CN103642797B

CN103642797B - A kind of Extraction method of total RNA of intermuscular bone of megalobrama amblycephala

Info

Publication number: CN103642797B
Application number: CN201310673534.6A
Authority: CN
Inventors: 高泽霞; 万世明; 易少奎; 钟嘉; 王卫民
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2013-12-11
Filing date: 2013-12-11
Publication date: 2016-01-20
Anticipated expiration: 2033-12-11
Also published as: CN103642797A

Abstract

The invention discloses a kind of method extracting Megalobrama amblycephala intermuscular bone total serum IgE, its step: be 1) material with Megalobrama amblycephala: get the mortar that its intermuscular bone is placed in sterilizing precooling, grinding, to intermuscular bone be ground to Powdered after proceed in centrifuge tube, add Trizol to shake up, room temperature is placed; 2) 4 DEG C, centrifugal, get supernatant liquor, add the chloroform of 1/5 volume, rock, fully emulsified is flat-white emulsion without layering, and room temperature is placed; 3) 4 DEG C, centrifugal, get supernatant liquor chloroform and repeat extracting; 4) get supernatant liquor, add isopyknic Virahol, Trisodium Citrate, sodium-chlor mixing ,-20 DEG C leave standstill; 5) after leaving standstill, 4 DEG C, centrifugal, abandon supernatant liquor and stay precipitation, add the ethanol of DEPC water configuration, concussion, repeated washing precipitates; 6) 4 DEG C, centrifugal, abandon supernatant liquor and stay precipitation, naturally dry, add DEPC water dissolution RNA, obtain total rna solution.Process is simple, and cost is low, and acquisition total rna concentration is high, purity is high, integrity good, meets further molecular biology research.

Description

A kind of Extraction method of total RNA of intermuscular bone of megalobrama amblycephala

Technical field

The present invention relates to a kind of extracting method of fish bone total serum IgE, specifically a kind of method being applicable to Megalobrama amblycephala intermuscular bone Total RNAs extraction.Present method is also applicable to the extraction of other fish intermuscular bone total serum IgE, and the total serum IgE of extraction can be used for further molecular biological cDNA synthesis and gene expression research.

Background technology

The flesh of fish is because nutrient composition content is high, and lipid content is low, is easy to digestion, is suitable for population and eats and the favor that is more and more subject to people.Megalobrama amblycephala ( megalobramaamblycephala) be under the jurisdiction of Cypriniformes ( cypriniformes), Cyprinidae ( cyprinidae), Culter subfamily ( culterinae), triangular bream belongs to ( megalobrama).It is one of distinctive important phytophage economic fish of China, because its feeding habits are wide, cost is low, growth is fast, surviving rate is high, easily fish for, and there is delicious, little, the advantage such as dressed fish is high, the bodily form good, specification is moderate, thus by as excellent herbivorous fishes kind at national popularity.Megalobrama amblycephala is promoted so far from cultivation, and cultured output increases year by year, occupies the 6th of China's freshwater aquiculture output, becomes the situation of selling well fish on the up-and-coming youngster of domestic freshwater aquiculture and market.But the existence of Megalobrama amblycephala intermuscular bone, causes very large disadvantageous effect to the processing of Megalobrama amblycephala with edible, so the molecular mechanism that research Megalobrama amblycephala intermuscular bone occurs, and then the Megalobrama amblycephala cultivated without intermuscular bone is significant.

The intermuscular bone (intermuscularbone) of fish also claims to sting between flesh, between muscle segment, is the membrane bone ossify by flesh diaphragm reticular tissue serial homology.Carry out Northernblot, cDNA clone, RT-PCR etc. of intermuscular bone gene expression research, all need to extract based on intermuscular bone total serum IgE.Intermuscular bone form of diverse, is distributed between muscle, is not easy to sampling; Volume is little, and relative surface area is large, and in sampling process, RNA easily degrades; Attachment muscle not easily removes, and easily causes protein contamination etc.Factors causes the difficulty extracting Megalobrama amblycephala intermuscular bone total serum IgE.

At present, the method extracting total serum IgE from osseous tissue is actually rare.Little from volume, easy degraded, the method being subject to extract total serum IgE intermuscular bone that mytolin pollutes not yet occur especially.Few by the intermuscular bone total serum IgE amount of traditional method for extracting, pollute high, further molecule experiments cannot be supported.

Summary of the invention

The object of the invention is the extracting method being to provide a kind of Megalobrama amblycephala intermuscular bone total serum IgE.Present method is the difficulty extracted for Megalobrama amblycephala intermuscular bone total tissue RNA of specificity first, provide a kind of implementation process simple, without the need to multiple plant and instrument, cost is low, the total rna concentration obtained is high, purity is high, integrity good, can meet the method for further molecular biology research.The reagent that present method uses, except Trizol, is all formulated with laboratory common drug; Instrument only has whizzer and aseptic operating platform; Used tool is basic molecule experiments instrument and dissection equipment.

To achieve these goals, present invention employs following technical measures:

A kind of Extraction method of total RNA of intermuscular bone of megalobrama amblycephala, the steps include:

1) take Megalobrama amblycephala as material: adopt 200-300g Megalobrama amblycephala, for anti-Hemostatic Oral Liquid is to the pollution of intermuscular bone, adopt asepsis injector from Megalobrama amblycephala tail venous puncture 1.5-2.0ml blood, after dead, 1-2min gets the mortar that its intermuscular bone is placed in sterilizing precooling, and back adds the grinding of liquid nitrogen limit, to intermuscular bone be ground to Powdered after proceed in 2ml centrifuge tube, every 50mg intermuscular bone powder adds 1mlTrizol, shake up, room temperature (20-25 DEG C, identical below) places 4-6min;

2) 4 DEG C, the centrifugal 4-6min of 12000rpm, gets supernatant liquor, adds the chloroform of 1/5 volume, acutely rocks 15sec, and fully emulsified is flat-white emulsion without layering, and room temperature places 4-6min;

3) 4 DEG C, the centrifugal 14-16min of 12000rpm, gets supernatant liquor chloroform and repeats extracting 1-2 time;

4) get supernatant liquor, add isopyknic Virahol, 200 μ l Trisodium Citrates (0.8mol/L), 200 μ l sodium-chlor (1.2mol/L) fully mix ,-20 DEG C of standing 3-5 hour;

5) after leaving standstill, 4 DEG C, the centrifugal 9-11min of 12000rpm, abandons supernatant liquor and stays precipitation, adds the 75%(volume ratio of DEPC water configuration) ethanol, gentle concussion, repeated washing precipitation 1-3 time;

6) 4 DEG C, the centrifugal 4-6min of 7500rpm, abandons supernatant liquor and stays precipitation, naturally dry, and adds 30 μ lDEPC water dissolution RNA, both obtains total rna solution.

The present invention compared with prior art, has the following advantages and effect:

From intermuscular bone, extracting directly total serum IgE can study the expression of intermuscular bone genes involved more targetedly, but not yet occurs before the extracting method of specificity for fish intermuscular bone total serum IgE.Because in intermuscular bone tissue, proteoglycan content is high, the intermuscular bone total serum IgE degraded of extracting with traditional cracking process is high, and protein residue is high.For these problems, the property of the present invention is directed to made solution, fully grind by intermuscular bone is separated with muscle rapidly and is placed in liquid nitrogen grinding, osteocyte is fully exposed, after add Trizol, the guanidinium isothiocyanate wherein contained has the function preventing RNA enzyme from playing a role, and cell can be made again to break rapidly, suppresses the release of nuclease.Reuse purity and integrity that chloroform ensure that RNA.While RNA is precipitated out by use Virahol, adds the salts solution of high density, make protein-polysaccharide solution modeling, reduce protein residue.

Concentration Testing, agarose gel electrophoresis, reverse transcription PCR inspection are carried out to extracted RNA, show that the intermuscular bone total rna concentration that the method is extracted is high, purity is high, integrity good, further molecular biology research can be met.

Accompanying drawing explanation

Fig. 1 is a kind of agarose gel electrophoresis detection figure of Megalobrama amblycephala intermuscular bone total serum IgE of extraction.

Fig. 2 is the agarose gel electrophoresis detection figure of cDNA template to specific gene pcr amplification result of a kind of Megalobrama amblycephala intermuscular bone total serum IgE reverse transcription.

In figure, band I is the electrophorogram of cDNA template to Megalobrama amblycephala internal reference β-actin gene PCR amplification; Band II is the electrophorogram of cDNA template to Megalobrama amblycephala RashomologgenefamilymemberAb gene PCR amplification; Band III is the electrophorogram of cDNA template to Megalobrama amblycephala transforminggrowthfactor β-2-like gene PCR amplification.

Embodiment

embodiment 1:

1) sample, it take Megalobrama amblycephala as material: the Megalobrama amblycephala sample fish three of random choose 200-300g, for anti-Hemostatic Oral Liquid is to the pollution of intermuscular bone, adopt asepsis injector from Megalobrama amblycephala tail venous puncture 1.5-2.0ml blood, after dead, 1-2min is separated intermuscular bone and picks clean muscle tissue from fish body muscle, be placed in the mortar of sterilizing precooling immediately, in the mortar of sterilizing precooling, limit adds the grinding of liquid nitrogen limit successively, to being ground into powder.Every bar fish gets 50mg intermuscular bone powder, puts the 2ml sterile centrifugation tube that 1mlTrizol is housed in advance respectively into, shakes up, and room temperature places 4 or 5 or 6min;

2) by place after centrifuge tube at 4 DEG C, after the centrifugal 5min of 12000rpm, Aspirate supernatant is transferred to an other 2ml sterile centrifugation tube, add the chloroform of supernatant liquor 1/5 volume, acutely rock 15sec, making mixed solution fully emulsified is the flat-white emulsion without layering, and room temperature places 4 or 5 or 6min;

3) by centrifuge tube at 4 DEG C, 12000rpm centrifugal 14 or 15 or 16min, gets supernatant liquor and repeats extracting 1 or 2 or 3 times;

4) extracting is again got supernatant liquor and is transferred to an other 2ml centrifuge tube after terminating, and add isopyknic Virahol, 200 μ l Trisodium Citrates (0.8mol/L), 200 μ l sodium-chlor (1.2mol/L), jog centrifuge tube fully mixes, and-20 DEG C leave standstill 4 hours;

5) after leaving standstill, by centrifuge tube 4 DEG C, 12000rpm centrifugal 9 or 10 or 11min, discards supernatant liquor and stays precipitation, adds the 75%(volume ratio of DEPC water configuration in centrifuge tube) ethanol, gentle concussion, fully washs;

6) repeated washing 1 or 2 or 3 times will be precipitated;

7), after last washing, by centrifuge tube 4 DEG C, 7500rpm centrifugal 4 or 5 or 6min, discards supernatant liquor and stays precipitation.After throw out dries naturally, add 30 μ lDEPC water dissolution RNA, obtain total rna solution;

8) take a morsel total rna solution for Concentration Testing, all the other-80 DEG C preservations, wait until subsequent experimental.

The agarose gel electrophoresis result of Megalobrama amblycephala intermuscular bone total serum IgE is as Fig. 1.

The cDNA template of Megalobrama amblycephala intermuscular bone total serum IgE reverse transcription to the agarose gel electrophoresis detected result of specific gene pcr amplification result as Fig. 2.Band I is the electrophorogram of cDNA template to Megalobrama amblycephala internal reference β-actin gene PCR amplification; Band II is the electrophorogram of cDNA template to Megalobrama amblycephala RashomologgenefamilymemberAb gene PCR amplification; Band III is the electrophorogram of cDNA template to Megalobrama amblycephala transforminggrowthfactor β-2-like gene PCR amplification.

In present method, the intermuscular bone total rna concentration of three sample fish extractions is followed successively by 452.9ng/ μ l, 370.5ng/ μ l, 470.1ng/ μ l, OD260/280 value is all greater than 2.00, further, subsequent detection experimental result is good, reaches the requirement of extracting intermuscular bone total serum IgE completely.

Claims

1. an Extraction method of total RNA of intermuscular bone of megalobrama amblycephala, the steps include:

1) with 200-300g Megalobrama amblycephala for material, adopt asepsis injector after tail venous puncture 1.5-2.0ml blood, from fish body muscle, be separated intermuscular bone and pick clean muscle tissue, being placed in the mortar of sterilizing precooling immediately, in mortar limit add liquid nitrogen limit grinding;

2) proceed in 2ml centrifuge tube by the intermuscular bone powder of grinding, every 50mg intermuscular bone powder adds 1mlTrizol, shakes up, and room temperature places 4-6min; 4 DEG C, the centrifugal 4-6min of 12000rpm, gets supernatant liquor, adds the chloroform of 1/5 volume, rocks 15sec, and fully emulsified is flat-white emulsion without layering, and room temperature places 4-6min; 4 DEG C, the centrifugal 15min of 12000rpm, gets supernatant liquor chloroform and repeats extracting 1-2 time;

3) get supernatant liquor, add isopyknic Virahol, 200 μ l0.8mol/L Trisodium Citrates, 200 μ l1.2mol/L sodium-chlor, fully mix ,-20 DEG C of standing 3-5 hour; After leaving standstill, 4 DEG C, the centrifugal 10min of 12000rpm, abandons supernatant liquor and stays precipitation, adds 75% volume ratio ethanol of DEPC water configuration, gentle concussion, repeated washing precipitation 1-3 time;

4) 4 DEG C, the centrifugal 4-6min of 7500rpm, abandons supernatant liquor and stays precipitation, naturally dry, and adds 30 μ lDEPC water dissolution RNA, obtains total rna solution.