CN105713897B - A kind of method of efficient river crab tissue preserration and RNA extraction - Google Patents

A kind of method of efficient river crab tissue preserration and RNA extraction Download PDF

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CN105713897B
CN105713897B CN201510579433.1A CN201510579433A CN105713897B CN 105713897 B CN105713897 B CN 105713897B CN 201510579433 A CN201510579433 A CN 201510579433A CN 105713897 B CN105713897 B CN 105713897B
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rna
tissue
added
river crab
centrifuged
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CN105713897A (en
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陈晓雯
王军
王成辉
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Shanghai Maritime University
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Shanghai Maritime University
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Abstract

It is respectively organized the invention discloses a kind of field preservation river crab and its highly effective extraction method of RNA, step includes:(1)Tissue sampling and preservation:Tissue is cut into 2mm or so tissue block with sterile scissors, is added appropriate RNAstore solution, after 4 DEG C of soaked overnights, -80 DEG C of refrigerators is transferred to and saves.(2)Total RNAs extraction:The tissue of storage is taken out from -80 DEG C of refrigerators, is put into Trizol, is ground broken, centrifuging and taking supernatant with tissue grinder on ice, chlorination is imitative, then centrifuging and taking supernatant, adds isopropanol, then be centrifuged, it abandons after supernatant plus 75% ethanol washing RNA, centrifugation is diluted after drying precipitated with RNase-free water.(3)RNA purifying:Utilize the DNA in DNA enzymatic removal RNA.The method of the present invention can respectively organize the river crab of field acquisition effectively to be saved and carry out high efficiency extraction to its RNA.The RNA of extraction is high-quality, integrality is strong, purity is high, can be used for various molecular biological analysis, especially in terms of transcript profile sequencing, there is very strong practical value.

Description

A kind of method of efficient river crab tissue preserration and RNA extraction
Technical field
The present invention relates to the methods of the preservation and high efficiency extraction RNA of river crab organizing field acquisition tissue samples, belong to skill Art application field.
Background technique
River crab, scientific name Eriocheir sinensis (Eriocheir sinensis), be under the jurisdiction of Arthropoda (Arthropoda), Crustachia (Crustacca), Decapoda (Decapoda), Grapsidae (Grapsidae), Eriocheir (Eriocheir), at me State is distributed widely in the coastal various regions lake in north and south, is distributed mainly on the Changjiang river, the Liaohe River and Oujiang River, is that China is mostly important One of aquaculture object and economic crab.In recent years, as the development of aquaculture and the continuous of cultivation intensive degree add Greatly, the degeneration of studies on germplasm in Chinese mitten crab, Erocheir sinensis, generation, the quickening of the sex premature frequency of occurrences of explosive disease etc. are bringing tremendous economic While loss, the sustainable development of its industry also seriously restrict.With the high throughput sequencing technologies of a new generation, with transcript profile For platform, every molecular mechanism that is deeper within the scope of full-length genome, more thoroughly studying Eriocheir sinensis, to healthy aquaculture, Disease control and genetic breeding have critically important practice significance.However, when carrying out these researchs to Eriocheir sinensis, often It need to be sampled in field.Due to being influenced by time, traffic condition, sample importance factor, reality cannot be taken back to whole living bodies It tests room to be sampled, and takes back laboratory after portion of tissue can only be taken to save and carry out the analysis such as RNA.If preservation is improper, can make At subsequent RNA extract it is unsuccessful or extract yield it is very low, be unable to satisfy requirement of experiment.Thus, develop effective tissue preserration Method, it is particularly significant for the extraction of subsequent RNA.
In addition, river crab RNA of the inventor in the longer term is extracted in test, discovery either flesh tissue still passes through one The tissue that the section time saves, difficulty are above other aquatic livestocks.Be in particular in RNA extract yield is low, degradation speed is fast, Vulnerable to DNA pollution, it is difficult to meet the needs of subsequent experimental.It can be seen that the RNA extraction method of exploitation river crab high quality is very Important.On the other hand, Eriocheir sinensis inhabits freshwater lake river throughout the year, but is required to return to river mouth brackish water during breeding Domain, and breed during river mouth brackish water domain acquisition tissue samples can not rapidly extracting RNA, this requires the river crab of effect RNA is saved and extracting method.
Summary of the invention
The continuous of water system river crab RNA extraction each to China gropes to find in the process the present inventor in recent years, by perfect The preserving type of tissue samples and its purification process after extraction can greatly improve and extract RNA purity, make it not by DNA, egg The pollution of white matter and inorganic reagent, and the RNA integrality extracted can be made high, it is adaptable to transcript profile sequencing experiment etc. and requires.
It is that applicant is grinding the purpose of the present invention is to provide the method for a kind of river crab tissue preserration and RNA high efficiency extraction Study carefully the stabilization technique to grow up in practice.The formedness that tissue samples save and the height that RNA is extracted can be ensured using this method Effect property, RNA purity is high, the integrality of extraction are good, high-quality, are able to satisfy the high requests RNA such as transcript profile sequencing.The present invention not only exists In science and technology, and has a very important significance and be worth in the research of river crab molecular biology.
Technical solution provided by the invention is:A kind of method of efficient river crab tissue preserration and RNA extraction, including it is following Step:
(1)Tissue samples save:Short-term flesh tissue sample is cut into tissue block with sterile scissors, is dipped in is mounted in rapidly It is saved in Trizol reagent in 1.5ml centrifuge tube;Or field river crab living tissue sample is cut into tissue block, according to 1: 10 (Quality: volume)Ratio, be added RNAstore be transferred to -20 DEG C or -80 DEG C medium-term and long-term preservations after 4 DEG C of soaked overnights;
(2)The extraction of total serum IgE, steps are as follows:
A. the sample of preservation is taken out, takes appropriate tissue to be put into Trizol, is ground with tissue grinder, vortex oscillation;
B. it is centrifuged, takes supernatant, chloroform is added;Centrifugation, takes supernatant to add chloroform;
C. it is centrifuged, takes supernatant that isopropanol is added;
D. it is centrifuged, abandons supernatant, precipitate the alcohol of addition 75% after RNA;Supernatant is abandoned in centrifugation, is added after precipitating RNA 100% alcohol.
E. supernatant is discarded after being centrifuged, and the dissolution of RNase-free water is added in precipitating in drying precipitated RNA.
It further comprises the purifying of total serum IgE, and steps are as follows:
A. appropriate DNA enzymatic is added in the RNA solution that step is extracted upwards, is incubated at room temperature.
B. it is added into acquired solutionβ- mercaptoethanol and dehydrated alcohol, are put into adsorption column, and waste liquid is abandoned in centrifugation.
C. dehydrated alcohol is added into adsorption column, is centrifuged;Abandon waste liquid, sky centrifugation 5min.
D. appropriate RNase-free water is added into adsorption column, is centrifuged to obtain the final product.
Preferably,(1)Organization material needs to be cut into the tissue block of 2mm in step.
Preferably, the vortex oscillation is the sample that will the save vortex oscillation 45s in centrifuge tube, is stored at room temperature 2 points Clock.
Preferably,(1)In step, river crab living body long-term field tissue samples are cut into the tissue block of 2mm, according to 1: 10 (Quality: volume)Ratio, be added RNAstore be transferred to -20 DEG C or -80 DEG C medium-term and long-term preservations after 4 DEG C of soaked overnights.
The invention has the advantages that:In the above method, the river crab saved is respectively organized to can be used for RNA extraction, and uses this The RNA purity that method is extracted is good, quality is high, integrality is good.The present invention can effectively save each tissue samples of river crab, solve field It acquires tissue samples RNA and extracts difficult problem;The tissue RNA extracted by the method is high-quality, is applicable to RNA demand It is low, of poor quality to solve preservation sample rna extraction yield for big different kinds of molecules biological experiment, the especially sequencing of transcript profile Problem.This method provides a technological means for the research of river crab different kinds of molecules mechanism, has great scientific meaning and reality With value.
Detailed description of the invention
Fig. 1 is the electrophoresis detection result that the river crab that the present invention extracts respectively organizes RNA.
Fig. 2 is the RNA electrophoretogram that RNAstore saves sample extraction.
Fig. 3 is 2100 testing result of Agilent for the river crab hepatopancrease RNA that the present invention extracts.
Fig. 4 is 2100 testing result of Agilent for the river crab optic stalk RNA that the present invention extracts.
Fig. 5 is 2100 testing result of Agilent for the river crab gill RNA that the present invention extracts.
Fig. 6 is 2100 testing result of Agilent for the river crab muscle RNA that the present invention extracts.
Fig. 7 is 2100 testing result of Agilent for the river crab ovary RNA that the present invention extracts.
Fig. 8 is 2100 testing result of Agilent for the river crab spermary RNA that the present invention extracts.
Specific embodiment
A specific embodiment of the invention is as follows.
One, experimental material prepares
1.5mL centrifuge tube is several, 70% ethanol solution, trash ice, Trizol reagent, RNAstore reagent, chloroform, isopropanol,β- mercaptoethanol, RNase-Free Spin Columns CR2 (TIANGEN), 4 DEG C of centrifuges, living body Eriocheir sinensis
Two, the acquisition that river crab is respectively organized
1. the preservation of short-term fresh sample:1mL Trizol reagent is added in 1.5mL centrifuge tube, by living body Sinensis It is immediately placed in Trizol after each tissue sampling of chela crab, is transferred to -80 DEG C of refrigerator storages.
2. the long-term preservation of field sample:Sterile RNase-free centrifuge tube is taken, will be protected with sterile scissors, tweezers The tissue deposited is cut into the tissue block of 2mm or so, then according to 1: 10(Quality: volume)Ratio, be added RNAstore, in 4 DEG C After soaked overnight, the extraction of RNA can not be influenced with long-term preservation by being transferred to -20 DEG C or -80 DEG C.
Three, the extraction of total serum IgE
1. 0.5ml Trizol solution is added in centrifuge tube, the sample of preservation is taken out, appropriate tissue is taken to be put into It in Trizol, is ground with tissue grinder, 0.5ml Trizol solution is being added, centrifuge tube is shaken in vortex oscillator 45s is sufficiently mixed each ingredient in solution uniformly, places 2 minutes at room temperature.
2. being put into 4 DEG C of centrifuges in advance to the cold, 13500 revs/min, it is centrifuged 5 minutes.
3. carefully taking supernatant into another centrifuge tube, the chloroform of 300uL is added, is acutely rocked with hand 15 seconds, fills it Divide emulsification, is stored at room temperature 5 minutes to its layering.
4. being put into 4 DEG C of centrifuge, 12000 revs/min, it is centrifuged 15 minutes.
5. drawing supernatant 500uL into another centrifuge tube, the chloroform of 300uL is added, mixing of turning upside down is stored at room temperature 2 Minute, it is put into 4 DEG C of centrifuge, 12000 revs/min, is centrifuged 5 minutes.
6. carefully drawing the supernatant of 200uL into another centrifuge tube, isometric isopropanol is added and turns upside down mixing 30 Second, it is stored at room temperature 10 minutes.
7. being put into 4 DEG C of centrifuge, 12000 revs/min, it is centrifuged 10 minutes.
8. discarding supernatant, 75% ethanol washing that 1mL is added into centrifuge tube precipitates RNA, and is softly blown and beaten with pipette tips, It is suspended in RNA precipitate in ethanol solution.
9. being put into 4 DEG C of centrifuge, 12000 revs/min, it is centrifuged 2 minutes.
10. discarding supernatant, 100% ethanol washing that 1mL is added into centrifuge tube precipitates RNA, and is softly blown with pipette tips It beats, is suspended in RNA precipitate in ethanol solution.
11. being put into 4 DEG C of centrifuge, 12000 revs/min, it is centrifuged 5 minutes.
12. carefully discard supernatant, in being stored at room temperature 2 minutes dry RNA precipitates.
13. the RNase-free ddH of 50 μ L is added2O dissolution, carries out RNA purifying immediately.
Four, RNA is purified
1. I storing liquid of DNase of 2.5ul is added into the RNA solution of extraction, it is incubated for 10 minutes at room temperature.
2. by being added 3.5uL's in acquired solutionβThe dehydrated alcohol of-mercaptoethanol and 250uL, mixes well.
3. solution is put into the adsorbing of RNase-Free Spin Columns CR2 centrifugal column, 12000 revs/min Centrifugation, discards the waste liquid in collecting pipe.
4. 500uL dehydrated alcohol is added into adsorption column, 12000 revs/min of centrifugations discard the waste liquid in collecting pipe
5. being walked on repeating.
6. 13500 revs/min, sky centrifugation 5 minutes removes remaining liquid.
7. appropriate RNase-free water is added into adsorption column, it is centrifuged, acquired solution is RNA solution after purification.
Five, RNA is detected
The detection of 2000 spectrophotometer of 1.Nanodrop
1 μ L of RNA solution is taken, is uniformly mixed, with 2000 spectrophotometer test sample of Nanodrop.If the RNA extracted is molten Its OD of liquid260 / OD280Ratio is between 1.8 ~ 2.2, OD260/OD 230Ratio is between 1.40 ~ 1.80(Such as table one), show to mention The RNA taken is few by the pollution of protein, DNA and other organic reagents, and it is real can to meet the different kinds of molecules biology such as transcript profile sequencing It tests.
2. electrophoresis detection
2.5 μ L of RNA solution is taken, carries out electrophoresis with 1.5% Ago-Gel(Voltage 150V, time 18min)Detection, solidifying The clarity of RNA band is observed in glue Image analysis system.
Through electrophoresis detection, RAN clearly discernible 28S, 18S that this method is extracted(Two)And tetra- RNA bands of 5S, and And other miscellaneous bands not spread.The integrality for illustrating that RNA is extracted is preferable, specifically as shown in Figure 1, and long with RNAstore solution The extracted RNA integrality of field sample that phase saves is also preferable, specific as shown in Figure 2.
3.Agilent 2100 is detected
The RNA solution that the river crabs such as hepatopancrease, optic stalk, muscle, the gill, ovary, spermary are respectively organized Agilent Bioanal Yzer carries out Capillary Electrophoresis (capillary electrophoresis), and with the RIN of software (RNA Integrity Number) score is assessed, and 10 is best for RNA integrality, and 0 is worst.
Through detecting, the RIN of the RNA of the tissues such as hepatopancrease, optic stalk, muscle, the gill, ovary, spermary of extraction 8.0 ~ 9.5 it Between(It is shown in Table one, Fig. 3 to Fig. 8), further illustrate that RNA integrality is very high.
Table one, each tissue RNA concentration, OD260 / OD280And OD260/OD 230Value
Crab is respectively organized Concentration(ng/uL) OD260 / OD280 OD260/OD 230 RIN value
Hepatopancrease 587.7 1.92 1.42 8.2
Optic stalk 211.8 2.12 1.68 9.2
Muscle 182.7 2.08 1.52 9.2
The gill 207.8 1.98 1.47 8.7
Ovary 219.5 2.14 1.59 8.3
Spermary 351.4 1.91 1.45 8.5

Claims (5)

1. a kind of method of the preservation and high efficiency extraction RNA of river crab tissue samples, it is characterised in that include the following steps:
(1)Tissue samples save:River crab living body long-term field tissue samples are cut into tissue block, according to quality: volume 1: 10 Ratio, be added RNAstore be transferred to -20 DEG C or -80 DEG C medium-term and long-term preservations after 4 DEG C of soaked overnights;
(2)The extraction of total serum IgE, steps are as follows:
A. the sample of preservation is taken out, takes appropriate tissue to be put into Trizol, grinds;
B. vortex oscillation;
C. it is centrifuged, takes supernatant, chloroform is added;Centrifugation, takes supernatant to add chloroform;
D. it is centrifuged, takes supernatant that isopropanol is added;
E centrifugation, abandons supernatant, precipitates the alcohol of addition 75% after RNA;
F. it is centrifuged, abandons supernatant, precipitate the alcohol of addition 100% after RNA;
Discard supernatant after centrifugation, the dissolution of RNase-free water is added in precipitating in drying precipitated RNA.
2. the method as described in claim 1, which is characterized in that it further comprises the purifying of total serum IgE, and steps are as follows:
A. appropriate DNA enzymatic is added in the RNA solution that step is extracted upwards, is incubated at room temperature;
B. it is added into acquired solutionβ- mercaptoethanol and dehydrated alcohol, are put into adsorption column, and waste liquid is abandoned in centrifugation;
C. dehydrated alcohol is added into adsorption column, is centrifuged;Abandon waste liquid, sky centrifugation 5min;
D. appropriate RNase-free water is added into adsorption column, is centrifuged to obtain the final product.
3. the method according to claim 1, wherein(1)Organization material needs to be cut into the tissue of 2mm in step Block.
4. the method according to claim 1, wherein the vortex oscillation is the sample that will save in centrifuge tube Middle vortex oscillation 45s is stored at room temperature 2 minutes.
5. the method according to claim 1, wherein(1)In step, by river crab living body long-term field tissue sample Originally it is cut into the tissue block of 2mm, according to quality: RNAstore is added in the ratio of volume 1: 10, after 4 DEG C of soaked overnights, turns Enter -20 DEG C or -80 DEG C medium-term and long-term preservations.
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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN104031910A (en) * 2014-06-27 2014-09-10 上海海洋大学 High-efficiency extraction method of total RNA (ribonucleic acid) in freshwater crab haemolymph
CN104212795A (en) * 2014-09-26 2014-12-17 北京市水产科学研究所 Method for extracting total ribonucleic acid (RNA) of fish brain tissue

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104031910A (en) * 2014-06-27 2014-09-10 上海海洋大学 High-efficiency extraction method of total RNA (ribonucleic acid) in freshwater crab haemolymph
CN104212795A (en) * 2014-09-26 2014-12-17 北京市水产科学研究所 Method for extracting total ribonucleic acid (RNA) of fish brain tissue

Non-Patent Citations (1)

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Title
RNA样品保存液;北京华越洋生物科技有限公司;《百度文库》;20150910;第1-2页 *

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