CN110577980A - messenger RNA extraction method - Google Patents

Abstract

The invention discloses a method for extracting messenger RNA, wherein, the method comprises the steps of transfecting a plasmid marked with ribosome into an embryo by an intrauterine embryo electrotransfection technology, and carrying out a translation ribosome affinity purification technology after the plasmid grows to a specific time to obtain purified messenger RNA. The invention combines the intrauterine embryo point transfection technology and the translation ribosome affinity purification technology, marks specific neurons on specific time and brain areas according to the time characteristics of the development of neurons of different laminae of cerebral cortex, performs experiments at different time points in the development process so as to obtain a dynamic molecule library in the development period to construct a translation spectrum, and lays a solid foundation for summarizing rules, finding specific key factors and screening effective genes.

Description

Messenger RNA extraction method

Technical Field

the invention relates to a method for extracting messenger RNA, belonging to the field of biotechnology.

Background

The mammalian neocortex of the brain is a complex, highly organized structure consisting of 6 layers of different types of laminae (layers) between which hundreds of different types of neuronal and glial cells are located. The neocortex of the brain is the region of the brain responsible for cognitive function, sensory perception and consciousness, and has undergone significant expansion and development during evolution. During development, each lamina has a fixed sequence and time of development and produces characteristic neurons of different laminae in chronological order. Neuronal development is based on a central principle, where genetic material is transcribed and translated to ultimately produce proteins that exert biological effects through complex choreography of their processes to achieve specific cell identities. The ultimate type of development and functional role that determines a particular neuron is controlled by the genome, but this type of study is largely hampered by the complexity of the neuronal development process in terms of time and space. Finding the origin of such diverse and specific neurons in the neocortex of the brain and finding the rule of forming and maintaining the diversity of neurons is a scientific problem to be solved urgently at present.

Most of the current research on the development process of neurons focuses on the transcriptional level, and the transcriptional spectroscopy analysis is performed by extracting RNA from the whole brain or the whole cortical tissue. Although some molecular change rules in the neural development process can be found from the transcription profile by bioinformatics analysis, at the same time it has some limitations, among which are important: firstly, due to the temporal and spatial characteristics of neuron development, the conventional tissue sample extraction method has a complicated sampling process, comprises a plurality of regions, cortex laminae and cell types, and cannot perform classified analysis research on a specific region, a specific lamina or a specific neuron subtype; second, the transcript profiling is a study of the level of transcription, which is the study of cellular total RNA containing both exons (exon) that can encode proteins during translation and non-introns (Intron) that do not respond well to the laws of protein changes that ultimately exert biological effects due to a series of biological responses including post-transcriptional, translational and post-translational modifications. And solving the challenge requires a high-quality neuron subclass purification method, and takes the dynamic and multilayer characteristics of neurons into consideration, and neurons are collected and integrated for analysis during transcription and translation. At the same time, the combined spectrum of multiple neuronal subtypes from the same tissue is also obtained to better understand the regulatory pathways that form complex reticular relationships. Currently, attempts have been made to explain the complexity of neocortex and the different problems of characterizing neuronal cell types by various genetic and surgical means, however, labeling and purification and definition of neuronal subtypes remains challenging, as specific markers are always dynamically changed during cortical development. Therefore, a method for accurately extracting a neuron sample is urgently needed, which facilitates accurate detection and research on neuron development.

Disclosure of Invention

in view of the above problems of the prior art, the present invention is directed to a method for extracting messenger RNA, which is based on fluorescent plasmid-labeled ribosomes and collects messenger RNA bound to the labeled ribosomes to extract messenger RNA undergoing translation.

In order to achieve one of the above objects, the technical scheme of the messenger RNA extraction method adopted by the invention is as follows:

The extraction method of the invention is to transfect the plasmid marked with ribosome into mouse embryo by intrauterine embryo electrotransfection technology (IUE), and to perform translation ribosome affinity purification Technology (TRAP) when the mouse grows to a specific time, so as to obtain purified messenger RNA (mRNA).

The method can extract and detect the intracellular ribosome of a specific object at a specific time by combining the intrauterine embryo electrotransfection technology and the translation ribosome affinity purification technology, takes the research of nerve development as an aim, takes the mRNA in a specific nerve layer as an extraction object to extract, and is only a conventional experiment object, but the method is not limited to the extraction of the mRNA in the nerve cells of the mouse.

The IUE technology can transfect the target plasmid in a living state, has high success rate, does not influence the growth and development of the transfected living body, and lays a firmer technical foundation for researching growth and development states and other scientific research directions. The technical advantages of TRAP are: the ability to extract mRNA in situ from a particular cell type without sorting the cells; TRAP extracts mRNA which is translated in cells, and can accurately represent the translation condition of protein in the cells compared with total RNA; TRAP technology extracts not only mRNA of neuronal cell bodies, but also mRNA of axons, dendrites and spinous processes.

Preferably, the ribosome-labeling plasmid is pUBC-EGFP-L10 a. The plasmid of the present invention uses human UBIQUITIN C highly expressed in neurons as a promoter followed by the addition of the EGFP-L10a domain.

Mouse intrauterine embryo electrotransfection technology adopts two plasmids for cotransfection. Because the EGFP-L10a plasmid is transfected alone, the fluorescence intensity is weak, and the success of in vivo transfection modeling cannot be detected under an anatomical fluorescence microscope, the pCAG-tdTomato plasmid (expressing red fluorescence) is selected for co-transfection, the tdTomato fluorescence intensity is easy to detect and can mark an area, and a range mark can be provided for microdissection of a specific brain area under fluorescence. And the efficiency of cotransfection of the two plasmids can reach more than 90 percent. Thus, preferred co-transfected plasmids are the pUBC-EGFP-L10a and pCAG-tdTomato plasmids.

in a preferred embodiment, a cell staining solution is added at the time of co-transfection.

More preferably, the cell staining solution is selected from the group consisting of: fast green, hematoxylin or methylene blue dye solution.

in order to directly observe that the plasmid solution is accurately and smoothly injected into the lateral ventricle of the brain of a mouse embryo, cell staining solution is added into the plasmid mixed solution. Fast green is preferred, which is blue in color, non-toxic and non-side effects, and can be dissipated cleanly in a few hours.

The translation ribosome affinity purification technology of the invention uses magnetic beads of anti-GFP antibody to adsorb mRNA translated on mouse cerebral cortex ribosome, and obtains mRNA after purification.

the extraction method mainly aims at the neurons in the mouse cerebral cortex layer II/III to extract mRNA, the layer of neurons are mainly generated in the embryonic period E15.5 days, therefore, the breeding period is utilized, IUE treatment is carried out on the mouse embryo of the E15.5 days, the neurons in the layer II/III which are required to be researched are specifically marked, the target plasmid pUBC-EGFP-L10a is transfected into the mouse embryo to be verified, and after the offspring mouse is born, the offspring mouse successfully modeled is selected to carry out subsequent verification experiments. Offspring mice are born at the embryonic stage of the mice from E19 to E20 days, and whether the modeling is successful and the modeling quality are detected under a dissecting fluorescence microscope immediately after the offspring mice are all born. After the model is completed and the mouse to be offspring is born, three time points of P0/P7/P14 are selected for subsequent TRAP experiments.

Specifically, the extraction method comprises the following steps:

a) taking a pregnant mouse to expose a mouse embryo, and injecting a plasmid solution into the exposed mouse embryo brain;

b) Clamping two sides of a mouse embryonic brain by the electrode head ends, stimulating the brain by an electrotransfer pulse, and recovering in vivo culture after stimulation;

c) Taking brains of the mice in an ice bath after birth, dissecting a target cortical region on ice, and performing cracking centrifugation to obtain a supernatant;

d) Taking the supernatant obtained in the step c) and antibody beads for co-immunoprecipitation, and incubating overnight at 4 ℃;

e) Beads were separated on ice using a magnetic device, and mRNA was separated from the beads.

Preferably, mouse layer II/III neurons are labeled with plasmid pUBC-EGFP-L10A.

Preferably, the plasmid solution comprises: pUBC-EGFP-L10A plasmid, pCAG-tdTomato plasmid and fast green dye.

Preferably, the electrotransformation instrument stimulates for 4-8 times, each time of stimulation lasts for 50ms, the intensity is 40V, and the interval between each time is 950 ms.

preferably, the mice are dissected out of the brain in ice dissection buffer and the lysis is eliminated in ice tissue lysate.

More preferably, dissection buffer (50 mL): 1 × HBSS 5 ml; 2.5mM HEPES-KOH 125. mu.L, 35mM glucose 700. mu.L, 4mM NaCO₃224 μ L; 43.901ml of RNase-free water; immediately before use, 50. mu.L of Cycloheximide at 100ug/ml was added.

fissure of tissuedigest (Tissue-lysine buffer) (10 ml): 20mM HEPES-KOH (pH 7.4) 200. mu.L, 150mM KCl 750. mu.L, 10mM MgCl₂100 μ L of RNase-free water 8.735 ml; adding EDTA-free protease inhibitors 1tablet before use; 0.5mM DTT 5. mu.L; 10 μ L of 100ug/ml Cycloheximide; 10 μ L/ml rRNase 100 μ L; superasin 100. mu.L.

low salt solution (Low-salt buffer) (10 ml): 20mM HEPES-KOH (pH 7.3) 200. mu.L, 150mM KCl 750. mu.L, 10mM MgCl₂100 mu L, 1% (vol/vol) NP-401 ml, RNase-free water 6.935 ml; EDTA-free protease inhibitors tablet, 0.5mM DTT 5. mu.L, 100ug/ml cycloheximide 10. mu.L was added just before use.

high-salt solution (High-salt buffer) (10 ml): 20mM HEPES-KOH (pH 7.3) 200. mu.L, 350mM KCl 1.75. mu.L; 10mM MgCl₂100 mu L, 1% (vol/vol) NP-401 ml, RNase-free water 6.935 ml; immediately before use, 5. mu.L of 0.5mM DTT, 10. mu.L of 100ug/ml cycloheximide was added.

Preferably, the amount of beads per time is 300. mu.L, the amount of biotinylated protein L is 120. mu.L, and the amount of GFP antibody (19C8 or 19F7) is 50. mu.L.

Preferably, the specific process of lysis comprises:

c1) Centrifugally separating the digested and cracked lysate, adding NP-40 accounting for 10% of the supernatant of 1/9 into the supernatant, and uniformly mixing to obtain a mixed solution;

c2) To the mixture from step c1) was added 300mM DHPC in the existing liquid amount 1/9 and the tube was inverted to allow thorough mixing, incubated on ice and centrifuged to remove the supernatant.

Preferably, the co-immunoprecipitation step in step d) specifically comprises:

d1) mixing and adding a prepared antibody-Protein L-Dynabeads compound into the supernatant finally obtained in the step (H) for a co-immunoprecipitation reaction, and incubating overnight at 4 ℃;

d2) The next day, after incubation, the suspension was placed on ice, separated with a magnetic device, and washed 4 times with high salt buffer; after the 4 th washing, all washing solutions were removed, and the Bead-mRNA mixture separated by the magnetic device was allowed to rewet at room temperature, then 100. mu.L of Nanoprep lysis buffer containing 0.7. mu.L of beta-ME was added to the Beads-mRNA complex using the Stratagene Absolutley RNA Nanoprep kit according to the instructions and mixed well, and incubated at room temperature for 10 minutes to allow mRNA to separate from Beads;

d3) then separating the Beads from the lysis Buffer containing mRNA by using a magnetic device, taking out the lysis Buffer and putting the lysis Buffer into a new centrifugal tube;

d4) RNA purification is carried out according to the steps instructed by a kit manufacturer, and after purification is finished, the final product mRNA is transferred to a new clean cryopreservation tube and stored in a low-temperature refrigerator at minus 80 ℃ for later use.

The mRNA collected and extracted according to the above method was tested at 260/280 of between 2.06 and 2.24, 260/230 of between 2.06 and 2.35, and RNA concentration of between 32 and 178.3 ng/. mu.L. The obtained mRNA is a high-quality RNA sample, and is sufficient and pure.

compared with the prior art, the invention marks specific neurons at specific time and in brain areas according to the development time characteristics of different cortical neurons of the brain, and performs experiments at different time points in the development process so as to obtain a dynamic molecular library in the development period to construct a translation spectrum, thereby laying a solid foundation for summarizing rules, finding specific key factors and screening effective genes. The invention combines the intrauterine embryo point transfection technology and the translation ribosome affinity purification technology to construct a development period translation spectrum of a specific neuron, the construction method is stable and reliable, the translation condition of protein in the development period neuron under a specific time and space can be accurately reflected, the genome expression change characteristic of the mouse brain neocortex specific neuron during the development period is revealed from the translation aspect, the constructed translation spectrum database can be used as a tool for screening specific interested targets or targets with special significance to research the action of the cells in the development process and the expression characteristic under the pathological condition, the direction is provided for the research of some rare diseases and rare diseases clinically, the complete mRNA of the neuron of a specific plate layer can be extracted, and the translation condition of the protein in a cell is reflected more accurately,

mRNA purified based on TRAP is matched with Microarray chip scanning (Microarray) to obtain the change rule of the molecular library expression level of layer II/III cone projection neurons at different time points in the development process at the translation level, potential important molecules influencing the development of the layer of neurons are found, and a molecular database for reference query is constructed for searching molecular mechanisms of certain congenital neural development defect diseases.

Drawings

FIG. 1 is a map of a pUBC-EGFP-L10a plasmid after insertion of the domains of the fusion protein and transfection according to the present invention;

FIG. 2 is a protein map of the fluorescently labeled region of a mouse following IUE intervention according to the present invention;

FIG. 3 is a graph showing the results of the detection of samples at each step of the TRAP assay of the present invention.

Detailed Description

The messenger RNA extraction method provided by the present invention will be described in detail and fully below with reference to examples. The following examples are illustrative only and are not to be construed as limiting the invention.

The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all commercially available unless otherwise specified.

The instruments and reagents used in this example were as follows:

Neuro2a cell line, purchased from ATCC;

CD-1 mice, purchased from Charles River Laboratories, USA;

Penicillin-streptomycin, purchased from gibco;

PolyJet^TMDNA in vitro cell transfection kit from Signagen Laboratories

BCA protein quantification kit, purchased from Thermo Scientific;

GFP antibodies (Htz-GFP-19F7, Htz-GFP-19C8), available from Melial Sloan-Kettering Monoclonal Antibody Facility.

in this example, the 2/3 th layer (layer II/III) of the mouse cerebral neocortex, which begins to grow at 15.5 days of the mouse embryonic stage, was studied intensively, so In this example, 15.5 days of the embryonic stage was selected as the intervention time point, the plasmid pUBC-EGFP-L10A labeled with ribosome was transferred into the layer II/III neurons specifically by using intrauterine embryo electrotransfection (IUE), and then mRNA of specific neurons was extracted at the predetermined time point by TRAP to lay a solid foundation for the subsequent development and translation profiling of microarray.

First, transfection of nerve cells

1.1Neuro2a cell culture

the subculture is carried out by culturing N2a cells in a bottom area of 75cm²In the flash (2), 5% CO at 37 ℃₂The cells were seeded in complete cell culture medium of high-glucose DMEM, and 5% or 10% FBS and 1% penicillin-streptomycin were added. When the cell growth reaches 80% -90%, 0.25% of Trypsin-EDTA is added to break up and separate the cell strain, the cell strain is suspended in a culture medium, and then the cell strain is separated and inoculated according to the proportion of 1:10 to 1: 20.

1.2Neuro2a cell chemical transfection

(1) planting N2a cells on a 6-well plate (Bection Dickinson, 353043) with 1ml of culture solution added to each well;

(2) When the cell growth density is reached, starting to prepare for chemical transfection of the target plasmid;

(3) adding a culture solution for fresh preheating of cells 30 minutes to 1 hour before transfection;

(4) The procedure for cytochemical transfection was performed using PolyJet cytochemical transfection kit according to the instructions of the manufacturer. The plasmid was prepared as follows: DMEM 50. mu.L was taken and placed in two fresh clean centrifuge tubes, labeled A and B, respectively. Adding PolyJet reagent into tube A, then adding 1ug of plasmid (pCAG-GFP is used as a control group, pUBC-EGFP-L10a is used as an experimental group, and N is 3), and fully mixing;

(5) Pouring the mixture in the tube A into the tube B at one time, fully and uniformly mixing, and then incubating at room temperature for about 15 minutes;

(6) Adding the uniformly mixed plasmid reagent mixture into each hole of the culture plate, and gently and fully mixing uniformly, wherein each hole cannot be too violent, and each plasmid of each hole cell gives 1 ug;

(7) Replacing the fresh culture solution after 5-12 hours;

(8) After 48-72 hours, cellular proteins were extracted for future use in subsequent experiments.

The pUBC-EGFP-L10a plasmid was tested using the neuro2a cell line. Cell protein lines were extracted from neuro2a cells 48 hours after transfection with pCAG-GFP as a control, as shown in FIG. 1, L: EGFP-L10 a; g: GFP, antibody selected from anti-GFP (abcam-ab290, concentration 1:10000), and a single EGFP-L10a fusion protein is stably expressed (molecular weight is about 51kd) in a plasmid group transfected with pUBC-EGFP-L10 a; in contrast, in the control group, only GFP protein (molecular weight: about 26kd) was expressed. The target plasmid can stably express the EGFP-L10a fusion protein in the neuron cells, and provides evidence support for the next stage of animal experiments.

1.3 extraction of cellular proteins

Extracting the cell protein in the step (8) 1.2, which comprises the following steps:

(s1) placing the cells on ice, removing the culture medium and washing the cells with ice 1 × PBS 3 times per well;

(s2), after washing, adding 300. mu.l of lysis buffer into each well of cells, shaking up slightly, and fully lysing the cells;

(s3) after sufficient lysis, transferring the cell lysate to a fresh 1.5ml centrifuge tube, and placing the centrifuge tube on a mixing instrument to shake for 10 minutes at 4 ℃;

(s4) subsequently transferring the tube to a low temperature centrifuge, 16000g at 4 ℃ for 20 minutes;

(s5) the centrifuged supernatant was retained and transferred to a fresh clean centrifuge tube for either immediate BCA protein assay or storage in a-80 ℃ cryofreezer for future use.

second, mouse intrauterine embryo electrotransfection (IUE)

plasmid solutions were prepared, including control GFP and experimental EGFP-L10a, both of which were 1.5. mu.g/. mu.l plasmid solutions. Because the EGFP-L10a plasmid is transfected alone, the fluorescence intensity is weak, and the success of in vivo transfection modeling cannot be detected under an anatomical fluorescence microscope, the pCAG-tdTomato plasmid (expressing red fluorescence) is selected for co-transfection, the tdTomato fluorescence intensity is easy to detect and can mark an area, and a range mark can be provided for microdissection of a specific brain area under fluorescence. And through early-stage test, the efficiency of cotransfection of the two plasmids can reach more than 90 percent. In addition, in order to directly observe that the plasmid solution is accurately and smoothly injected into the lateral ventricle of the brain of a mouse embryo, fast green is added into the plasmid mixed solution together, and the mixed solution is blue, has no toxic or side effect, and can be completely dispersed within hours. The specific operation process is as follows:

2.1 mouse treatment

CD-1 pregnant mice with pregnancy duration E15.5.5 were used to continuously inhale anesthetized mice with isoflurane (concentration 2.8% at induction, 2.5% at the beginning of surgery) and anesthesia satisfaction was assessed with needle-prick toes; after anesthesia is satisfied, the abdomen of the mouse is shaved and is fixed on a heat preservation operation table in a supine position. The eye ointment is applied on the glasses to prevent the cornea from being dried and damaged during the anesthesia operation; spreading sterile gauze, making abdominal median incision (skin incision 1.5-2 cm, muscle peritoneum incision 1-1.5cm), and cutting skin and peritoneum layer by layer to obtain mouse uterus and string embryo; the uterus was gently hooked out of the abdominal cavity with loop forceps, the mouse embryos were exposed and kept moist with sterile 0.9% normal saline.

2.2 plasmid solution injection and electrotransfection

A. slowly injecting the prepared plasmids into the ventriculus cerebri of the embryo by a glass capillary needle;

B. to transfect the plasmid into neuronal progenitors, the tips of the electrodes were then clamped at the appropriate angle to both sides of the embryonic brain of the mouse using a transilluminator (BTX) and given 5 electrical pulse stimuli each lasting 50ms with an intensity of 40V and 950ms apart.

2.3 post-electrotransfer and post-operative treatment

Recovering embryo, suturing peritoneum and skin tightly, sterilizing locally, and stopping pain with drinking water containing ibuprofen after reviving.

2.4 postnatal examination of mice

After the modeling operation is completed, the mother mouse can grow the mouse at about the embryonic period E19-20 days, and whether the modeling is successful or not and the modeling quality are checked under a fluorescence microscope on the day of the birth. And immediately detecting whether the modeling is successful and the modeling quality under a dissecting fluorescence microscope after all the offspring mice of one litter are born. As the skull of the just born mouse is not developed, the light transmittance is good, and the fluorescence is easy to present. The plasmid was electroporated into radial glial cells at the periphery of the lateral ventricle and migrated to a predetermined lamina (layer II/III) of the cortex. After the offspring mouse is born, an anatomical fluorescence microscope is used for detecting a modeling result, visible red fluorescence is located in the left cerebral cortex, the fluorescence labeling cells migrate and are located on the upper layer of the cortex and are close to the surface of the brain, so that fluorescence color development is strong, then under the anatomical fluorescence microscope, the brain tissue with the fluorescence in the regional cortex is subjected to microdissection and is taken out, tissue protein is extracted, and then Western Blot detection is carried out. As shown in FIG. 2, the stably expressed EGFP-L10a fusion protein was found in the group of IUE electrotransfected pUBC-EGFP-L10a, which had a molecular weight of about 51kd, whereas no such fusion protein was expressed in the control Group (GFP). The results prove that the target plasmid and the cotransfection are stably and effectively expressed in vivo, and lay the foundation for the next stage TRAP experiment.

three time points are selected and respectively grouped, and the next experiment is carried out: i.e. the day after birth (P0), day 7 after birth (P7) and day 14 after birth (P14). The mouse P0 stage corresponds to a new born state, similar to the late embryonic stage of human embryos, in which neuronal migration is not completely completed and no regularity is formed in the upper cortex; 7 days after birth (P7), neuron migration is basically finished, neurons reach a preset fixed position and begin to mature, dendrite development is increased, the morphology is complicated, and connection with the periphery and far away is started to play a role, which is similar to the neonatal period of human beings; then the neuron continues to develop and mature, the dendrites continue to develop and grow, the branches become more, the complexity is enhanced, the fiber network continues to expand, the dendritic spines and synapses begin to form and rapidly increase in quantity, the dendrites basically develop at P14, at the moment, the dendritic spines begin to develop, the synapses are produced and formed in quantity, and the connection with the surroundings is established, which is similar to the adolescence of human beings.

2.5 brain tissue fixation, sectioning and validation of results

(b1) anesthetizing the offspring mice after successful modeling, and then sacrificing, and perfusing the offspring mice successfully modeled at different time points with 1 XPBS solution containing 4% PFA;

(b2) taking off the brain rapidly by microdissection, taking care of protecting the integrity of cerebral cortex, fixing in 1 × PBS containing 4% PFA overnight, and changing to 1 × PBS overnight the next day;

(b3) After the fixation is completed satisfactorily, placing the brain in 2-3% agarose gel for protection and fixation;

(b4) Using a vibrating microtome to perform coronal section on the brain of the mouse, wherein the thickness of each layer is 100 um;

(b5) after finishing, loading, flatly paving the brain slice on a glass slide, wiping off redundant PBS, covering the brain tissue slice with a Prolong Gold with DAPI (Invitrogen) capable of staining nuclei to protect fluorescence, completely covering with a cover glass, sealing the periphery of the cover glass by applying nail polish, and airing for later use;

(b6) pictures were taken using FV1000 confocal fluorescence microscope (Olympus) laser scanning. Cortical structures and cellular changes were observed. After IUE was performed at E15.5, P0 started to mark cells to be located in the upper cortex of the upper layer (layer II/III), the layer II/III was obviously thickened at P7 with the age of the mice, the cells became mature and complicated, the neurons continued to mature at P14, the regularity of the whole cell sheet layer was enhanced, and the fiber connection was increased. EGFP-L10a was green and expressed in pyramidal neurons of the cerebral cortex layer II/III, P0-P14. The pCAG-tdTomato for co-transfection labeled fluorescence is located at a position basically overlapping with green, which can indicate that the co-transfection efficiency is extremely high, and transfected cells have td-Tomato and EGFP-L10a together. DAPI is 4',6-diamidino-2-phenylindole (4',6-diamidino-2-phenylindole), can permeate through an intact cell membrane, quickly enter living cells, is strongly combined with DNA, is blue under a fluorescence microscope, can be used for living cell nuclear staining, and can clearly distinguish the basic structure condition of each neocortex layer after cortical lamella nuclear staining. As the wavelength of GFP, tomato and DAPI excitation light is different, the color of divergent light after excitation is also different, and therefore, the single sample can be subjected to multiple fluorescent staining detection verification.

2.6 extraction of histones

(e1) Placing a 60mm culture plate on ice, placing precooled 1 x PBS and fixing under a dissecting microscope or a fluorescence dissecting microscope;

(e2) Isoflurane anesthetized mice;

(e3) after satisfactory anesthesia, cutting off the head, quickly taking out the mouse brain from the precooled PBS, carrying out microdissection on a target cortical area of the mouse under a microscope or carrying out microdissection on a fluorescent area marked by IUE modeling of the mouse cerebral cortex under a fluorescence microscope, putting the mouse brain cortical area into a 1.5ml cryopreservation tube, quickly freezing the mouse cortical area in liquid nitrogen, and storing the mouse cortical area at-80 ℃ for later use; or directly putting the mixture into a precooled lysbuffer to directly enter the step of cracking and extracting protein;

(e4) Adding a proper amount of (300-;

(e5) Placing the centrifugal tube on a mixing oscillator and fully shaking for 10 minutes at 4 ℃;

(e6) Placing the centrifugal tube into a high-speed centrifuge, centrifuging for 10 minutes at the temperature of 4 ℃ and at 16000 g;

(e7) The supernatant protein extract is transferred to a fresh and clean freezing storage tube, and BCA protein determination is carried out immediately or the supernatant protein extract is stored in a low-temperature refrigerator at minus 80 ℃ for standby.

Third, Translational Ribosome Affinity Purification (TRAP)

The progeny mice after successful modeling were divided into 3 groups (P0, P7, P14), with males and females, and each experiment was repeated three times. The TRAP technology adopts improved steps, working solution is prepared one day in advance according to the operation steps, and a specific reagent is added before use. All tissues were sampled fresh without freezing. The operation needs to be rapid, the tissue is taken down to clean the blood, and the blood is immediately put into a special lysis solution, so that the dissection sampling time and the exposure time in the air are reduced, and the RNA degradation by the RNase in each step in the whole process is prevented from being reduced to the greatest extent, and the yield and the quality are prevented from being influenced. After the experiment was completed according to the procedure, the final TRAPmRNA product obtained was immediately stored in a refrigerator at-80 ℃ for further analysis.

3.1 preparation of working solution

Dissection buffer (separation buffer) (50 mL): 1 × HBSS 5 ml; 2.5mM HEPES-KOH 125. mu.L, 35mM glucose 700. mu.L, 4mM NaCO₃224 μ L; 43.901ml of RNase-free water; immediately before use, 50. mu.L of Cycloheximide at 100ug/ml was added.

tissue lysate (Tissue-lysis buffer) (10 ml): 20mM HEPES-KOH (pH 7.4) 200. mu.L, 150mM KCl 750. mu.L, 10mM MgCl₂100 μ L of RNase-free water 8.735 ml; adding EDTA-free protease inhibitors 1tablet before use; 0.5mM DTT 5. mu.L; 10 μ L of 100ug/ml Cycloheximide; 10 μ L/ml rRNase 100 μ L; superasin 100. mu.L.

low salt solution (Low-salt buffer) (10 ml): 20mM HEPES-KOH (pH 7.3) 200. mu.L, 150mM KCl 750. mu.L, 10mM MgCl₂100 mu L, 1% (vol/vol) NP-401 ml, RNase-free water 6.935 ml; EDTA-free protease inhibitors tablet, 0.5mM DTT 5. mu.L, 100ug/ml cycloheximide 10. mu.L was added just before use.

High-salt solution (High-salt buffer) (10 ml): 20mM HEPES-KOH (pH 7.3) 200. mu.L, 350mM KCl 1.75. mu.L; 10mM MgCl₂100 mu L, 1% (vol/vol) NP-401 ml, RNase-free water 6.935 ml; immediately before use, 5. mu.L of 0.5mM DTT, 10. mu.L of 100ug/ml cycloheximide was added.

3.2 preparation of affinity matrices

gently mix Streptavidin MyOne T1Dynabeads into a homogeneous suspension; the beads used in each purification were 300. mu.L, biotinylated protein L120. mu.L, and GFP antibody (19C8 or 19F7) 50. mu.L. Selecting different antibodies according to experimental groups, calculating the total amount of beads, taking out the antibodies by a liquid transfer machine, transferring the antibodies into a fresh and clean centrifugal tube, separating the magnetic beads on a magnetic operation plate, and washing the magnetic beads once by 1 multiplied by PBS (phosphate buffer solution); the beads were collected on a magnetic bench and dissolved in an appropriate amount of 1 XPBS, followed by addition of Protein L at a target concentration of 1 ug/. mu.L and incubation at room temperature for 35 minutes; the Protein L-beads complexes were collected on a magnetic bench and washed 5 times with 1 XPBS containing 3% IgG and protease-free BSA; after cleaning, collecting beads, adding low-salt buffer, adding GFP antibody according to a proportion, and incubating for 1 hour at room temperature; after incubation, the antibody beads complexes were washed 3 times with low-salt buffer, followed by addition of the appropriate amount of low-salt buffer for subsequent co-immunoprecipitation.

3.3 preparation of target tissue lysate (all manipulations were performed on ice):

i. After the offspring mice are anesthetized by isoflurane, the heads are cut off, and the brains are quickly dissected out in an ice disuse buffer;

ii, carrying out micro-dissection on the cerebral cortex of the required target area according to the fluorescence range under a fluorescence microscope, and cleaning residual blood;

immediately thereafter placing the obtained target tissue in a lysine buffer, after thorough trituration and digestion, adding the lysate to a fresh, clean 1.5ml centrifuge tube;

Centrifugation at 2000g for 10 min at 4 ℃;

v. taking out the supernatant, transferring the supernatant into a new centrifugal tube, adding 1/9 NP-40 accounting for 10% of the supernatant, and turning the freezing tube gently to mix fully;

add 300mM DHPC in the existing liquid volume of 1/9 and quickly and gently invert the centrifuge tube, mix well and incubate on ice for 5 minutes;

Subsequently the suspension is placed in a centrifuge and centrifuged at 20,000g for 10 minutes at 4 ℃;

Taking out the supernatant and placing the supernatant in a new centrifuge tube; (50. mu.L of the supernatant was additionally retained as Input, and after adding an equal sample buffer, it was boiled for 5 minutes for later verification).

3.4mRNA affinity purification

h1) (viii) mixing and adding a prepared antibody-Protein L-Dynabeads compound into the supernatant finally obtained in the step (viii) for co-immunoprecipitation, and incubating overnight at 4 ℃;

h2) the next day, after incubation, the suspension was placed on ice, separated with a magnetic device, and washed 4 times with high salt buffer; after the 4 th washing, all washing solutions were removed, and the Bead-mRNA mixture separated by the magnetic device was allowed to rewet at room temperature, then 100. mu.L of Nanoprep lysis buffer containing 0.7. mu.L of beta-ME was added to the Beads-mRNA complex using the Stratagene Absolutley RNA Nanoprep kit according to the instructions and mixed well, and incubated at room temperature for 10 minutes to allow mRNA to separate from Beads;

h3) then separating the Beads from the lysis Buffer containing mRNA by using a magnetic device, taking out the lysis Buffer and putting the lysis Buffer into a new centrifugal tube; (for confirmation, the remaining magnetic particle material was boiled for 5 minutes after adding 50. mu.L of sample buffer, and the supernatant was separated

h4) RNA purification operation is carried out according to the steps instructed by a kit manufacturer, and after purification is finished, the final product mRNA is transferred to a new clean cryopreservation tube and stored in a low-temperature refrigerator at minus 80 ℃ for later use;

h5) And reserving 1-2 mu L of sample for RNA quality detection so as to meet the requirements of the next microarray experiment on the sample. We measured 260/230 values, 260/280 values and mRNA concentrations of the samples using a Nanodrop 1000 spectrophotometer. The results are shown in table 1:

TABLE 1

table 1 shows the RNA quality data measured by Nanodrop, 260/280 is basically between 2.06 and 2.24, 260/230 is between 2.06 and 2.35, and the RNA concentration is between 32 and 178.3 ng/. mu.L. All the RNA samples are high-quality RNA samples, except that the 260/230 value of the P7 sample 2 is slightly low, but the 260/280 value is good, and the purity and the concentration are normal, so the overall change trend of downstream analysis is not influenced, and the result is not influenced. Usually, the absorbance of RNA solution is measured to determine RNA purity, and three indexes, 260/280, 260/230 and mRNA concentration of sample, are mainly detected to meet the sample quality requirement for microarray scanning analysis. The absorbance of the sample at 280nm represents the absorbance of nucleic acid, 260nm represents the absorbance of organic substances such as protein, and 230nm represents the salt concentration. 260/280 and 260/230 can reflect RNA purity very accurately, and high purity and high quality RNA is usually about 2 for A260/A280 and about 2 for A260/A230. In conclusion, the TRAP mRNA obtained in this example was pure and sufficient.

3.5TRAP procedure validation

western Blot is carried out on samples in all stages by using anti-GFP, as shown in figure 3, Input is supernatant of whole cell tissue lysate before adding anti-GFP magnetic beads, and IP is protein cleaved from the magnetic beads after completing the TRAP whole process. The EGFP-L10a fusion protein was visible in the Input experimental group (EGFP-L10a), whereas only GFP expression was detectable in the control Group (GFP). After the co-immunoprecipitation reaction, the fusion protein in the supernatant of the lysate is combined by magnetic beads through antigen-antibody reaction, after the reaction is finished, the mRNA-ribosome (EGFP-L10a fusion protein expression) -anti-GFP magnetic bead compound is separated by a magnet, the mRNA is separated from the ribosome by using a kit, and the final product mRNA is purified and stored. The EGFP-L10 a-anti-GFP magnetic bead complex was subsequently dissociated and examined by Western Blot experiments. In the verification of the products after the reaction (anti-GFP), the presence of EGFP-L10a fusion protein was clearly detected in the experimental group, whereas only GFP band was present in the control group and no fusion protein was expressed (as shown in FIG. 3), whereas no GFP or EGFP-L10a was expressed in the lysate (IgG) after the IP reaction. The anti-GFP magnetic bead can be specifically combined with ribosome protein, the translation ribosome-mRNA compound is effectively separated, and the experimental process is efficient, specific and reliable.

Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Claims (10)

1. a method for extracting messenger RNA is characterized in that: the extraction method comprises the steps of transfecting the plasmid marked with the ribosome into an embryo by an intrauterine embryo electrotransfection technology, and carrying out a translation ribosome affinity purification technology when the plasmid grows to a specific time to obtain purified messenger RNA.

2. the method for extracting messenger RNA according to claim 1, wherein: the plasmid for marking the ribosome is pUBC-EGFP-L10 a.

3. The method for extracting messenger RNA according to claim 1, wherein: mouse intrauterine embryo electrotransfection technology adopts two plasmids for cotransfection.

4. The method for extracting messenger RNA according to claim 3, wherein: the co-transfected plasmids were pUBC-EGFP-L10a and pCAG-tdTomato plasmid.

5. the method for extracting messenger RNA according to claim 2, wherein: and adsorbing the mRNA translated on the ribosome of the cerebral cortex of the mouse by using the magnetic beads of the anti-GFP antibody, and purifying to obtain the mRNA.

6. the method for extracting messenger RNA according to claim 1, characterized in that it comprises the following steps:

a) Taking a pregnant mouse to expose a mouse embryo, and injecting a plasmid solution into the exposed mouse embryo brain;

b) clamping two sides of a mouse embryonic brain by the electrode head ends, stimulating the brain by an electrotransfer pulse, and recovering in vivo culture after stimulation;

c) Taking brains of the mice in an ice bath after birth, dissecting a target cortical region on ice, and performing cracking centrifugation to obtain a supernatant;

d) taking the supernatant obtained in the step c) and antibody beads for co-immunoprecipitation, and incubating overnight at 4 ℃;

e) beads were separated on ice using a magnetic device, and mRNA was separated from the beads.

7. The method for extracting messenger RNA according to claim 6, wherein: mouse layer II/III neurons were labeled with plasmid pUBC-EGFP-L10A.

8. The method for extracting messenger RNA according to claim 6, wherein the plasmid solution comprises: pUBC-EGFP-L10A plasmid, pCAG-tdTomato plasmid and fast green dye.

9. The method for extracting messenger RNA according to claim 6, wherein: the electrotransformation instrument stimulates for 4-8 times, each time of stimulation lasts for 50ms, the intensity is 40V, and the interval between each time of stimulation is 950 ms.

10. The method for extracting messenger RNA according to claim 6, wherein the specific process of cleavage comprises:

c1) Centrifugally separating the digested and cracked lysate, adding NP-40 accounting for 10% of the supernatant of 1/9 into the supernatant, and uniformly mixing to obtain a mixed solution;

c2) To the mixture from step c1) was added 300mM DHPC in the existing liquid amount 1/9 and the tube was inverted to allow thorough mixing, incubated on ice and centrifuged to remove the supernatant.