol, DNA is dissolved in organic faciess, and RNA is dissolved in aqueous phase,
And protein is then in mesophase；Thus effectively by RNA and DNA/ protein separately.The method advantage is economical, flexibly.Shortcoming
It is to use phenol, chloroform toxicant.Another kind is silicagel column（Spin Columns）Purification technique, this is RNA extraction purification
Second milestone of technology.Nucleic acid extraction purification is become a kind of simple filter operation by silicagel column, to replace conventional solution
The centrifugation of type extraction technique.Its Purification: Principles is：The Purification: Principles of silicagel column are exactly using a kind of special glass fibre
Filter membrane, this filter membrane is in high chaotropic agent（Guanidine thiocyanate, guanidine hydrochloride, NaI etc.）Under conditions of, with RNA, adsorption reaction can occur,
Protein and other impurity will not be adsorbed or rinse removal simultaneously, and then under low-salt conditions, RNA again can be from filter membrane
Discharge, thus reaching the purpose of purification of nucleic acid.The advantage of the method is the chloroform toxicant without phenol, simple to operate,
Speed is fast.Shortcoming is that silicagel column can adsorb RNA/DNA simultaneously, is therefore easily caused DNA residual, causes purity low.
Osseous tissue is hard, osseous tissue is hard, osteocyte density is low and periphery substrate contain mucoprotein in a large number（Albumen is many
Sugar）It is difficult to separate with RNA it is impossible to carry out high-quality extraction with traditional Trizol method.During extracting osseous tissue RNA, often
Because extraction time is long, the reason such as RNA enzyme pollution causes RNA to degrade so that follow-up test cannot be carried out.Therefore, adopt
A kind of method of rapid extraction RNA is most important for the related experiment carrying out RNA.
Content of the invention
It is an object of the invention to provide a kind of test kit quickly effectively extracting osseous tissue RNA, this test kit is using solely
Special buffer system and double silica column purification system solve DNA residue problem, can obtain purity height, the good osseous tissue of integrity
RNA.
The present invention solves its technical problem and is adopted the technical scheme that：
Osseous tissue RNA rapid extraction test kit, including at least one genomic DNA remove post, at least one RNA adsorption column and
The lysate using cooperatively with genomic DNA removing post and the eluent using cooperatively with RNA adsorption column.
As optimize, described lysate includes lysate RLT Plus and lysate CLB, and described eluent is no ribose
Nuclease water.
As optimize, in described lysate RLT Plus containing the guanidinium isothiocyanate for 2-5mol/L for the concentration, PH it is
The Tris-HCl buffer solution of 6.0-8.5, the sodium lauryl sulphate of 1.5-3.0g/L；Concentration is contained in described lysate CLB
Guanidinium isothiocyanate for 2-5mol/L, the cetyl trimethylammonium bromide of 2-2.5g/L, PH are that the Tris-HCl of 6.0-8.5 delays
Rush solution, the Polyvinylpyrrolidone of 2-10g/L, the sodium chloride for 10-100mmol/L for the concentration.
As optimize, described osseous tissue RNA rapid extraction test kit also includes rinsing liquid RW, extraction aid PLANT
Aid, protein liquid removal RW1.
As optimize, in described rinsing liquid RW containing PH be 6.0-8.5, concentration be 5-50mol/L trihydroxy methyl ammonia
Methylmethane-hydrochloric acid buffer solution, concentration are 10-100mmol/L sodium chloride.
As optimize, the dithiothreitol, DTT containing 0.5-1.0mmol/L, 0.5- in described extraction aid PLANT aid
2.0% tween 20, the Polyvinylpyrrolidone of 3-15%.
As optimize, the guanidinium isothiocyanate containing 2-5mol/L in described protein liquid removal RW1, volume fraction 60-80%
Ethanol.
As optimize, it is silicon fiml adsorption column that described genomic DNA removes post and RNA adsorption column.
As optimize, described osseous tissue RNA rapid extraction test kit extracts RNA, at least includes following two steps：
Step A, removes post with genomic DNA and removes genomic DNA；RNA is adsorbed by step B with RNA adsorption column.
As optimize, described osseous tissue RNA rapid extraction test kit extracts RNA, comprises the steps：
（1）Using lysate CLB, cracking is carried out to sample and obtain lysate；
（2）Lysate is proceeded to centrifuge tube, centrifugation, collect supernatant, add 0.5 times of dehydrated alcohol of its volume in supernatant,
Mix；
（3）Step A, removes post with genomic DNA and removes genomic DNA：Supernatant is added to genomic DNA remove on post
（Remove post to be placed in collecting pipe）, it is centrifuged and discards waste liquid, genomic DNA removing post is placed in centrifuge tube, adds lysate RLT
Plus, is collected by centrifugation filtrate；
（4）To step（3）The dehydrated alcohol adding 0.5 times of its volume in the filtrate obtaining mixes；
（5）RNA is adsorbed by step B with RNA adsorption column：By step（4）The mixture obtaining adds in a RNA adsorption column, from
The heart, discards filtrate；
（6）Add protein liquid removal RW1, centrifugation in RNA adsorption column, discard waste liquid；
（7）Add rinsing liquid RW, centrifugation in RNA adsorption column, discard waste liquid, come again；
The invention has the beneficial effects as follows：This test kit adopts the exclusive lysate without phenol/chloroform formula, and adds multiple
Composition removes osseous tissue proteoglycan.Unique gene group DNA is removed column technology and can effectively be removed genomic DNA simultaneously（gDNA）
Residual, the RNA obtaining does not need dnase digestion, can be used for the experiment such as reverse transcriptional PCR, quantitative fluorescent PCR, and single sample operates
Typically can complete in 35 minutes.
Brief description
Fig. 1, osseous tissue RNA electrophoretogram.
In figure five swimming lanes from left to right are respectively swimming lane 1, swimming lane 2, swimming lane 3, swimming lane 4, swimming lane 5；Wherein swimming lane 1 is
Molecular weight marker, swimming lane 2 is rat femur head, and swimming lane 3 is rabbit intervertebral disc, and swimming lane 4 is rabbit knee cartilage, and swimming lane 5 is mice
Tibia.
Specific embodiment
Equipment before experiment：
Take 1ml lysate CLB (if CLB has precipitation or precipitation need to first be placed in 65 °C of water-baths and again dissolve to centrifuge tube）,
Add 100 μ l PLANTaid in lysate CLB, overturn and mix preheating in rear 65 °C of water-baths.Multiple samples proportionally amplify
Prepare.
Experimental procedure：
1. liquid nitrogen grinding method:
A. bone forceps puts into mortar after being caught broken osseous tissue, repeatedly grinds to form fine powder, constantly mend after noting liquid nitrogen vaporization after adding liquid nitrogen
Plus preservation liquid nitrogen exists always.
B. transfer 100mg fine powder adds to the lysate CLB of preheating（Added with PLANTaid）In centrifuge tube.Violent immediately
Vortex 30-60 second or mix cracking with suction nozzle piping and druming and directly obtain satisfied homogenate result (or electronic homogenate 30 seconds), can cut
Cut DNA, reduce viscosity and improve yield.
C. the step 3 connecing operating procedure at once.
2. other osseous tissue breaking methods：
A. 100mg osseous tissue is taken to add the lysate CLB of 1ml preheating（Added with PLANTaid）Homogenate pulverized by high speed homogenization instrument.
Or take the bone that 100mg liquid nitrogen freezing embedded section is pulverized to add lysate CLB（Added with PLANTaid）Pulverize homogenate.
B. the step 3 connecing operating procedure at once.
3. put back in short-term in 65 °C of water-baths（10 min）, centre overturns once in a while 1-2 time and helps cracking.
4. lysate 13,000 rpm is centrifuged 10 minutes, precipitates the fragment that can not crack.
5. take lysate supernatant（More supernatants can be taken in the case of removing post ability less than genomic DNA,
Yield so can be improved）Go to a new centrifuge tube.Add the dehydrated alcohol (0.5 volume) of supernatant volume half, now may be used
Precipitation can occur, but not affect extraction process, piping and druming immediately mixes, and must not be centrifuged.If there is floating thing on supernatant surface, use suction nozzle
Teasing draws following liquid.
6. by mixture（It is less than 720 μ l every time, how can add at twice）A genome is added to remove in post,（Clearly
Except post is put in collecting pipe）13,000 rpm are centrifuged 2 minutes, discard waste liquid.After guaranteeing centrifugation, liquid all go by filtration, and film does not have
There is residual, if it is necessary, centrifugal force and centrifugation time can be increased.
7. genomic DNA removing pillar is placed in a clean 2ml centrifuge tube（Without RNAse free or DEPC
Process, typically clean new centrifuge tube.Also the new clean collecting pipe of column kit can be adsorbed using RNA）, clear in genome
Except in post plus 500 μ l lysate RLT Plus, 13,000 rpm are centrifuged 30 seconds, collect filtrate（RNA is in filtrate）, with micro
Pipettor more accurately estimates filtered solution volume（It is usually 450-500 μ l, when filtration, loss volume should deduct）, add
The dehydrated alcohol of 0.5 times of volume, is now likely to occur precipitation, but does not affect extraction process, and piping and druming immediately mixes, Bu Yaoli
The heart.
8. at once mixture (being less than 720 μ l every time, how can add at twice) is added in an adsorption column RNA,
（Adsorption column is put in collecting pipe）13,000 rpm are centrifuged 2 minutes, discard waste liquid.After guaranteeing centrifugation, liquid all go by filtration, film
On do not remain, if it is necessary, centrifugal force and centrifugation time can be increased.
9. add 700 μ l protein liquid removal RW1, room temperature is placed 1 minute, 13,000rpm centrifugations 30 seconds discard waste liquid.
10. add in 500 μ l rinsing liquid RW containing PH be 7.0, concentration be the Tris-HCl buffer solution, dense of 25mol/L
Spend for 10-100mmol/L sodium chloride, 13,000 rpm are centrifuged 30 seconds, discard waste liquid.Add 500 μ l rinsing liquid RW, repeat one
Time.
Adsorption column RNA is put back in sky collecting pipe by 11., and 13,000 rpm are centrifuged 2 minutes, remove rinsing liquid as far as possible, with
Exempt from residual ethanol suppression downstream reaction in rinsing liquid.
12. taking-up adsorption column RNA, put in a deoxyribonuclease centrifuge tube, according to expected RNA yield in absorption
The middle part of film adds 30-50 μ l RNase free water（In 70-90 DEG C of water-bath, heating can improve yield in advance）,
Room temperature is placed 1 minute, and 12,000 rpm are centrifuged 1 minute.
13. if it is expected that RNA yield>30 μ g, plus 30-50 μ l deoxyribonuclease water repeat step 12, merge and wash twice
Liquid, or it is added back to adsorption column repeat step one time (high if necessary to RNA concentration) using primary eluent.Twice of eluting
RNA eluate concentration high, at twice eluting merge eluent RNA yield high by 15 30% than the former, but concentration is low, use
Family selects as needed.
This test kit can be additionally used in the RNA cleaning after deoxyribonuclease I is processed.
Specific embodiment：
Rat femur head, rabbit intervertebral disc, rabbit knee cartilage, four kinds of osseous tissues of mouse tibia are proceeded as follows：
1. bone forceps puts into mortar after being caught broken osseous tissue, repeatedly grinds to form fine powder after adding liquid nitrogen, keeps liquid nitrogen to exist always.Turn
Move 100mg fine powder to add in the lysate CLB centrifuge tube of preheating.Described lysate contains the isothiocyanic acid that concentration is 3mol/L
Guanidine, the cetyl trimethylammonium bromide of 2g/L, PH be 7.0 Tris-HCl buffer solution, the Polyvinylpyrrolidone of 2g/L,
Concentration is the sodium chloride of 50mmol/L.Acutely it is vortexed 45 seconds immediately.Put in 65 °C of water-baths（10 min）, middle reverse 2 sides
Help cracking.
2. lysate is centrifuged 10 minutes, precipitates the fragment that can not crack, take supernatant, add 1/2 volume of supernatant
Dehydrated alcohol mix to proceed to genome and remove and in post, carry out absorption centrifugation, discard waste liquid.It is silicon fiml that genome removes post
Adsorption column.
3. genomic DNA removing pillar is placed in a clean centrifuge tube, removes in post plus lysate in genome
RLT Plus, delays containing the Tris-HCl that the guanidinium isothiocyanate for 3mol/L for the concentration, PH are 7.0 in described lysate RLT Plus
Rush the sodium lauryl sulphate of solution, 2g/L.Filtrate is collected by centrifugation, adds the dehydrated alcohol of 1/2 volume of filtrate to mix.
3. add mixture in the adsorption column RNA being positioned in collecting pipe, centrifugation discards waste liquid.Plus protein liquid removal
RW1, the guanidinium isothiocyanate containing 3mol/L, the ethanol of volume fraction 70% in described RW1.Room temperature is placed 1 minute, and centrifugation discards
Waste liquid.Add rinsing liquid RW, in described rinsing liquid RW containing PH be 7.0, concentration be 5-50mol/L Tris-HCl buffer molten
Liquid, concentration are 10-100mmol/L sodium chloride.Centrifugation discards waste liquid, comes again.
4. adsorption column RNA is put back in sky collecting pipe, be centrifuged off rinsing liquid.
5. take out adsorption column RNA, put in a deoxyribonuclease centrifuge tube, add in the middle part of adsorbed film
RNase free water, room temperature is placed 1 minute, centrifugation.
6. repeat step 5, merge washing liquid twice, respectively obtain the rat femur head of needs, rabbit intervertebral disc, rabbit knee
Cartilage, mouse tibia osseous tissue RNA.
The rat femur obtaining head, rabbit intervertebral disc, rabbit knee cartilage, four kinds of osseous tissue RNA of mouse tibia are carried out electricity
Swimming, obtains electrophoretogram as shown in Figure 1.
Swimming lane 1 be molecular weight marker, swimming lane 2,3,4,5 be with osseous tissue RNA rapid extraction test kit extract respectively big
Mus femoral head, rabbit intervertebral disc, rabbit knee cartilage, four kinds of osseous tissue electrophoretograms of mouse tibia, adopt this as can be seen from the picture
The osseous tissue RNA noresidue DNA that method is extracted, extraction effect is good.
Operation principle：Unique rapid cell lysis of lysate and killed cells RNase, centrifugation goes removing protein many
Sugar and secondary metabolite, then cleavage mixture ethanol regulation RNA combination is adsorbed onto genomic DNA and removes post, then RNA
Filtered by selectivity eluting, absorption genomic DNA remove post on residual DNA cannot eluting abandon together with pillar thus
Dispose DNA.Filtration RNA with ethanol adjust conjugation condition after, RNA height under sequence salt state selective absorption in centrifugal column
Interior silicon substrate plasma membrane, then a series of step by quick rinsing-centrifugations, protein liquid removal and rinsing liquid are by cell metabolite, egg
White wait Impurity removal, the deoxyribonuclease water of the last less salt eluting from silicon substrate plasma membrane by pure RNA.
Above-mentioned specific embodiment is only the concrete case of the present invention, and the scope of patent protection of the present invention includes but is not limited to
The product form of above-mentioned specific embodiment and style, any osseous tissue RNA rapid extraction meeting claims of the present invention
Suitable change or modification that test kit and any person of an ordinary skill in the technical field are done to it, all should fall into the present invention
Scope of patent protection.
Claims (10)
1. osseous tissue RNA rapid extraction test kit it is characterised in that：Including at least one genomic DNA remove post, at least one
RNA adsorption column and the lysate using cooperatively with genomic DNA removing post and the eluent using cooperatively with RNA adsorption column.
2. osseous tissue RNA rapid extraction test kit according to claim 1 it is characterised in that：Described lysate includes splitting
Solution liquid RLT Plus and lysate CLB, described eluent is deoxyribonuclease water.
3. osseous tissue RNA rapid extraction test kit according to claim 2 it is characterised in that：Described lysate RLT
It is the Tris-HCl buffer solution of 6.0-8.5,1.5-3.0g/L containing the guanidinium isothiocyanate for 2-5mol/L for the concentration, PH in Plus
Sodium lauryl sulphate；In described lysate CLB containing the guanidinium isothiocyanate for 2-5mol/L for the concentration, 2-2.5g/L ten
Six alkyl trimethyl ammonium bromides, PH are the Tris-HCl buffer solution of 6.0-8.5, the Polyvinylpyrrolidone of 2-10g/L, concentration
Sodium chloride for 10-100mmol/L.
4. osseous tissue RNA rapid extraction test kit according to claim 1 it is characterised in that：Also include rinsing liquid RW, help
Carry agent PLANT aid, protein liquid removal RW1.
5. osseous tissue RNA rapid extraction test kit according to claim 4 it is characterised in that：Contain in described rinsing liquid RW
Have PH to be 6.0-8.5, concentration be the Tris-HCl buffer solution of 5-50mol/L, concentration be 10-100mmol/L sodium chloride.
6. osseous tissue RNA rapid extraction test kit according to claim 4 it is characterised in that：Described extraction aid PLANT
Dithiothreitol, DTT containing 0.5-1.0mmol/L, the tween 20 of 0.5-2.0%, the Polyvinylpyrrolidone of 3-15% in aid.
7. osseous tissue RNA rapid extraction test kit according to claim 4 it is characterised in that：Contain in protein liquid removal RW1
The guanidinium isothiocyanate of 2-5mol/L, the ethanol of volume fraction 60-80%.
8. osseous tissue RNA rapid extraction test kit according to claim 1 it is characterised in that：Described genomic DNA is removed
Post and RNA adsorption column are silicon fiml adsorption column.
9. osseous tissue RNA rapid extraction test kit according to claim 1 it is characterised in that：At least include following two
Individual step：Step A, removes post with genomic DNA and removes genomic DNA；RNA is adsorbed by step B with RNA adsorption column.
10. osseous tissue RNA rapid extraction test kit according to claim 9 it is characterised in that：Comprise the steps：
（1）Using lysate CLB, cracking is carried out to sample and obtain lysate；
（2）Lysate is proceeded to centrifuge tube, centrifugation, collect supernatant, add 0.5 times of dehydrated alcohol of its volume in supernatant,
Mix；
（3）Step A, removes post with genomic DNA and removes genomic DNA：Supernatant is added to and is positioned in collecting pipe
Genomic DNA is removed on post, and centrifugation discards waste liquid, genomic DNA removing post is placed in centrifuge tube, adds lysate RLT
Plus, is collected by centrifugation filtrate；
（4）To step（3）The dehydrated alcohol adding 0.5 times of its volume in the filtrate obtaining mixes；
（5）RNA is adsorbed by step B with RNA adsorption column：By step（4）The mixture obtaining adds in a RNA adsorption column, from
The heart, discards filtrate；
（6）Add protein liquid removal RW1, centrifugation in RNA adsorption column, discard waste liquid；
（7）Add rinsing liquid RW, centrifugation in RNA adsorption column, discard waste liquid, come again；
（8）Obtain RNA eluent with anhydrous ribonucleic acid water elution.
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| CN110373311A (en) * | 2019-08-21 | 2019-10-25 | 佛山科学技术学院 | A kind of bone tissue RNA milling and extracting device |
| CN110479450A (en) * | 2019-08-21 | 2019-11-22 | 佛山科学技术学院 | A kind of bone tissue RNA milling and extracting method and grinding device |
| CN110804609A (en) * | 2019-09-29 | 2020-02-18 | 杭州联科生物技术股份有限公司 | Whole blood RNA rapid lysis solution and application |
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| CN110479450A (en) * | 2019-08-21 | 2019-11-22 | 佛山科学技术学院 | A kind of bone tissue RNA milling and extracting method and grinding device |
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Application publication date: 20170222 |
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