CN105385680A - Reagent for simultaneous extraction of DNA and RNA, extraction method and application - Google Patents
Reagent for simultaneous extraction of DNA and RNA, extraction method and application Download PDFInfo
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Abstract
The invention relates to the field of gene engineering, in particular to a reagent for simultaneous extraction of DNA and RNA, a method for simultaneously extracting the DNA and the RNA and application of the reagent. The reagent comprises a natural saccharide compound, a heavy metal salt compound, a neutral salt compound, a thiocyanic acid compound, an alcoholic compound, an organic solvent compound, a phenolic compound, N-lauroylsarcosine and a salt compound used for adjusting the acidity or alkalinity of a solution. The reagent for extracting the DNA and the RNA is optimized, protection to the DNA and the RNA is enhanced, the whole extraction process can be conducted at room temperature, cooling treatment does not need to be conducted, and the needs of follow-up second-generation sequencing and third-generation sequencing can be completely met. The operation method is simple and rapid, the quantity demand of samples is less, and the purity and the concentration of extracted DNA and RNA are high.
Description
Technical field
The present invention relates to genetically engineered field, be specifically related to a kind of reagent simultaneously extracted for DNA and RNA, for the method that DNA and RNA extracts simultaneously, and the purposes of this reagent.
Background technology
RNA is a kind of macromolecular substance in viable cell and much virus, be condensed into the long chain molecule of strand by ribonucleotide through phosphide key, a ribonucleic acid molecule is made up of phosphoric acid, ribose and base (A VITAMIN B4, G guanine, C cytosine(Cyt), U uridylic).RNA is used in the building-up process of viable cell all proteins, and carries the genetic information of a lot of virus.
At present, order-checking is divided into genome sequencing, and exon checks order, and the diverse ways such as specific region order-checking, need to adopt different treatment processs to sample respectively, can not realize adopting same reagent to extract DNA and RNA, and can meet the needs of follow-up order-checking.
Summary of the invention
The present invention needs the problem of prior art solved to be: existing reagent can only be used for DNA extraction or RNA extracts, and its complex operation step, do not have in existing reagent simultaneously for the reagent that DNA and RNA extracts, the difficulty extracting high quality RNA in a small amount of sample can be faced with simultaneously.
In order to solve the problem, the invention provides a kind of reagent, extracting method and purposes for extracting DNA and RNA in tissue sample simultaneously.
Specifically, the invention provides a kind of reagent simultaneously extracted for DNA and RNA in sample,
Described reagent comprises natural sugar compounds, heavy metallic salt compounds, neutral salt compounds, thiocyanic acid compounds, alcohol compound, organic solvent compounds, phenolic compound, N-lauroyl sarkosine and the salt compounds for regulator solution potential of hydrogen.
Preferably, in described reagent, the concentration of each compound is respectively:
(1) natural sugar compounds is 0.1-0.4mol/L, is preferably 0.2-0.3mol/L;
(2) heavy metallic salt compounds is 0.05-0.5mol/L, is preferably 0.1-0.2mol/L;
(3) neutral salt compounds 1.0-2.0mol/L, is preferably 1.0-1.4mol/L;
(4) thiocyanic acid compounds 0.9-3.0mol/L, is preferably 0.9-1.5mol/L;
(5) alcohol compound 4-15% (v/v), preferably 4-6% (v/v);
(6) organic solvent compounds 1.55-7.5mol/L, preferably 1.55-3.1mol/L;
(7) phenolic compound 35%-65% (v/v), is preferably 35%-45% (v/v);
(8) N-lauroyl sarkosine 0.01-0.07mol/L, is preferably 0.03-0.06mol/L;
(9) for the salt compounds 0.8-2.5mol/L of regulator solution potential of hydrogen, 0.8-1.2mol/L is preferably.
Preferably, described natural sugar compounds is selected from more than one or more in trehalose, isotrehalose, neotrehalose;
Described heavy metallic salt compounds is selected from more than one or more in Silver Nitrate, copper sulfate, iron protochloride, zinc chloride, is preferably copper sulfate or Silver Nitrate;
Described neutral salt compounds is selected from more than one or more in sodium-chlor, Repone K, magnesium chloride, calcium chloride, is preferably sodium-chlor or Repone K;
Described organic solvent compounds is selected from more than one or more in chloroform, acetone, methyl alcohol, ethanol, is preferably the composition of chloroform and ethanol;
Described alcohol compound to be carbon atom number the be alcohols of 3-4, is selected from more than one or more in glycerine, n-propyl alcohol, Virahol, propyl carbinol, ethylene glycol, is preferably glycerine or n-propyl alcohol;
Described thiocyanic acid compounds is selected from more than one or more in guanidine thiocyanate, ammonium thiocyanate, is preferably the composition of guanidine thiocyanate and ammonium thiocyanate;
Described phenolic compound is selected from more than one or more in phenol, p-cresol, Ortho Cresol, m-cresol, Phloroglucinol, is preferably phenol;
The salt compounds of described regulator solution potential of hydrogen is selected from more than one or more in sodium-acetate, Trisodium Citrate, is preferably the composition of Trisodium Citrate and sodium-acetate.
Preferably, described reagent comprises trehalose, copper sulfate, sodium-chlor, guanidine thiocyanate, ammonium thiocyanate, ethanol, glycerine, chloroform, phenol, N-lauroyl sarkosine, Trisodium Citrate and sodium-acetate.
Preferably, in described reagent, the concentration of each component is respectively:
Trehalose 0.1-0.4mol/L, preferably 0.2-0.3mol/L; Copper sulfate 0.05-0.5mol/L, preferably 0.1-0.2mol/L; Sodium-chlor 1.0-2.0mol/L, preferably 1.0-1.4mol/L; Guanidine thiocyanate 0.6-2.0mol/L, preferably 0.6-1.0mol/L; Ammonium thiocyanate 0.3-1.0mol/L, preferably 0.3-0.5mol/L; Glycerine 4%-15% (v/v), preferably 4-6% (v/v); Chloroform 1.5-5.0mol/L, preferably 1.5-2.0mol/L; Ethanol 0.05-2.5mol/L, preferably 0.05-1.1mol/L; Phenol 35%-65% (v/v), preferably 35-45% (v/v); N-lauroyl sarkosine 0.01-0.07mol/L, is preferably 0.03-0.06mol/L; Trisodium Citrate 0.7-1.5mol/L, preferably 0.7-1.0mol/L; Sodium-acetate 0.1-1.0mol/L, preferably 0.1-0.2mol/L.
Invention also provides the method that the reagent described in above any one extracts for DNA and RNA, comprise the steps:
(1) reagent described in above any one is added in sample, centrifugal after concussion, be divided into supernatant liquid and lower floor's liquid;
(2) wherein, in supernatant liquid for the extraction of RNA;
(3) lower floor's liquid is used for the extraction of DNA.
Preferably, in step (1), the concussion time is 5-10min, and centrifugal speed is 10000g-15000g, and centrifugation time is 10-20 minute.
Preferably, step (2) adds room temperature concussion after Virahol at the middle and upper levels in liquid, centrifugal, discards liquid; Then add the ethanol of 75%, room temperature is centrifugal, removes liquid, obtains RNA.
Preferably, shake 10-20 minute after adding Virahol, concussion speed is 50rpm-200rpm; Centrifugal speed 10000g-15000g, centrifugation time is 10-20 minute.
Preferably, after step (2) adds 75% ethanol, centrifugal speed is 10000g-15000g, and centrifugation time is 10-20 minute.
Preferably, after adding dehydrated alcohol in step (3) in lower floor's liquid, room temperature concussion is centrifugal, removes liquid; Then add Trisodium Citrate ethanolic soln, the centrifugal rear removal liquid of room temperature concussion, obtains DNA.
Preferably, after adding dehydrated alcohol in step (3), the concussion time is 5-10min, and concussion speed is 50rpm-200rpm;
After adding Trisodium Citrate ethanolic soln, the concussion time is 5-10 minute, and concussion speed is 50rpm-200rpm.
Preferably, the concentration of the Trisodium Citrate in described Trisodium Citrate ethanolic soln is 0.05M-0.2M.
The method described in the reagent described in above any one or more any one of present invention also offers is for the application in the content of DNA and RNA that extracts and/or in working sample.
Preferably, DNA and RNA of this application fetches can be used as genome sequencing, exon order-checking, specific region order-checking.
Preferably, the sample in this application comprises fresh pathological tissues, tumor tissues case wax stone, tumor tissues case paraffin section, whole blood, blood plasma, serum, hydrothorax or fresh biopsy sample.
Reagent normal temperature provided by the invention is preserved, and the quality guaranteed period is 1 year.
Agents useful for same of the present invention may be used for measuring and variously needs that genome sequencing, exon check order, the tissue samples of specific region order-checking, if when simultaneously the amount of tissue samples is more than 1g, after needing at-20 degrees Celsius, tissue to be shredded, grind 2-10 minute namely passable.
The invention has the beneficial effects as follows:
(1) reagent of the present invention simultaneously for the extraction of DNA and RNA in sample, can strengthen the protection of DNA and RNA simultaneously, and a small amount of sample can meet extraction requirement, and can meet the needs of follow-up two generations order-checking and three generations's order-checking completely.
(2) leaching process of the whole reagent of the present invention, room temperature can complete, without the need to carrying out subzero treatment, working method simple and fast, sample requirements is few, DNA and the RNA purity of extraction and concentration high.
Below in conjunction with each embodiment, the present invention and Advantageous Effects thereof are described in detail, wherein:
Embodiment
As mentioned above, the object of the invention is to: provide a kind of simultaneously for the reagent that DNA and RNA extracts, and extract method and the purposes of DNA and RNA simultaneously.
Specifically; the invention provides a kind of reagent simultaneously extracted for DNA and RNA in tissue sample, described reagent comprises natural sugar compounds, heavy metallic salt compounds, neutral salt compounds, thiocyanic acid compounds, alcohol compound, organic solvent compounds, phenolic compound, N-lauroyl sarkosine and the salt compounds for regulator solution potential of hydrogen.
Wherein, in the preferred embodiment of the present invention, described reagent comprises trehalose, copper sulfate, sodium-chlor, guanidine thiocyanate, ammonium thiocyanate, glycerine, ethanol, chloroform, phenol, N-lauroyl sarkosine, Trisodium Citrate and sodium-acetate.
In the concrete preferred implementation of one of the present invention, described reagent comprises trehalose, copper sulfate, sodium-chlor, guanidine thiocyanate, ammonium thiocyanate, ethanol, glycerine, chloroform, phenol, N-lauroyl sarkosine, Trisodium Citrate and sodium-acetate, in described reagent, the concentration of each component is respectively: trehalose 0.1-0.4mol/L, copper sulfate 0.05-0.5mol/L, sodium-chlor 1.0-2.0mol/L, guanidine thiocyanate 0.6-2.0mol/L, ammonium thiocyanate 0.3-1.0mol/L, glycerine 4%-15% (v/v), ethanol 0.05-2.5mol/L, chloroform 1.5-5.0mol/L, phenol 35%-65% (v/v), N-lauroyl sarkosine 0.01-0.07mol/L, Trisodium Citrate 0.7-1.5mol/L, sodium-acetate 0.1-1.0mol/L.
In the concrete preferred implementation of another kind of the present invention, described reagent comprises trehalose, copper sulfate, sodium-chlor, guanidine thiocyanate, ammonium thiocyanate, ethanol, glycerine, chloroform, phenol, N-lauroyl sarkosine, Trisodium Citrate and sodium-acetate, in described reagent, the concentration of each component is respectively: trehalose 0.2-0.3mol/L, copper sulfate 0.1-0.2mol/L, sodium-chlor 1.0-1.4mol/L, guanidine thiocyanate 0.6-1.0mol/L, ammonium thiocyanate 0.3-0.5mol/L, glycerine 4-6% (v/v), ethanol 0.05-1.1mol/L, chloroform 1.5-2.0mol/L, phenol 35-45% (v/v), N-lauroyl sarkosine 0.03-0.06mol/L, , Trisodium Citrate 0.7-1.0mol/L, sodium-acetate 0.1-0.2mol/L.
The extraction of high-quality RNA is the committed step of carrying out many molecular biology experiments.And the extraction of high-quality RNA in a small amount of sample, check order even more important for the order-checking of modern genetic two generation and three generations.The invention provides a kind of instant reagent extracted for RNA in cell or tissue, the DNA in sample can be extracted simultaneously, meet the order-checking of two generations and three generations greatly and to check order industrialization demand.
Reagent of the present invention can protect the integrity of RNA molecule when being used as tissue homogenate, destroy cellularstructure, cell mixing assembly simultaneously.After centrifugal, because the density of different substances is different in reagent, solution can be divided into organic phase and aqueous phase.Phenolic compound, with the organic solvent mixing in solution, effectively makes the protein denaturation in sample.The wherein mixing of phenol and chloroform, especially can reduce the formation of the undissolvable RNA-albumen composition of interface effectively.And chloroform can reduce the amount of the phenol remained in aqueous phase, thus reduce the degradation amount of RNA, improve the output of RNA.
Under normal circumstances, solute is more easily dissolved in the solvent with its structural similitude, namely similarly mixes.A kind of dissolving power of solvent entirety depends on its polarity.Such as, the solute (as urea) that a kind of polarity is stronger is very easily dissolved in the solvent (as water) stronger than its polarity, be insoluble in the solvent (as alcohol) of working as with its polar phase, be dissolved in hardly in non-polar solvent (as chloroform, ether).Nucleic acid is polarity macromole (because it has electronegative phosphate backbones), so it is soluble in the aqueous phase of lower floor, and is insoluble to organic phase (water is stronger than the polarity of phenol).On the contrary, protein is made up of the charged of different ratios and uncharged structural domain, produces wetting ability and water repellent region respectively.Water repellent region and phenol interact, and produce protein precipitation, polymkeric substance (carbohydrate containing) is gathered in the layering interfaces (being generally white flock) of two solvents, or is dissolved in the organic phase of lower floor together with lipid.
The PH of solution decides being separated of organic phase and DNA with RNA in aqueous phase.In neutrality or weakly alkaline environment (PH7-8), the phosphodiester bond in nucleic acid is electronegative, and at this moment DNA and RNA is all present in aqueous phase.When PH drops to 4.8, the mobility of DNA in aqueous phase reaches maximum.In this sour environment, most protein and little DNA (<10kb) are gathered in organic phase, and large DNA molecular and a part of protein aggregation are in layering place of organic phase and aqueous phase interface.The acidic conditions of solution, can make RNA stay in aqueous phase, make DNA move to organic phase.Sour environment also can make RNase activity reduce.Thiocyanic acid compounds can be used for reducing the impact of nuclease on experiment.
Natural sugar compounds, it act as provides protection to DNA and RNA, the damage of DNA or the RNA chain preventing reagent from may bring.
Heavy metallic salt compounds, it act as and plays restraining effect to the activity of RNA enzyme and DNA enzymatic, can ensure that leaching process can carry out, without the need to cold operation in room temperature.
Below in conjunction with embodiment, the present invention is further detailed explanation.
Reagent used in the embodiment of the present invention and device information as follows:
The reagent used in the present invention is the reagent that those skilled in the art commonly use, and the reagent wherein in embodiment and comparative example is all purchased from SIGMA, and its purity is analytical pure.Wherein trehalose is with 1 by two glucose molecules, the nonreducing sugar that 1-glycosidic link is formed, there are 3 kinds of isomer and trehalose (α, α), isotrehalose (β, β) with neotrehalose (α, β), the trehalose used in the embodiment of the present invention and comparative example is (α, α) type.
The instrument that DNA and RNA concentration determination is used is Nanodrop2000 ultramicrospectrophotometer, purchased from ThermoScientific.
Wherein, the compound method of 0.1M Trisodium Citrate ethanolic soln is as follows:
Accurately take 29.41g Trisodium Citrate (template with rolling well), drop in 200ml beaker and dissolve with appropriate distilled water, lysate transfers to the volumetric flask of 1000ml, adds 100% ethanol 10ml, with distilled water constant volume to 1000ml.Transfer in clean reagent bottle, labeling is for subsequent use.The compound method of 0.05M and the 0.2M Trisodium Citrate ethanolic soln in embodiment two and embodiment three is prepared with reference to the compound method of 0.1M Trisodium Citrate ethanolic soln.
The formula of agents useful for same in each embodiment of table 1 and comparative example
Wherein, in table 1, "-" representative is do not add corresponding component, and namely the content of respective components is zero.
Embodiment one
(1) process of tissue sample
Get 10mg tissue sample to be measured (mouse liver tissue) and after homogenate, add as the reagent 1ml in table 1 embodiment one in liquid nitrogen, after room temperature 200rpm shakes 5 minutes, centrifugal 15 minutes of room temperature 12000g, is divided into two-layer.Wherein supernatant liquid is next for the extraction of RNA, and lower floor's liquid is next for the extraction of DNA.The density of purified petroleum benzin phenol is 1.07g/cm
3, when mixing with water, be positioned at the lower floor of aqueous phase.Chloroform makes the layering of two kinds of solvents more obvious, because chloroform and phenol dissolve each other, and has larger density 1.47g/cm
3.
(2) RNA extracts:
(1) carefully taken out by supernatant liquid, dropwise add Virahol, after room temperature 100rpm slowly shakes 10 minutes, centrifugal 15 minutes of room temperature 12000g, discards whole liquid; Virahol can mix with the water of arbitrary proportion, therefore adds Virahol and can capture moisture around RNA or DNA, and therefore RNA can assemble and precipitates, thus reaches the object of precipitated rna.
(2) add 75% ethanol 1ml of 4 DEG C of precoolings, centrifugal 15 minutes of room temperature 12000g, blots whole liquid with vacuum pump, opens centrifuge tube room temperature and places 5 minutes; Its objective is to remove remaining DNA, making solvent evaporates, dry RNA simultaneously.
(3) add 10ulDEPC water, dissolve RNA.
Adopt Nanodrop2000 ultramicrospectrophotometer to measure RNA concentration, its concentration is 51ug/ul.Through RNA agarose gel denaturation electrophoretic analysis obvious 28,18s band and lighter 5s band as seen.
(3) extraction of DNA
(1) careful sucking-off lower floor liquid, add 0.2ml dehydrated alcohol, room temperature 100rpm shakes 5 minutes, and centrifugal 5 minutes of 2000g, carefully blots whole liquid with vacuum pump; Object is to obtain DNA precipitation.
(2) add 0.1M Trisodium Citrate ethanolic soln 0.5ml, after room temperature 100rpm shakes 10 minutes, centrifugal 5 minutes of 2000g, carefully blots liquid with vacuum pump; Its objective is to remove impurity, DNA is cleaned.
(3) add 0.5ml8mmol/L sodium hydroxide solution, centrifugal 10 minutes of 12000rpm, is transferred to supernatant in new centrifuge tube, and adopt HEPS that pH value is adjusted to 7.0, object obtains stable DNA solution.
Adopt Nanodrop2000 ultramicrospectrophotometer to measure its concentration, its concentration is: 52ug/ul.
Embodiment two
(1) process of tissue sample
Get 10mg tissue sample to be measured (mouse liver tissue) and after homogenate, add as the reagent 1ml in table 1 embodiment two in liquid nitrogen, after room temperature 100rpm shakes 10 minutes, centrifugal 10 minutes of room temperature 15000g, is divided into two-layer.Wherein supernatant liquid is next for the extraction of RNA, and lower floor's liquid is next for the extraction of DNA.
(2) RNA extracts
(1) carefully taken out by supernatant liquid, dropwise add Virahol, after room temperature 200rpm slowly shakes 15 minutes, centrifugal 10 minutes of room temperature 15000g, discards whole liquid.
(2) add 75% ethanol 1ml of 4 DEG C of precoolings, centrifugal 10 minutes of room temperature 15000g, blots whole liquid with vacuum pump, opens centrifuge tube room temperature and places 5 minutes.
(3) add 10ulDEPC water, dissolve RNA.
Adopt ultramicrospectrophotometer to measure its concentration, its concentration is 44ug/ul.Through RNA agarose gel denaturation electrophoretic analysis obvious 28,18s band and lighter 5s band as seen.
(3) extraction of DNA
(1) careful sucking-off lower floor liquid, add 0.2ml dehydrated alcohol, room temperature 200rpm shakes 10 minutes, and centrifugal 5 minutes of 2000g, carefully blots whole liquid with vacuum pump.
(2) add 0.05M Trisodium Citrate ethanolic soln 0.5ml, after room temperature 200rpm shakes 5 minutes, centrifugal 5 minutes of 2000g, carefully blots liquid with vacuum pump.
(3) add 0.5ml8mmol/L sodium hydroxide solution, centrifugal 5 minutes of 15000rpm, is transferred to supernatant in new centrifuge tube, adopts HEPS that pH value is adjusted to 7.0, obtains stable DNA solution.
The concentration adopting ultramicrospectrophotometer to measure its DNA is 38ug/ul.
Embodiment three
(1) process of tissue sample
Get 10mg tissue sample to be measured (mouse liver tissue) and after homogenate, add as the reagent 1ml in table 1 embodiment three in liquid nitrogen, after room temperature 200rpm shakes 5 minutes, centrifugal 20 minutes of room temperature 10000g, is divided into two-layer.Wherein supernatant liquid is next for the extraction of RNA, and lower floor's liquid is next for the extraction of DNA.
(2) RNA extracts:
(1) carefully taken out by supernatant liquid, dropwise add Virahol, after the slow 50rpm of room temperature shakes 20 minutes, centrifugal 20 minutes of room temperature 10000g, discards whole liquid.
(2) add 75% ethanol 1ml of 4 DEG C of precoolings, centrifugal 20 minutes of room temperature 10000, blots whole liquid with vacuum pump, opens centrifuge tube room temperature and places 5 minutes.
(3) add 10ulDEPC water, dissolve RNA.
Adopt ultramicrospectrophotometer to measure its concentration, its concentration is 33ug/ul.Through RNA agarose gel denaturation electrophoretic analysis obvious 28,18s band and lighter 5s band as seen.
(3) extraction of DNA
(1) careful sucking-off lower floor liquid, add 0.2ml dehydrated alcohol, room temperature 50rpm shakes 10 minutes, and centrifugal 5 minutes of 2000g, carefully blots whole liquid with vacuum pump.
(2) add 0.2M Trisodium Citrate ethanolic soln 0.5ml, room temperature 50rpm shook placement after 10 minutes, and centrifugal 5 minutes of 2000g, carefully blots liquid with vacuum pump.
(3) add 0.5ml8mmol/L sodium hydroxide solution, centrifugal 20 minutes of 10000rpm, is transferred to supernatant in new centrifuge tube, adopts HEPS that pH value is adjusted to 7.0, obtains stable DNA solution.
The concentration adopting ultramicrospectrophotometer to measure its DNA is 35ug/ul.
Embodiment four
Getting 10mg tissue sample to be measured (mouse liver tissue) in liquid nitrogen, after homogenate, adds as the reagent 1ml in table 1 embodiment four, then carries out the extraction of DNA and RNA according to the step of embodiment one.Adopt ultramicrospectrophotometer to measure the concentration of RNA, its concentration is: 25ug/ul, through RNA agarose gel denaturation electrophoretic analysis obvious 28,18s band and lighter 5s band as seen.The concentration measuring DNA is 27ug/ul.
Embodiment five
Getting 10mg tissue sample to be measured (mouse liver tissue) in liquid nitrogen, after homogenate, adds as the reagent 1ml in table 1 embodiment five, then carries out the extraction of DNA and RNA according to the step of embodiment one.Adopt ultramicrospectrophotometer to measure the concentration of RNA, its concentration is: 28ug/ul, and through RNA agarose gel denaturation electrophoretic analysis obvious 28,18s band and lighter 5s band as seen, the concentration measuring DNA is 22ug/ul.
Embodiment six
Getting 10mg tissue sample to be measured (mouse liver tissue) in liquid nitrogen, after homogenate, adds as the reagent 1ml in table 1 embodiment six, then carries out the extraction of DNA and RNA according to the step of embodiment one.Adopt ultramicrospectrophotometer to measure the concentration of RNA, its concentration is: 24ug/ul, and through RNA agarose gel denaturation electrophoretic analysis obvious 28,18s band and lighter 5s band as seen, the concentration measuring DNA is 23ug/ul.
Comparative example one
Getting 10mg tissue sample to be measured (mouse liver tissue) in liquid nitrogen, after homogenate, adds as the reagent 1ml in table 1 comparative example one, then carries out the extraction of DNA and RNA according to the step of embodiment one.
Adopt ultramicrospectrophotometer to measure the concentration of RNA, its concentration is: 8ug/ul, through the electrophoretic analysis of RNA agarose gel denaturation, has no obvious RNA band, and show that RNA degraded is serious, the concentration measuring DNA is 5ug/ul.
Comparative example two
Getting 10mg tissue sample to be measured (mouse liver tissue) in liquid nitrogen, after homogenate, adds as the reagent 1ml in table 1 comparative example two, then carries out the extraction of DNA and RNA according to the step of embodiment one.Adopt ultramicrospectrophotometer to measure the concentration of its RNA, its concentration is: 4ug/ul, through the electrophoretic analysis of RNA agarose gel denaturation, has no obvious RNA band, and show that RNA degraded is serious, the concentration measuring DNA is 3ug/ul.
Comparative example three
Getting 10mg tissue sample to be measured (mouse liver tissue) in liquid nitrogen, after homogenate, adds as the reagent 1ml in table 1 comparative example three, then carries out the extraction of DNA and RNA according to the step of embodiment one.Adopt ultramicrospectrophotometer to measure the concentration of its RNA, its concentration is: 3ug/ul, through the electrophoretic analysis of RNA agarose gel denaturation, has no obvious RNA band, and show that RNA degraded is serious, the concentration measuring DNA is 2ug/ul.
Comparative example four
Getting 10mg tissue sample to be measured (mouse liver tissue) in liquid nitrogen, after homogenate, adds as the reagent 1ml in table 1 comparative example four, then carries out the extraction of DNA and RNA according to the step of embodiment one.Adopt ultramicrospectrophotometer to measure the concentration of its RNA, its concentration is 3ug/ul, through the electrophoretic analysis of RNA agarose gel denaturation, has no obvious RNA band, and show that RNA degraded is serious, the concentration measuring DNA is 2ug/ul.
Comparative example five
Getting 10mg tissue sample to be measured (mouse liver tissue) in liquid nitrogen, after homogenate, adds as the reagent 1ml in table 1 comparative example five, then carries out the extraction of DNA and RNA according to the step of embodiment one.Adopt ultramicrospectrophotometer to measure the concentration of its RNA, its concentration is: 1ug/ul, through the electrophoretic analysis of RNA agarose gel denaturation, has no obvious RNA band, and show that RNA degraded is serious, the concentration measuring DNA is 2ug/ul.
In a word; by the corresponding data in above comparative example and embodiment; can find out; the present invention is optimized RNA and DNA extraction reagent; strengthen the protection of DNA and RNA; room temperature in whole leaching process, without the need to carrying out cooling process, and can meet the needs of follow-up two generations order-checking and three generations's order-checking completely.Working method simple and fast, sample requirements is few, DNA and the RNA purity of extraction and concentration high.
The foregoing is only present pre-ferred embodiments, and be not used in limitation the present invention, all amendments done within the spirit and principles in the present invention, equivalent to replace and improvement etc., within the protection domain all needing to be included in invention.
Claims (16)
1., for the reagent that DNA and RNA in sample extracts simultaneously, it is characterized in that,
Described reagent comprises natural sugar compounds, heavy metallic salt compounds, neutral salt compounds, thiocyanic acid compounds, alcohol compound, organic solvent compounds, phenolic compound, N-lauroyl sarkosine and the salt compounds for regulator solution potential of hydrogen.
2. reagent according to claim 1, is characterized in that, in described reagent, the concentration of each compound is respectively:
(1) natural sugar compounds is 0.1-0.4mol/L, is preferably 0.2-0.3mol/L;
(2) heavy metallic salt compounds is 0.05-0.5mol/L, is preferably 0.1-0.2mol/L;
(3) neutral salt compounds 1.0-2.0mol/L, is preferably 1.0-1.4mol/L;
(4) thiocyanic acid compounds 0.9-3.0mol/L, is preferably 0.9-1.5mol/L;
(5) alcohol compound 4-15% (v/v), preferably 4-6% (v/v);
(6) organic solvent compounds 1.55-7.5mol/L, preferably 1.55-3.1mol/L;
(7) phenolic compound 35%-65% (v/v), is preferably 35%-45% (v/v);
(8) N-lauroyl sarkosine 0.01-0.07mol/L, is preferably 0.03-0.06mol/L;
(9) for the salt compounds 0.8-2.5mol/L of regulator solution potential of hydrogen, 0.8-1.2mol/L is preferably.
3. reagent according to claim 1 and 2, is characterized in that, described natural sugar compounds is selected from more than one or more in trehalose, isotrehalose, neotrehalose;
Described heavy metallic salt compounds is selected from more than one or more in Silver Nitrate, copper sulfate, iron protochloride, zinc chloride, is preferably copper sulfate or Silver Nitrate;
Described neutral salt compounds is selected from more than one or more in sodium-chlor, Repone K, magnesium chloride, calcium chloride, is preferably sodium-chlor or Repone K;
Described organic solvent compounds is selected from more than one or more in chloroform, acetone, methyl alcohol, ethanol, is preferably the composition of chloroform and ethanol;
Described alcohol compound to be carbon atom number the be alcohols of 3-4, is selected from more than one or more in glycerine, n-propyl alcohol, Virahol, propyl carbinol, ethylene glycol, is preferably glycerine or n-propyl alcohol;
Described thiocyanic acid compounds is selected from more than one or more in guanidine thiocyanate, ammonium thiocyanate, is preferably the composition of guanidine thiocyanate and ammonium thiocyanate;
Described phenolic compound is selected from more than one or more in phenol, p-cresol, Ortho Cresol, m-cresol, Phloroglucinol, is preferably phenol;
The salt compounds of described regulator solution potential of hydrogen is selected from more than one or more in sodium-acetate, Trisodium Citrate, is preferably the composition of Trisodium Citrate and sodium-acetate.
4. the reagent according to any one of claim 1-3; it is characterized in that, described reagent comprises trehalose, copper sulfate, sodium-chlor, guanidine thiocyanate, ammonium thiocyanate, ethanol, glycerine, chloroform, phenol, N-lauroyl sarkosine, Trisodium Citrate and sodium-acetate.
5. reagent according to claim 4, is characterized in that, in described reagent, the concentration of each component is respectively:
Trehalose 0.1-0.4mol/L, preferably 0.2-0.3mol/L; Copper sulfate 0.05-0.5mol/L, preferably 0.1-0.2mol/L; Sodium-chlor 1.0-2.0mol/L, preferably 1.0-1.4mol/L; Guanidine thiocyanate 0.6-2.0mol/L, preferably 0.6-1.0mol/L; Ammonium thiocyanate 0.3-1.0mol/L, preferably 0.3-0.5mol/L; Glycerine 4%-15% (v/v), preferably 4-6% (v/v); Chloroform 1.5-5.0mol/L, preferably 1.5-2.0mol/L; Ethanol 0.05-2.5mol/L, preferably 0.05-1.1mol/L; Phenol 35%-65% (v/v), preferably 35-45% (v/v); N-lauroyl sarkosine 0.01-0.07mol/L, is preferably 0.03-0.06mol/L; Trisodium Citrate 0.7-1.5mol/L, preferably 0.7-1.0mol/L; Sodium-acetate 0.1-1.0mol/L, preferably 0.1-0.2mol/L.
6. the reagent described in any one of claim 1-5 is used for the method that DNA and RNA extracts, and it is characterized in that, comprises the steps:
(1) reagent described in any one of claim 1-5 is added in sample, centrifugal after concussion, be divided into supernatant liquid and lower floor's liquid;
(2) wherein, in supernatant liquid for the extraction of RNA;
(3) lower floor's liquid is used for the extraction of DNA.
7. method according to claim 6, is characterized in that, in step (1), the concussion time is 5-10min, and centrifugal speed is 10000g-15000g, and centrifugation time is 10-20 minute.
8. the method according to claim 6 or 7, is characterized in that, step (2) adds room temperature concussion after Virahol at the middle and upper levels in liquid, centrifugal, discards liquid;
Then add the ethanol of 75%, room temperature is centrifugal, removes liquid, obtains RNA.
9. method according to claim 8, is characterized in that, shakes 10-20 minute after adding Virahol, and concussion speed is 50rpm-200rpm; Centrifugal speed 10000g-15000g, centrifugation time is 10-20 minute.
10. the method according to claim 7 or 8, is characterized in that, after step (2) adds 75% ethanol, centrifugal speed is 10000g-15000g, and centrifugation time is 10-20 minute.
11. methods according to any one of claim 6-10, is characterized in that, after adding dehydrated alcohol in step (3) in lower floor's liquid, room temperature concussion is centrifugal, removes liquid; Then add Trisodium Citrate ethanolic soln, the centrifugal rear removal liquid of room temperature concussion, obtains DNA.
12. methods according to claim 11, is characterized in that, after adding dehydrated alcohol in step (3), the concussion time is 5-10min, and concussion speed is 50rpm-200rpm;
After adding Trisodium Citrate ethanolic soln, the concussion time is 5-10 minute, and concussion speed is 50rpm-200rpm.
13. methods according to any one of claim 11 or 12, it is characterized in that, the concentration of the Trisodium Citrate in described Trisodium Citrate ethanolic soln is 0.05M-0.2M.
Reagent described in 14. any one of claim 1-5 or the method described in any one of claim 6-13 are for the application in the content of DNA and RNA that extracts and/or in working sample.
15. application according to claim 14, DNA and RNA of this application fetches can be used as genome sequencing, exon order-checking, specific region order-checking.
16. application according to claim 14, the sample in this application comprises fresh pathological tissues, tumor tissues case wax stone, tumor tissues case paraffin section, whole blood, blood plasma, serum, hydrothorax or fresh biopsy sample.
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