CN105969763A - Extraction method of blueberry RNA - Google Patents
Extraction method of blueberry RNA Download PDFInfo
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- CN105969763A CN105969763A CN201610345498.4A CN201610345498A CN105969763A CN 105969763 A CN105969763 A CN 105969763A CN 201610345498 A CN201610345498 A CN 201610345498A CN 105969763 A CN105969763 A CN 105969763A
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Abstract
The invention discloses an extraction method of blueberry RNA. The method comprises the following steps: 1) grinding blueberry tissues into blueberry powder, mixing the blueberry powder with an extraction solvent, and incubating to obtain an incubation mixed solution; 2) carrying out extraction treatment on the incubation mixed solution to obtain an extraction supernate; 3) mixing the extraction supernate with a lithium chloride solution, standing over night, and centrifuging to obtain a total RNA precipitate; 4) dissolving the total RNA precipitate in DEPC (diethylpyrocarbonate)-treated ultrapure water, carrying out extraction treatment in a chloroform/isoamyl alcohol mixed solution, and carrying out centrifugation to obtain a centrifugal supernate; 5) mixing the centrifugal supernate with a sodium acetate solution, adding isopropanol, standing, and centrifuging to obtain a precipitate; 6) cleaning the precipitate with an ethanol solution to obtain a clean precipitate; and 7) putting the clean precipitate in the open container, and adding DEPC-treated ultrapure water to obtain the blueberry RNA. The method can be used for extracting blueberry RNA with excellent purity at high extraction rate.
Description
Technical field
The present invention relates to the extracting method of RNA, in particular it relates to the extracting method of blue berry RNA.
Background technology
Blue berry (Vaccinium spp.) is Ericaceae Vaccinium fruit shrubs.Blueberry is by the United Nations
Health organization is classified as the fruit that best nutritional is worth, and is chosen as the big brain-care foods in the world ten, quilt by the U.S. simultaneously
International food and agricultural organization is classified as one of big health food of the mankind five.So the social required quantity of blue berry increases day by day
Long, various countries' introducing and planting in succession.Since 2009, there is fast development in China's blueberry industry, currently mainly
It is distributed in five regions: Changbai Mountain and Xing'an Mountains region, Liaodong Peninsula, Jiaodong Peninsula, the Changjiang river stream
Territory, river, Yunnan-Guizhou and South China.Along with the continuous rising of cultivated area and yield, related application research and
Applied basic research increasingly receives publicity.Transcription group is carried out more on other garden crops, but
Research on blue berry is few.
In the histoorgan such as Blueberry and blade, the Secondary Metabolite Contents such as anthocyanidin is of a relatively high, adopts
Being not readily available successfully by conventional RNA extraction method (including that RNA extracts test kit), this becomes
The bottleneck of follow-up transcription group research.
Summary of the invention
It is an object of the invention to provide the extracting method of a kind of blue berry RNA, can be extracted by the method
Blue berry RNA and extraction ratio that purity is excellent are high.
To achieve these goals, the invention provides the extracting method of a kind of blue berry RNA, this extraction
Method includes:
1) first blueberry tissue is ground to form in the presence of liquid nitrogen blue berry powder, then by blue berry powder with take out
Extraction solvent mixes and hatches to obtain hatching mixed liquor；
2) mixed liquor will be hatched be stripped processing to obtain extracting supernatant in chloroform/isoamyl alcohol mixed liquor
Liquid；
3) extracting supernatant is mixed with lithium chloride solution, then carry out standing overnight process, then enter
Row centrifugal treating is to obtain total serum IgE precipitation；
4) total serum IgE is precipitated and dissolved in the ultra-pure water that DEPC (pyrocarbonic acid diethyl ester) processes, connects
And be stripped processing in chloroform/isoamyl alcohol mixed liquor, be then centrifuged processing to obtain centrifugal supernatant
Liquid；
5) centrifuged supernatant is mixed with sodium acetate solution, be subsequently added into isopropanol, then stand
Process, be finally centrifuged processing to be precipitated thing；
6) it is carried out obtaining cleaning deposit by ethanol solution by precipitate；
7) by uncovered for cleaning deposit placement, it is subsequently adding the ultra-pure water of DEPC process to obtain blue berry
RNA；
Wherein, extraction solvent contains CTAB (cetyl trimethylammonium bromide) extract and β-sulfydryl
Ethanol；It is stripped being processed as: by thing to be extracted and chloroform/isoamyl alcohol in chloroform/isoamyl alcohol mixed liquor
Mixed liquor mixes, and then carries out ice bath, is finally centrifuged process and takes supernatant；The cleaning of ethanol solution
For: thing to be cleaned is dissolved in ethanol solution, is then centrifuged for process and takes lower sediment.
By technique scheme, the present invention synergism by above steps, thus successfully from
Blue berry material has extracted blue berry RNA；Further, this easy extraction, and then meet
The order-checking of follow-up transcript profile and the requirement of correlational study.Meanwhile, the extraction ratio of this extracting method is higher.
Other features and advantages of the present invention will be described in detail in detailed description of the invention part subsequently.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and constitutes the part of description, with
Detailed description below is used for explaining the present invention together, but is not intended that limitation of the present invention.?
In accompanying drawing:
Fig. 1 is to detect sample indicatrix in detection example 1；
Fig. 2 is the agarose gel electrophoresis testing result figure detecting sample in detection example 1；
Fig. 3 is the testing result figure of the chip biological analyser of sample WGC055871 in detection example 1；
Fig. 4 is the testing result figure of the chip biological analyser of sample WGC055872 in detection example 1；
Fig. 5 is the testing result figure of the chip biological analyser of sample WGC055873 in detection example 1；
Fig. 6 is the testing result figure of the chip biological analyser of sample WGC055874 in detection example 1.
Detailed description of the invention
Hereinafter the detailed description of the invention of the present invention is described in detail.It should be appreciated that this place is retouched
The detailed description of the invention stated is merely to illustrate and explains the present invention, is not limited to the present invention.
The invention provides the extracting method of a kind of blue berry RNA, this extracting method includes:
1) first blueberry tissue is ground to form in the presence of liquid nitrogen blue berry powder, then by blue berry powder with take out
Extraction solvent mixes and hatches to obtain hatching mixed liquor；
2) mixed liquor will be hatched be stripped processing to obtain extracting supernatant in chloroform/isoamyl alcohol mixed liquor
Liquid；
3) extracting supernatant is mixed with lithium chloride solution, then carry out standing overnight process, then enter
Row centrifugal treating is to obtain total serum IgE precipitation；
4) total serum IgE is precipitated and dissolved in the ultra-pure water that DEPC processes, then in chloroform/isoamyl alcohol
Mixed liquor is stripped process, is then centrifuged processing to obtain centrifuged supernatant；
5) centrifuged supernatant is mixed with sodium acetate solution, be subsequently added into isopropanol, then stand
Process, be finally centrifuged processing to be precipitated thing；
6) it is carried out obtaining cleaning deposit by ethanol solution by precipitate；
7) by uncovered for cleaning deposit placement, it is subsequently adding the ultra-pure water of DEPC process to obtain blue berry
RNA；
Wherein, extraction solvent contains CTAB extract and beta-mercaptoethanol；In chloroform/isoamyl alcohol mixing
Liquid is stripped be processed as: mixed with chloroform/isoamyl alcohol mixed liquor by thing to be extracted, then carries out ice bath,
Finally it is centrifuged process and takes supernatant；The cleaning of ethanol solution is: thing to be cleaned is dissolved in ethanol solution
In, it is then centrifuged for process and takes lower sediment.
In the present invention, the constituent content of extraction solvent can select in wide scope, but in order to enable
Enough extraction ratios improving RNA further, it is preferable that in extraction solvent, CTAB extract and β-
The volume ratio of mercaptoethanol is 100:1-2；Wherein, CTAB extract particular make-up can also be wide
In the range of select, but in order to improve the extraction ratio of RNA further, it is highly preferred that CTAB extracting
Liquid contain CTAB, 95-105mM of 1.8-2.2 weight ratio % Tris-HCl buffer solution,
The EDTA-Na of NaCl, 22-28mmol/L of 1.8-2.2mol/L2The ultra-pure water processed with DEPC.
It addition, extraction solvent can be that configuration is the most standby in advance, this extraction solvent can preserve one at 3-5 DEG C
My god, but in order to prevent extraction solvent from going bad, the most now with the current.
Meanwhile, in the present invention, the consumption of each component in chloroform/isoamyl alcohol mixed liquor can be wide
In the range of select, but be able to improve further the extraction ratio of RNA, it is preferable that at chloroform/different
In amylalcohol mixed liquor, chloroform is 22-26:1 with the volume ratio of isoamyl alcohol.
It addition, in extracting processes, the consumption of chloroform/isoamyl alcohol mixed liquor can select in wide scope
Select, but in order to improve the extraction ratio of RNA, it is preferable that relative to 750 μ L things to be extracted, chloroform/
The consumption of isoamyl alcohol mixed liquor is 700-800 μ L.Meanwhile, in extracting processes, condition of ice bath can also
Select in wide scope, but in order to improve the extraction ratio of RNA, it is highly preferred that ice bath is the fullest
Be enough to lower condition: ice bath temperature is-25～-15 DEG C, and the ice bath time is 8-12min.
Step 1 at said method) in, the temperature of extraction solvent, the time of mixing and the tool hatched
Concrete conditions in the establishment of a specific crime all can select in wide scope, but in order to improve the extraction ratio of RNA further, excellent
Selection of land, in step 1) in, the temperature of extraction solvent is 63-68 DEG C, and the time of mixing is not less than 30s,
Hatching and at least meet following condition: incubation temperature is 63-68 DEG C, incubation time is 8-12min.
Additionally, in the step 1 of said method) in, CTAB extract and the concrete consumption of blue berry powder
Can select in wide scope, but in order to improve the extraction ratio of RNA, it is preferable that relative to
The CTAB extract of 750 μ L, the consumption of blue berry powder is 80-110mg.This is because, blue berry powder
End can reduce lytic effect too much, affects final result.But it is relatively low for rna contents such as fruits
Material, should be scaling up sample and extract consumption.
In the present invention, step 2) in the number of times that processes of extracting can change in wide scope, but
It is the extraction ratio in order to improve RNA, it is preferable that in step 2) in, the number of times that extracting processes
For 2-3 time.
Step 3 in the present invention) in, the volume ratio of extracting supernatant and lithium chloride solution can be wide
In the range of select, but in order to improve RNA extraction ratio, it is preferable that in step 3) in,
Extracting supernatant is 3-5:1 with the volume ratio of lithium chloride solution.
Step 3 in the present invention) in, the actual conditions standing overnight process can select in wide scope
Select, but in order to improve RNA extraction ratio, it is preferable that stand overnight process at least meet with
Lower condition: stand 8-16h at 3-5 DEG C or stand more than 6h at-22～-18 DEG C.
Step 3 in the present invention) in, the concentration of lithium chloride solution can select in wide scope, but
It is the extraction ratio in order to improve RNA, it is preferable that the concentration of lithium chloride solution is 8-12mol/L.
Step 4 in the present invention) in, the ultra-pure water that DEPC processes is permissible with the consumption that total serum IgE precipitates
Select in wide scope, but in order to improve the extraction ratio of RNA, it is preferable that in step 4)
In, the total serum IgE precipitation prepared relative to thing to be extracted described in 750 μ L, it is super that described DEPC processes
The consumption of pure water is 450-550 μ L.
Step 5 in the present invention) in, the consumption of each material can select in wide scope, but is
Improve the extraction ratio of RNA, it is preferable that in step 5) in, relative to the vinegar of 1 parts by volume
Acid sodium solution, the consumption of centrifuged supernatant is 8-12 parts by volume, and the consumption of isopropanol is 8-12 parts by volume.
Step 5 in the present invention) in, the actual conditions that standing processes can select in wide scope,
But in order to improve the extraction ratio of RNA, it is preferable that standing processes and at least meets following condition:
Dwell temperature is-25～-15 DEG C, and time of repose is 2-3h.
Step 5 in the present invention) in, sodium acetate solution and concentration and the temperature of isopropanol can be
Select in wide scope, but in order to improve the extraction ratio of RNA, the concentration of sodium acetate solution is
2-4mol/L, isopropanol carries out precooling treatment before use.
Step 6 in the present invention) in, the consumption of each material can select in wide scope, but is
Improve the extraction ratio of RNA, it is preferable that in step 6) in, treat described in 750 μ L
The precipitate that extract prepares, the consumption of described ethanol solution is 700-800 μ L.
Step 6 in the present invention) in, the number of times of cleaning can select in wide scope, but in order to
Improve the extraction ratio of RNA, it is preferable that the number of times of cleaning is 2-3 time.
Step 7 in the present invention) in, the actual conditions of uncovered placement can select in wide scope,
But in order to improve the extraction ratio of RNA, it is preferable that in step 7) in, uncovered it is placed as:
25-35min is placed at 3-5 DEG C.
Step 7 in the present invention) in, the consumption of each material can select in wide scope, but is
Improve the extraction ratio of RNA, it is preferable that relative to the cleaning deposit of 8.4-9.9 μ g, DEPC
The consumption of the ultra-pure water processed is 20-50 μ L.
In the present invention, the actual conditions of centrifugal treating can select in wide scope, but in order to carry
The high extraction ratio improving RNA, it is preferable that centrifugal treating at least meets following condition: centrifuging temperature is
3-5 DEG C, rotating speed is 11000-13000rpm, and centrifugation time is 0.5-20min.
On the basis of the above, the concrete kind of blueberry tissue can also select in wide scope,
Preferably, one or more in the root of blue berry, stem, leaf, flower, fruit and seed of blueberry tissue.
Hereinafter will be described the present invention by embodiment.In following example, should be noted that with
Lower item:
1) CTAB extract (containing 2 weight %CTAB, 100mmol/LTris-HCl buffer solution,
2mol/LNaCl solution, 25mmol/L EDTA-Na2The ddH that solution and DEPC process2O), second
Acid sodium solution (3mol/L, pH 5.2), lithium chloride solution (10mol/L), chloroform/isoamyl alcohol mixing is molten
Liquid (24:1, v/v) solution is purchased from Shanghai Jiang Lai (Shanghai Jianglai) company limited, and all
For RNase-free (without RNase)；The 1.5ml centrifuge tube of RNase-free, the PCR of 200 μ L
Pipe is purchased from Kirgen company purchased from Thermofisher company, the suction nozzle of 10-1000 μ L.
2) configuration of the ultra-pure water that DEPC processes: the DEPC of addition 0.1% in ultra-pure water, overnight,
Autoclaving removes DEPC, and the DEPC being RNase-free processes water.At unpasteurized DEPC
Reason water and DEPC reagent are forbidden for being tested, and sterilized DEPC processes water and can not be used for processing suction again
First-class article, to avoid being contaminated.
3) device such as agents useful for same, suction nozzle, centrifuge tube is RNase-free, concrete process step
For: by the ultra-pure water (ddH of addition 0.1% (v/v) DEPC such as suction nozzle, centrifuge tube2O) soak
Overnight, take out autoclaving and remove DEPC, be RNase-free, can be used for RNA experiment.CTAB
It should be noted that check for precipitation before extract uses, if there being precipitation, then should re-use in 65 DEG C of dissolvings.
4) 4h is toasted in 180 DEG C before mortar, spoon etc. use, to remove RNase.Mortar, spoon
Application Liquid nitrogen precooler before using.Process of lapping should add liquid nitrogen at any time, it is ensured that powder is adding extract
Do not melt before, RNA otherwise may be caused to degrade.
5) with 75% ethanol and absorbent cotton wiping superclean bench before embodiment starts, by required pipettor,
The things such as suction nozzle, mortar, spoon are placed in table top, alcohol burner on point, open superclean bench, and ultraviolet disappears
Use after poison 30min；Alcohol burner is kept not go out during work.
6) embodiment is in implementation process, glove Ying Qinhuan, when touching door handle, refrigerator doors, reality
Glove should be changed when testing the surface such as room table top, tables and chairs surface；Keep lab-gown clean and tidy, during work, avoid people
Member walks up and down, and closes the doors and windows, and reduces talk etc. to reduce RNase (RNase) and pollutes probability.
7) owing to containing a large amount of polyphenols in blueberry tissue, the brown material after oxidation sticks to grind
Be difficult on alms bowl remove, and under the influence of RNA once extract, can seriously reduce yield；Therefore use at mortar
Pouring 95 weight % ethanol after complete immediately into, soak in a moment, ethanol rinses twice, dry with water punching；If
The most divisible brown material, cleans mortar, notes making after soaking one day by the NaOH solution of 40g/L
NaOH is not had to remain with front.
Embodiment 1
1) beta-mercaptoethanol of the CTAB extract and 10 μ L that take 750 μ L joins 1.5mL and is centrifuged
To be configured to extraction solvent in pipe, then it is placed in 65 DEG C of baking oven preheatings；Then by blue berry material (such as 2-3
Blade) put in the mortar of Liquid nitrogen precooler, and abundant by liquid nitrogen grinding, become white powder.Take
100mg powder proceeds in the CTAB extract of above-mentioned preheating, immediately with the vibration mixing of vortex vortex mixer extremely
Few 30s, finally places in 65 DEG C of baking ovens and hatches 10min to obtain hatching mixed liquor.
2) by above-mentioned hatch mixed liquor take out shake up, be cooled to 25 DEG C, then in centrifuge tube add
The chloroform of 750 μ L/isoamyl alcohol mixed solution, softly mixes with hands, and ice bath 10min (is placed in-20 DEG C
In refrigerator)；Then at 4 DEG C, under the rotating speed of 12000rpm, it is centrifuged 10min；Last Aspirate supernatant,
Process with the extracting in the same manner of chloroform/isoamyl alcohol again and once obtain extracting supernatant；Wherein, draw
During supernatant, pollute for reducing impurity, should avoid being drawn onto lower floor's solute, with second layer liquid bottom the superiors
The liquid feed of face contact is not drawn.
3) above-mentioned supernatant is joined in the centrifuge tube of another 1.5ml, add supernatant volume
The LiCl solution of the 10mol/L of 1/4 volume, softly mixes；It is subsequently placed in 4 DEG C of left overnight 12h；
Finally, take out centrifuge tube at 4 DEG C, under the rotating speed of 12000rpm, be centrifuged 20min, abandon supernatant, it is thus achieved that thick
The total serum IgE precipitation carried.Wherein, for preventing RNA from precipitating the most completely, supernatant should transfer to one newly
The centrifuge tube without RNase in, guarantee that RNA abandons this supernatant after precipitating completely more later.
4) ultra-pure water of the DEPC process that above-mentioned total serum IgE is deposited in 500 μ L dissolves and puts
Put 2min, add the chloroform of 500 μ L/isoamyl alcohol mixed solution extracting process 1 time (concrete steps with
Step 2) in extracting process identical)；At 4 DEG C, finally under the rotating speed of 12000rpm, it is centrifuged 10min
To obtain centrifuged supernatant.
5) centrifuged supernatant is transferred in a new 1.5ml centrifuge tube, adds the 1/10 of supernatant volume
The NaAc solution of the 3mol/L of volume, after mixing, is subsequently added into the isopyknic pre-cooling of centrifuged supernatant
Isopropanol, then precipitates 2.5h at-20 DEG C；Last centrifugal under the rotating speed of 12000rpm at 4 DEG C
20min, abandons supernatant and is precipitated thing.
6) above-mentioned precipitate is dissolved in the ethanol solution of 75 weight % of 700 μ L, then at 4 DEG C
Under the rotating speed of 12000rpm, centrifugal 10min, carefully pours out supernatant.Then, the most again
Wash once with ethanol solution, pour out ethanol, centrifugal 30s under the rotating speed of 12000rpm at 4 DEG C, finally
With the remaining ethanol of the careful sucking-off of liquid-transfering gun.
7) above-mentioned centrifuge tube is uncapped at 4 DEG C, place 30min；After ethanol volatilizees totally completely, use
The described cleaning deposit of 9.0 μ g is dissolved by the ultra-pure water that the DEPC of 40 μ L processes, and sealed packaging obtains
Blue berry RNA sample WGC055871.Wherein, it is dispensed into after dissolving in the PCR pipe of 200 μ L, inspection
Save backup at-20 DEG C surveyed；That checks order tightly wraps the mouth of pipe with plastic packaging film, puts in cryopreservation tube, so
After liquid nitrogen at-70 DEG C preserves.
Embodiment 2
1) beta-mercaptoethanol of the CTAB extract and 7.5 μ L that take 750 μ L joins 1.5mL and is centrifuged
To be configured to extraction solvent in pipe, then it is placed in 63 DEG C of baking oven preheatings；Then by blue berry material (such as 2-3
Blade) put in the mortar of Liquid nitrogen precooler, and abundant by liquid nitrogen grinding, become white powder.Take 80mg
Powder proceeds in the CTAB extract of above-mentioned preheating, mixes at least 30s with the vibration of vortex vortex mixer immediately,
Finally place in 63 DEG C of baking ovens and hatch 8min to obtain hatching mixed liquor.
2) by above-mentioned hatch mixed liquor take out shake up, be cooled to 25 DEG C, then in centrifuge tube add
The chloroform of 700 μ L/isoamyl alcohol mixed solution, softly mixes with hands, and ice bath 10min (is placed in-25 DEG C
In refrigerator)；Then at 4 DEG C, under the rotating speed of 12000rpm, it is centrifuged 10min；Last Aspirate supernatant,
Process with the extracting in the same manner of chloroform/isoamyl alcohol again and once obtain extracting supernatant；Wherein, draw
During supernatant, pollute for reducing impurity, should avoid being drawn onto lower floor's solute, with second layer liquid bottom the superiors
The liquid feed of face contact is not drawn.
3) above-mentioned supernatant is joined in the centrifuge tube of another 1.5ml, add supernatant volume
The LiCl solution of the 10mol/L of 1/3 volume, softly mixes；It is subsequently placed in 4 DEG C of left overnight 8h；
Finally, take out centrifuge tube at 4 DEG C, under the rotating speed of 12000rpm, be centrifuged 20min, abandon supernatant, it is thus achieved that thick
The total serum IgE precipitation carried.Wherein, for preventing RNA from precipitating the most completely, supernatant should transfer to one newly
The centrifuge tube without RNase in, guarantee that RNA abandons this supernatant after precipitating completely more later.
4) ultra-pure water of the DEPC process that above-mentioned total serum IgE is deposited in 500 μ L dissolves and puts
Put 2min, add the chloroform of 500 μ L/isoamyl alcohol mixed solution extracting process 1 time (concrete steps with
Step 2) in extracting process identical)；At 4 DEG C, finally under the rotating speed of 12000rpm, it is centrifuged 10min
To obtain centrifuged supernatant.
5) centrifuged supernatant is transferred in a new 1.5ml centrifuge tube, adds the 1/8 of supernatant volume
The NaAc solution of the 3mol/L of volume, after mixing, is subsequently added into the isopyknic pre-cooling of centrifuged supernatant
Isopropanol, then precipitates 2h at-25 DEG C；At 4 DEG C, finally under the rotating speed of 12000rpm, it is centrifuged 20min,
Abandon supernatant and be precipitated thing.
6) above-mentioned precipitate is dissolved in the ethanol solution of 75 weight % of 700 μ L, then at 4 DEG C
Under the rotating speed of 12000rpm, centrifugal 10min, carefully pours out supernatant.Then, the most again
Wash once with ethanol solution, pour out ethanol, centrifugal 30s under the rotating speed of 12000rpm at 4 DEG C, finally
With the remaining ethanol of the careful sucking-off of liquid-transfering gun.
7) above-mentioned centrifuge tube is uncapped at 3 DEG C, place 25min；After ethanol volatilizees totally completely, use
The described cleaning deposit of 8.4 μ g is dissolved by the ultra-pure water that the DEPC of 20 μ L processes, and sealed packaging obtains
Blue berry RNA sample WGC055872.Wherein, it is dispensed into after dissolving in the PCR pipe of 200 μ L, inspection
Save backup at-20 DEG C surveyed；That checks order tightly wraps the mouth of pipe with plastic packaging film, puts in cryopreservation tube, so
After liquid nitrogen at-70 DEG C preserves.
Embodiment 3
1) beta-mercaptoethanol of the CTAB extract and 7.5 μ L that take 750 μ L joins 1.5mL and is centrifuged
To be configured to extraction solvent in pipe, then it is placed in 68 DEG C of baking oven preheatings；Then by blue berry material (such as 2-3
Blade) put in the mortar of Liquid nitrogen precooler, and abundant by liquid nitrogen grinding, become white powder.Take
110mg powder proceeds in the CTAB extract of above-mentioned preheating, immediately with the vibration mixing of vortex vortex mixer extremely
Few 30s, finally places in 68 DEG C of baking ovens and hatches 12min to obtain hatching mixed liquor.
2) by above-mentioned hatch mixed liquor take out shake up, be cooled to 25 DEG C, then in centrifuge tube add
The chloroform of 800 μ L/isoamyl alcohol mixed solution, softly mixes with hands, and ice bath 10min (is placed in-15 DEG C
In refrigerator)；Then at 4 DEG C, under the rotating speed of 12000rpm, it is centrifuged 10min；Last Aspirate supernatant,
Process with the extracting in the same manner of chloroform/isoamyl alcohol again and once obtain extracting supernatant；Wherein, draw
During supernatant, pollute for reducing impurity, should avoid being drawn onto lower floor's solute, with second layer liquid bottom the superiors
The liquid feed of face contact is not drawn.
3) above-mentioned supernatant is joined in the centrifuge tube of another 1.5ml, add supernatant volume
The LiCl solution of the 10mol/L of 1/5 volume, softly mixes；It is subsequently placed in 4 DEG C of left overnight 16h；
Finally, take out centrifuge tube at 4 DEG C, under the rotating speed of 12000rpm, be centrifuged 20min, abandon supernatant, it is thus achieved that thick
The total serum IgE precipitation carried.Wherein, for preventing RNA from precipitating the most completely, supernatant should transfer to one newly
The centrifuge tube without RNase in, guarantee that RNA abandons this supernatant after precipitating completely more later.
4) ultra-pure water of the DEPC process that above-mentioned total serum IgE is deposited in 500 μ L dissolves and puts
Put 2min, add the chloroform of 500 μ L/isoamyl alcohol mixed solution extracting process 1 time (concrete steps with
Step 2) in extracting process identical)；At 4 DEG C, finally under the rotating speed of 12000rpm, it is centrifuged 10min
To obtain centrifuged supernatant.
5) centrifuged supernatant is transferred in a new 1.5ml centrifuge tube, adds the 1/12 of supernatant volume
The NaAc solution of the 3mol/L of volume, after mixing, is subsequently added into the isopyknic pre-cooling of centrifuged supernatant
Isopropanol, then precipitates 3h at-15 DEG C；At 4 DEG C, finally under the rotating speed of 12000rpm, it is centrifuged 20min,
Abandon supernatant and be precipitated thing.
6) above-mentioned precipitate is dissolved in the ethanol solution of 75 weight % of 700 μ L, then at 4 DEG C
Under the rotating speed of 12000rpm, centrifugal 10min, carefully pours out supernatant.Then, the most again
Wash once with ethanol solution, pour out ethanol, centrifugal 30s under the rotating speed of 12000rpm at 4 DEG C, finally
With the remaining ethanol of the careful sucking-off of liquid-transfering gun.
7) above-mentioned centrifuge tube is uncapped at 5 DEG C, place 35min；After ethanol volatilizees totally completely, use
The described cleaning deposit of 9.9 μ g is dissolved by the ultra-pure water that the DEPC of 50 μ L processes, and sealed packaging obtains
Blue berry RNA sample WGC055873.Wherein, it is dispensed into after dissolving in the PCR pipe of 200 μ L, inspection
Save backup at-20 DEG C surveyed；That checks order tightly wraps the mouth of pipe with plastic packaging film, puts in cryopreservation tube, so
After liquid nitrogen at-70 DEG C preserves.
Embodiment 4
Carry out preparing blue berry RNA sample WGC055874 according to the method for embodiment 1, except for the difference that will
Step 2) in stand overnight particularly as follows: at-20 DEG C place 8h.
Detection example 1
Blue berry RNA sample WGC055871-WGC055874 is detected, concrete detection sample
Index is shown in Fig. 1, wherein, agarose gel electrophoresis testing result see Fig. 2 (Mlll is molecular weight marker,
Stripe size is followed successively by from top to bottom: 200,500,800,1200,2000,3000 and 4500bp),
The testing result of Agilent 2100Bioanalyzer chip biological analyser is shown in Fig. 3-Fig. 6, by Fig. 2-
Fig. 6 understands, and the blue berry total serum IgE electrophoretic band using this method to extract is clear, integrity preferable, energy
Enough meet the needs of downstream tests.The preferred embodiment of the present invention described in detail above, but, this
The detail that invention is not limited in above-mentioned embodiment, in the technology concept of the present invention, can
So that technical scheme is carried out multiple simple variant, these simple variant belong to the guarantor of the present invention
Protect scope.
It is further to note that each the concrete technology described in above-mentioned detailed description of the invention is special
Levy, in the case of reconcilable, can be combined by any suitable means, in order to avoid need not
The repetition wanted, various possible compound modes are illustrated by the present invention the most separately.
Additionally, combination in any can also be carried out between the various different embodiment of the present invention, as long as its
Without prejudice to the thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1. the extracting method of a blue berry RNA, it is characterised in that described extracting method includes:
1) first blueberry tissue is ground to form in the presence of liquid nitrogen blue berry powder, then by described blue berry powder
Mix with extraction solvent and hatch to obtain hatching mixed liquor；
2) mixed liquor will be hatched be stripped processing to obtain extracting supernatant in chloroform/isoamyl alcohol mixed liquor
Liquid；
3) described extracting supernatant is mixed with lithium chloride solution, then carry out standing overnight process, so
After carry out centrifugal treating with obtain total serum IgE precipitation；
4) described total serum IgE is precipitated and dissolved in the ultra-pure water that DEPC processes, then in chloroform/different
Amylalcohol mixed liquor is stripped process, is then centrifuged processing to obtain centrifuged supernatant；
5) described centrifuged supernatant is mixed with sodium acetate solution, be subsequently added into isopropanol, then carry out
Standing processes, and is finally centrifuged processing to be precipitated thing；
6) it is carried out obtaining cleaning deposit by ethanol solution by described precipitate；
7) by uncovered for described cleaning deposit placement, it is subsequently adding the ultra-pure water of DEPC process to obtain
Blue berry RNA；
Wherein, extraction solvent contains CTAB extract and beta-mercaptoethanol；Be set forth in chloroform/isoamyl alcohol
Mixed liquor is stripped be processed as: mixed with chloroform/isoamyl alcohol mixed liquor by thing to be extracted, then carry out
Ice bath, is finally centrifuged process and takes supernatant；The cleaning of described ethanol solution is: by molten for thing to be cleaned
In ethanol solution, it is then centrifuged for process and takes lower sediment.
Extracting method the most according to claim 1, wherein, in described extraction solvent, described
CTAB extract is 100:1-2 with the volume ratio of beta-mercaptoethanol, in described chloroform/isoamyl alcohol mixing
In liquid, chloroform is 22-26:1 with the volume ratio of isoamyl alcohol；
Preferably, described CTAB extract contains CTAB, 95-105mM of 1.8-2.2 weight ratio %
Tris-HCl buffer solution, the EDTA-Na of NaCl, 22-28mmol/L of 1.8-2.2mol/L2With
The ultra-pure water that described DEPC processes；
In described extracting processes, relative to thing to be extracted described in 750 μ L, described chloroform/isoamyl alcohol mixes
The consumption closing liquid is 700-800 μ L；Described ice bath at least meets following condition: ice bath temperature for-25～
-15 DEG C, the ice bath time is 8-12min.
Extracting method the most according to claim 1 and 2, wherein, in step 1) in, described in take out
The temperature of extraction solvent is 63-68 DEG C, the time of described mixing be not less than 30s, described in hatch the most satisfied
Following condition: incubation temperature is 63-68 DEG C, incubation time is 8-12min；
Preferably, relative to the CTAB extract of 750 μ L, the consumption of described blue berry powder is
80-110mg。
Extracting method the most according to claim 3, wherein, in step 2) in, at described extracting
The number of times of reason is 2-3 time.
Extracting method the most according to claim 4, wherein, in step 3) in, in described extracting
Clear liquid is 3-5:1 with the volume ratio of lithium chloride solution；
Preferably, stand overnight process described in and at least meet following condition: at 3-5 DEG C, stand 8-16h
Or at-22～-18 DEG C, stand more than 6h；
It is highly preferred that the concentration of described lithium chloride solution is 8-12mol/L.
6. according to the extracting method described in claim 4 or 5, wherein, in step 4) in, relative to
The total serum IgE precipitation that thing to be extracted described in 750 μ L prepares, the consumption of the ultra-pure water that described DEPC processes
For 450-550 μ L.
Extracting method the most according to claim 6, wherein, in step 5) in, relative to 1
The described sodium acetate solution of parts by volume, the consumption of described centrifuged supernatant is 8-12 parts by volume, institute
The consumption stating isopropanol is 8-12 parts by volume；
Preferably, described standing processes and at least meets following condition: dwell temperature is-25～-15 DEG C, quiet
The time of putting is 2-3h；
It is highly preferred that the concentration of described sodium acetate solution is 2-4mol/L, described isopropanol enters before use
Row precooling treatment.
Extracting method the most according to claim 7, wherein, in step 6) in, relative to 750 μ L
The precipitate that described thing to be extracted prepares, the consumption of described ethanol solution is 700-800 μ L；
Preferably, the number of times of described cleaning is 2-3 time.
9. according to the extracting method described in claim 7 or 8, wherein, in step 7) in, described spacious
Mouth is placed as: place 25-35min at 3-5 DEG C；
Preferably, relative to the described cleaning deposit of 8.4-9.9 μ g, it is ultrapure that described DEPC processes
The consumption of water is 20-50 μ L.
Extracting method the most according to claim 1 and 2, wherein, described centrifugal treating is the fullest
Be enough to lower condition: centrifuging temperature is 3-5 DEG C, rotating speed is 11000-13000rpm, and centrifugation time is
0.5-20min；
Preferably, during described blueberry tissue is selected from the root of blue berry, stem, leaf, flower, fruit and seed
Plant or multiple.
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| CN110157702A (en) * | 2019-05-24 | 2019-08-23 | 中国水产科学研究院南海水产研究所深圳试验基地 | It is a kind of for extracting the acquisition station of coral RNA sample |
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