CN111440787A - Method for obtaining animal tissue RFPs by using molecular sieve centrifugal column - Google Patents

Method for obtaining animal tissue RFPs by using molecular sieve centrifugal column Download PDF

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CN111440787A
CN111440787A CN202010256695.5A CN202010256695A CN111440787A CN 111440787 A CN111440787 A CN 111440787A CN 202010256695 A CN202010256695 A CN 202010256695A CN 111440787 A CN111440787 A CN 111440787A
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molecular sieve
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rfps
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何慧琼
董鸣
彭龙
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Wuhan Yongding Medical Technology Co ltd
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Abstract

The invention provides a method for obtaining animal tissue RFPs by using a molecular sieve centrifugal column, which comprises the following steps: carrying out pretreatment such as blood removal, freezing, grinding and the like on animal tissues, and then cracking cell lysate to obtain a pretreatment substance; carrying out staged high-speed centrifugal treatment on the pretreated substance, and collecting supernatant; adding the supernatant into a pretreated molecular sieve centrifugal column, and then centrifuging at a low speed to obtain a primary extract RNC; adding nuclease into the primary extract RNC for enzyme digestion, and then adding a reaction stopping reagent and an RNA extraction reagent to obtain the animal tissue RFPs. Compared with other extraction methods, the animal tissue RFPs obtained by the method, particularly the RFPs in animal heart tissues, can save a large amount of extraction time, has high available effective data quality and low ribosome occupation ratio, and can eliminate the interference of invalid data on the research of cardiovascular diseases in the aspect of the translatomics.

Description

Method for obtaining animal tissue RFPs by using molecular sieve centrifugal column
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for obtaining animal tissue RFPs by using a molecular sieve centrifugal column.
Background
The translatomics, which connect transcriptomes and proteomes, are an important pivotal point in the hierarchical transmission process of bioinformatics. Translation is not simple to transmit gene expression information, but widely and finely regulates and controls effective information in the transmission process, is a regulation step with the largest amplitude, and plays a vital role in protein folding, protein positioning, functions and yield. The research on the aspect also increasingly draws more and more attention from scientists and experts in the same industry. In 2009, the Weissman project group first proposed the ribosol profiling (rib-seq) technique. From this time, the Ribo-seq technology has gradually occupied an important position in the scientific research field. The Ribo-seq technology mainly adopts the second generation sequencing technology to detect RNA small fragment of about 22-30bp protected by Ribosome, also called Ribosome Focprins (RFPs). Generally, in the methods for studying Ribosome profiling, tissue or cells are basically lysed, after non-Ribosome-enriched free mRNA is removed by enzyme digestion, a sucrose density gradient centrifugation is adopted to separate a single Ribosome, and then different methods for removing Ribosome RNA are adopted according to different experimental requirements and then arranged for sequencing.
However, there are many problems in the application of the Ribo-seq technology, such as complicated operation, high requirement for instruments, long time consumption, and poor effect. In the process of tissue or cell prophase cracking treatment, the concentration and time of ribonuclease (RNase) are not controlled, the condition of over-enzyme digestion or under-enzyme digestion can occur, and the enzyme digestion conditions under different conditions are difficult to unify, so that the final sequencing result is invalid, and even the research cannot be continuously completed. In addition, in the case of sucrose density gradient centrifugation, expensive instruments such as an ultra-high speed centrifuge are required, which not only increases the cost but also makes the operation very cumbersome. In addition, during the operation process of sucrose density gradient centrifugation, the sucrose solution cannot be sterilized to cause RNase pollution, and impurity pollution often exists in the centrifugation process, so that the degradation of sample nucleic acid is finally caused, and the recovery rate is low. In addition, in the final rRNA removal procedure, a probe for removing rRNA is usually designed for intact rRNA, and after the cleavage, the cleaved rRNA cannot be effectively bound to the probe, resulting in low rRNA removal efficiency. The challenges in the application of these Ribo-seq techniques have greatly hindered the development of Riboseq techniques in laboratory and commercial applications.
In order to find out the characteristics of the Riboseq technology in the process of extracting rRNA from heart tissues, according to the previous RIBO-seq sequencing experiments of different tissue samples of animals (mice), the ribosome rRNA accounts for a very high percentage (60-80%) in heart tissue sequencing data after the ribosome rRNA is enucleated after the traditional sucrose density gradient centrifugation method is adopted, and effective data are greatly reduced. The heart is used as the central hub of human body functions, the protein expression in heart tissues is abnormally rich, and the expression of the proteins is also regulated and controlled by translation. The change of the translation regulation mode can further influence the change of the protein content and the function of the protein, and further cause cardiovascular diseases such as myocardial infarction, heart failure, myocardial hypertrophy and the like. Therefore, whether RFPs in animal heart tissues can be rapidly and completely acquired and the content and change of the RFPs can be measured is an important prerequisite for understanding the occurrence, development and treatment effects of cardiovascular diseases; the technical problem of the Riboseq technology (the ribosome rRNA occupation ratio is still very high) results in that the experimental data obtained by the Riboseq technology cannot be used for judging cardiovascular diseases.
When the research progress of the main extraction method of RFPs in the Riboseq technology is tracked, the conventional method is also referred, after animal tissues are cracked, non-specific nuclease is used for removing non-ribosome-covered free mRNA, and a sucrose density gradient centrifugation method is used for extraction, but the technical problems of high instrument requirement, high cost, easy pollution, complex operation and the like are still not overcome in the extraction process, and according to the previous research data, the method has the defects of low data quality and high ribosome proportion in the extraction of the RFPs in the animal heart tissues, and plays an interference role in the research of the cardiovascular diseases in the aspect of the translational omics.
Therefore, there is a need to provide an improved technical solution to overcome the technical problems in the prior art.
Disclosure of Invention
In order to solve the above problems in the prior art, the present invention provides a method for obtaining animal tissue RFPs by using a molecular sieve column centrifuge, and the animal tissue RFPs obtained by the method of the present invention, particularly the RFPs in animal heart tissue, can save a lot of extraction time compared with other extraction methods, and wherein the available effective data quality is high, the ribosome occupation ratio is low, and interference of invalid data on the research of cardiovascular diseases in the aspect of the transcriptomics can be eliminated.
The invention provides a method for obtaining animal tissue RFPs by using a molecular sieve centrifugal column, which comprises the following steps:
step one, animal tissue pretreatment: cleaning collected animal tissues with blood, freezing the animal tissues in liquid nitrogen for at least 2-5 min, grinding the frozen animal tissues into powder with the granularity of 5-10 meshes, adding cell lysate, and cracking the animal tissues at the temperature of 10-35 ℃ for 20-30 min to obtain a pretreated substance;
step two, centrifuging the pretreated substance obtained in the step one at a high speed, collecting supernatant, then dropwise adding the supernatant into a pretreated molecular sieve centrifugal column to obtain a treated molecular sieve centrifugal column, centrifuging the treated molecular sieve centrifugal column at a low temperature and a low speed, and collecting filtrate to obtain a primary extract RNC;
step three, adding nuclease into the primary extract RNC for enzyme digestion, wherein the enzyme digestion temperature is 20-30 ℃, and the reaction time is 10-60 min, wherein the addition volume V of the nuclease is = (A260 RNC-A260 buffer) × VE × 800/VE, the A260buffer and the A260RNC are respectively an enzymolysis buffer solution of the nuclease measured by using a spectrophotometer and the light absorption value of the primary extract RNC obtained in the step two at the position of 260nm, VE is the volume (m L) of the primary extract RNC, and CE is the concentration (U/ml) of the nuclease;
and step four, adding an RNA extraction reagent into the product obtained in the step three to obtain RFPs.
Through the technical scheme, the molecular sieve centrifugal column subjected to pre-sedimentation reaction and enzyme digestion reaction under the action of the molecular sieve can effectively release and store high-quality mRNA (messenger ribonucleic acid), particularly mRNA subjected to translation in heart tissues, in a short time.
Further, in the method, the adding volume of the cell lysate in the first step is 3-20 times of the volume of the frozen animal tissue, and the cell lysate comprises 20mM monopotassium phosphate (pH7.4), 15mM MgCl2, 200mM KCl, 2mM DTT, 100ug/m L cycloheximide and 1% (v/v) Triton X-100.
Further, in the method, the temperature of the high-speed centrifugation in the second step is 0-5 ℃, the speed of the high-speed centrifugation in the second step is 13000-24000 g, and the time of the high-speed centrifugation in the second step is 3-30 min; the temperature of the low-temperature low-speed centrifugation in the second step is 0-5 ℃, the speed of the low-temperature low-speed centrifugation in the second step is 300-800 g, and the time of the low-temperature low-speed centrifugation in the second step is 1-3 min. The inventor finds that through the technical scheme, through the action of high-speed centrifugation, the crushed cells are precipitated and separated by high-mass organelles and other impurities; after the molecular sieve action of the centrifugal column, the low-speed centrifugation can further separate and screen the new peptide chain compound.
Further, in the method, the pretreated molecular sieve centrifugal column in the second step comprises a pretreated MicroSpin S400 HR-GE molecular sieve centrifugal column, and the fourth step further comprises: and (4) adding an enzyme digestion reaction stopping reagent into the product obtained in the step three. The inventor adopts the technical scheme that the screening of the molecular sieve centrifugal column subjected to liquid-passing sedimentation treatment in advance is used for activating matrix substances in the centrifugal column, so that the later-stage separation and screening are facilitated; and (4) adding an enzyme digestion reaction termination reagent to avoid the risk of invalidation of experimental results due to over digestion after the enzyme digestion reaction in the step four is finished.
Further, in the method, the nuclease comprises micrococcal nuclease (Micrococcus nuclease), and the RNA extraction reagent comprises Trizol. Through the technical scheme, the inventor realizes the following technical effects: treating with added micrococcal nuclease, performing enzyme digestion reaction on the nascent peptide chain complex (RNC) to obtain short-chain RNA (ribonucleic acid) combined with ribosome, namely RFP (Ribose nucleic acid) which is translated instantly, and then adding a normal RNA extraction reagent Trizol to extract RNA.
Further, in the method, the preparation method of the pretreated molecular sieve centrifugal column comprises the steps of adjusting the temperature of the molecular sieve centrifugal column to 10-35 ℃, adding 2.5-6 m L molecular sieve centrifugal column balance buffer solution without RNase to perform column balance, standing at the temperature of 2-6 ℃ for 12-18 hours, centrifuging at the centrifugal speed of 300-800 g for 1-5 minutes, removing liquid in the column, and obtaining the pretreated molecular sieve centrifugal column.
Further, in the method, the high-speed centrifugation in step two is performed at least twice, and the molecular sieve column equilibration buffer comprises: 20mM monopotassium phosphate (pH7.4), 15mM MgCl2200mM KCl, sterile water without RNase and sterile instruments are not needed when the molecular sieve centrifugal column balance buffer solution is prepared, and the molecular sieve centrifugal column balance buffer solution is prepared without sterile treatment. Through this technical scheme, through centrifugation and aseptic processing, can prevent to cause the pollution of final material because of the interference of other bacterium or other materials.
Further, in the method, the sequence length of the RFPs does not exceed 40 bp. Through the technical scheme, the achievable technical effects are as follows: RNA which is not combined with ribosome is removed through enzyme digestion, and a tightly combined RNA fragment with the size not more than 40bp theoretically is obtained.
The invention also provides a nucleic acid prepared according to the method.
The invention also provides the use of a nucleic acid according to the above in the biological, medical or chemical field.
The beneficial effects created by the invention are as follows: compared with other extraction methods, the animal tissue RFPs obtained by the method, particularly the RFPs in animal heart tissues, can save a large amount of extraction time, has high available effective data quality and low ribosome occupation ratio, and can eliminate the interference of invalid data on the research of cardiovascular diseases in the aspect of the translatomics.
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In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to these drawings without inventive effort.
FIG. 1 is a nucleic acid size measurement chart after digestion of kidney tissue (S) and heart tissue (C) in the experimental group (C1, S1) and the control group (C2, S2).
Detailed Description
The experimental methods of the following examples, which are not specified under specific conditions, are generally determined according to national standards. If there is no corresponding national standard, it is carried out according to the usual international standards, to the conventional conditions or to the conditions recommended by the manufacturer.
The features mentioned with reference to the invention or the features mentioned with reference to the embodiments can be combined. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.
In the present invention, all the technical features mentioned herein and preferred features may be combined with each other to form a new technical solution, if not specifically stated.
In order to make the technical means, the original characteristics, the achieved purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments, but the invention includes but is not limited to the embodiments.
Molecular sieve spin columns (Microspin S400 HR-GE, available from GE Healthcare);
nanodrop ultramicro spectrophotometer (available from Thermo Fish);
tissue grinder (TIANGEN (OSE-Y50));
balb/c mice (purchased from the research center for laboratory animals in Hubei province);
trizol (Invitrogen corporation);
NEB small RNA library kit (from New England corporation);
Micrococcalnuclean (from New England Biolabs);
cycloheximide mother liquor (purchased from Sigma-Aldrich);
EGTA stop buffer (from New England Biolabs);
other reagents or equipment are all common products sold in the market.
Before the experiment, the solutions used in the examples were prepared as follows:
(1) the concentration of the cycloheximide mother liquor is 10mg/m L.
(2) Cell lysis solution: 20mM monopotassium phosphate (pH7.4), 15mM MgCl2200mM KCl, 2mM DTT, 100ug/m L cycloheximide and 1% (v/v) Triton X-100, wherein sterile water without RNase and sterile instruments are used for preparation, and the solution needs to be sterilized by filtration after preparation.
(3) Molecular sieve spin column equilibration buffer: 20mM monopotassium phosphate (pH7.4), 15mM MgCl2200 MKCl, sterile and RNase-free water and sterile instruments are used for preparation, and the solution needs to be filtered and sterilized after preparation.
(4) Nuclease:Micrococcalnuclean, sterile and RNase-free water and sterile instruments are used for preparation, and after the solution is prepared, filtration sterilization is carried out.
Comparative example 1 extraction method of ribosome nascent-chain complex (RNC) from animal kidney and heart tissue
This comparative example used the method described in patent CN108624651A to extract ribosomal nascent peptide chain complexes (RNCs), labeled as control RNCs, from the same batch of mouse kidney and heart tissue of example 1 using sucrose density gradient ultracentrifugation.
Example 1 extraction method of animal Kidney and Heart tissues RNC
This example describes a method for extracting ribosome nascent-chain complex (RNC) from animal kidney and heart tissue, comprising the following steps:
(1) and (3) balancing the molecular sieve centrifugal column, namely taking out the molecular sieve centrifugal column (MicroSpin S400 HR-GE or under the same condition), recovering to room temperature (10-35 ℃), adding 3m L RNase-free molecular sieve centrifugal column balancing buffer solution, performing column balancing, vertically placing in a refrigerator at 4 ℃, and performing overnight settlement or settlement for 12-18 h.
(2) Dissecting 4-week-old Balb/c mice, collecting kidney and heart tissues of the mice, rapidly cleaning blood with PBS solution, placing into liquid nitrogen, and rapidly freezing for 3 min.
(3) And (3) putting the quick-frozen kidney and heart tissues into a grinding tube, grinding by using a tissue grinder until the granularity is 6 meshes, collecting the ground powder into a sterile EP tube, adding 1m L of the cell lysate prepared above, and performing lysis at 4 ℃ for 25 min.
(4) Collecting mouse kidney and heart tissue lysate, placing in a precooled 4 ℃ centrifuge, centrifuging at 17000 × g for 5min to remove large debris, carefully sucking the supernatant, continuing to place in the centrifuge, centrifuging at 17000 × g for 15min to remove small debris, carefully sucking the supernatant, and transferring to a clean sterile EP tube.
(5) And taking out the molecular sieve which has been settled or settled overnight, centrifuging for 2min at the rotating speed of 600 × g, and removing liquid in the column to obtain the prepared molecular sieve centrifugal column.
(6) And C, quickly taking out the 50u L transferred cell lysis supernatant obtained in the step four, carefully and dropwise adding the cell lysis supernatant to the surface of a molecular sieve centrifugal column bed without scattering the surface of the column bed, centrifuging for 1min at the rotating speed of 600 × g, and collecting filtrate, wherein the filtrate is a purified RNC sample and is marked as an experimental group RNC.
Example 2 method for rapidly obtaining animal tissue RFPs Using molecular Sieve spin columns
This example mainly describes a method for rapidly acquiring RFPs by using a molecular sieve centrifugal column, comprising the following steps:
(1) nuclease was added to the RNC sample of the experimental group obtained in example 1, and the value of a260 in the RNC solution was measured using a Nanodrop ultramicro spectrophotometer according to the formula V = (a 260)RNC-A260buffer)×VE×800/CEConfirming the amount of nuclease added, and reacting at room temperature for 30min, wherein V is the volume amount of nuclease added (m L), A260RNCAbsorbance at 260nm for RNC, A260bufferAbsorbance at 260nm of the buffer, VEVolume of RNC solution (m L), CEIs the concentration of MicrococcalNuclean (gel U/ml), wherein the buffer solution is nuclease enzymolysis buffer solution, and the specific components are Tris-HC L (the range is 40-100 mM) with pH7.650 mM and CaCl 5mM2(1-10mM), 6mM MgCl2(1-10mM), 100ug/m L cycloheximide solution (50-200 ug/m L), and 100ug/m L BSA solution (50-200 ug/m L).
(2) After the enzyme digestion time is finished, EGTA stop solution with the final concentration of 50mM is added into the system.
(3) RFPs were obtained by extracting RNA from the solution using Trizol (Invitrogen) which is an RNA extraction reagent according to the manual of instructions.
The control group RNC obtained in comparative example 1 and the experimental group RNC obtained in example 1 were extracted by the method described in this example to obtain the control group RFP-RNA and the experimental group RFP-RNA respectively.
Example 3 identification of animal tissue RFPs extracted by different methods
In order to detect the method of the invention, undegraded RFPs can be extracted from different tissues, the RFP-RNA of the control group and the RFP-RNA of the experimental group obtained in the embodiment 2 are subjected to library construction by using a NEB small RNA library construction kit, and a 120-bp band is cut for second-generation sequencing. The obtained data are mapped to a mouse genome reference sequence, an rRNA reference sequence and a CDS reference sequence respectively by adopting a FANSe3 algorithm (specific parameters, refer to the application of an ultrahigh-precision large-scale sequencing mapping algorithm FANSe in non-model organisms in research literature of Caoxin and Zhang Tokyo), the gene with Read count larger than 10 is taken as an identified gene, the result is shown in figure 1, the sizes of detected fragments of RFPs extracted from a detected sample are all 20-35bp through detection, main fragments are concentrated at about 28-29 bp, the size characteristics of the RFPs are met, and correct extraction is proved.
Example 4 analysis of sequence information of animal tissue RFPs extracted by different methods
In order to detect the undegraded RFPs extracted from different tissues by the method of the present invention, the control RFP-RNA and the experimental RFP-RNA obtained in example 2 were subjected to high-throughput secondary sequencing, and the results are shown in Table 1. The experimental result shows that the extraction method of the invention adopts the molecular sieve centrifugal column method to obtain better experimental effect on heart tissues, the ribosome occupation ratio is obviously reduced, the CDS region mapping rate is obviously higher than that of a control group, the number of identified genes is also larger than that of the control group, and the reaction time and the loss cost are obviously reduced, as shown in Table 1.
TABLE 1 sequencing alignment data after restriction enzyme digestion of Kidney and Heart tissues in Experimental and control groups
Figure 228050DEST_PATH_IMAGE001
In some embodiments of the present invention, the inventor finds that the method of the present invention can be used for not more than 3 hours only by extracting the RNC. The original ultra-high speed centrifugation method is adopted, including the ultra-high speed centrifugation method, the experiment time is about 5-12 hours, impurities are easily mixed, and the quality of the product obtained by the experiment result is influenced.
Example 5A method of obtaining animal tissue RFPs Using a molecular Sieve spin column
This example describes a method for obtaining animal tissue RFPs using a molecular sieve spin column.
Firstly, preparing a pretreated molecular sieve centrifugal column, wherein the preparation method comprises the steps of restoring a molecular sieve centrifugal column (MicroSpin S400 HR-GE or the same condition) to room temperature (10-35 ℃), adding 2.5m L RNase-free molecular sieve centrifugal column balance buffer solution, carrying out column balance, vertically placing in a refrigerator at 2 ℃, carrying out overnight settlement or settlement for 12 hours, then centrifuging at the centrifugal speed of 300g for 5min, and removing liquid in the column to obtain the pretreated molecular sieve centrifugal column;
then, starting to obtain animal tissue RFPs by using a molecular sieve centrifugal column method, which comprises the following steps:
step one, animal tissue pretreatment: dissecting a Balb/c mouse with the age of 4 weeks, collecting the kidney of the mouse, quickly cleaning blood by adopting a PBS (phosphate buffer solution), quickly freezing for 2min after putting into liquid nitrogen, grinding the frozen animal tissue to powder with the granularity of 5 meshes to obtain ground animal tissue powder, then adding a cell lysate (the volume of which is 3 times of the ground animal tissue powder), and cracking for 30min at the temperature of 10 ℃ to obtain a pretreated substance;
step two, placing the pretreated substance obtained in the step one in a precooled 0 ℃ centrifugal machine, centrifuging for 5min at the rotating speed of 13000 × g to remove large fragments, sucking the supernatant, continuing to place the supernatant in the centrifugal machine, centrifuging for 25min at the rotating speed of 13000 × g to remove small fragments, sucking the supernatant, transferring the supernatant into a clean sterile EP tube, collecting the supernatant, and then dropwise adding the supernatant into a pretreated molecular sieve centrifugal column to obtain the treated molecular sieve centrifugal column;
step three, centrifuging the treated molecular sieve centrifugal column at 0 ℃ at the centrifugal speed of 300 × g for 3min, and collecting filtrate to obtain a primary extract RNC;
step four, adding nuclease into the primary extract RNC for enzyme digestion, wherein the enzyme digestion temperature is 20 ℃, and the reaction time is 10 min; the addition volume of the nuclease V = (A260)RNC－A260buffer）×VE×800／VE，A260bufferAnd A260RNCRespectively measuring the absorbance value V at 260nm of the enzymolysis buffer solution of the nuclease obtained by using a spectrophotometer and the primary extract RNC obtained in the step twoEVolume of primary carrier RNC (m L), CEConcentration of nuclease (U/ml); and step five, adding an enzyme digestion reaction stopping reagent (EGTA stop solution with the final concentration of 50 mM) into the product obtained in the step four, and then extracting RNA in the solution by using Trizol (Invitrogen company) according to an instruction manual to obtain the RFPs.
Example 6A method of obtaining animal tissue RFPs Using a molecular Sieve spin column
This example describes a method for obtaining animal tissue RFPs using a molecular sieve spin column.
Firstly, preparing a pretreated molecular sieve centrifugal column, wherein the preparation method comprises the steps of restoring a molecular sieve centrifugal column (MicroSpin S400 HR-GE or the same condition) to room temperature (10-35 ℃), adding 6m L RNase-free molecular sieve centrifugal column balance buffer solution for column balance, vertically placing the column in a 6 ℃ refrigerator for overnight settlement or settlement for 18 hours, then centrifuging at the centrifugal speed of 800g for 1min, and removing liquid in the column to obtain the pretreated molecular sieve centrifugal column;
then, starting to obtain animal tissue RFPs by using a molecular sieve centrifugal column method, which comprises the following steps:
step one, animal tissue pretreatment: dissecting a Balb/c mouse with the age of 4 weeks, collecting the kidney of the mouse, quickly cleaning blood by adopting a PBS (phosphate buffer solution), quickly freezing for 5min after putting into liquid nitrogen, grinding the frozen animal tissue to powder with the granularity of 10 meshes to obtain ground animal tissue powder, then adding a cell lysate (the volume of which is 20 times of the ground animal tissue powder), and cracking for 20min at the temperature of 35 ℃ to obtain a pretreated substance;
step two, placing the pretreated substance obtained in the step one in a precooled 5 ℃ centrifugal machine, centrifuging for 1min at the rotating speed of 24000 × g to remove large fragments, sucking the supernatant, continuing to place the supernatant in the centrifugal machine, centrifuging for 2min at the rotating speed of 24000 × g to remove small fragments, sucking the supernatant, transferring the supernatant into a clean sterile EP tube, collecting the supernatant, and then dropwise adding the supernatant into a pretreated molecular sieve centrifugal column to obtain the treated molecular sieve centrifugal column;
step three, centrifuging the treated molecular sieve centrifugal column at 5 ℃ at the centrifugation speed of 800 × g for 1min, and collecting filtrate to obtain a primary extract RNC;
step four, adding nuclease into the primary extract RNC for enzyme digestion, wherein the enzyme digestion temperature is 30 ℃, and the reaction time is 60 min; the addition volume of the nuclease V = (A260)RNC－A260buffer）×VE×800／VE，A260bufferAnd A260RNCRespectively measuring the absorbance value V at 260nm of the enzymolysis buffer solution of the nuclease obtained by using a spectrophotometer and the primary extract RNC obtained in the step twoEVolume of primary carrier RNC (m L), CEConcentration of nuclease (U/ml);
and step five, adding an enzyme digestion reaction stopping reagent (EGTA stop solution with the final concentration of 40 mM) into the product obtained in the step four, and then extracting RNA in the solution by using Trizol (Invitrogen company) according to an instruction manual to obtain the RFPs.
Example 7A method of obtaining animal tissue RFPs Using a molecular Sieve spin column
This example describes a method for obtaining animal tissue RFPs using a molecular sieve spin column.
Firstly, preparing a pretreated molecular sieve centrifugal column, wherein the preparation method comprises the steps of restoring a molecular sieve centrifugal column (MicroSpin S400 HR-GE or the same condition) to room temperature (10-35 ℃), adding a molecular sieve centrifugal column balance buffer solution with the concentration of 4m L and without RNase, carrying out column balance, vertically placing the column in a refrigerator with the temperature of 4 ℃, carrying out overnight settlement or settlement for 15 hours, then centrifuging at the centrifugal speed of 500g for 2 minutes, and removing liquid in the column to obtain the pretreated molecular sieve centrifugal column;
then, starting to obtain animal tissue RFPs by using a molecular sieve centrifugal column method, which comprises the following steps:
step one, animal tissue pretreatment: dissecting a Balb/c mouse with the age of 4 weeks, collecting the kidney of the mouse, quickly cleaning blood by adopting a PBS (phosphate buffer solution), quickly freezing for 4min after putting into liquid nitrogen, grinding the frozen animal tissue to powder with the granularity of 8 meshes to obtain ground animal tissue powder, then adding a cell lysate (the volume of which is 10 times of the ground animal tissue powder), and cracking for 25min at the temperature of 25 ℃ to obtain a pretreated substance;
step two, placing the pretreated substance obtained in the step one in a precooled 4 ℃ centrifugal machine, centrifuging for 4min at the rotating speed of 20000 × g to remove large fragments, sucking the supernatant, continuing to place the supernatant in the centrifugal machine, centrifuging for 12min at the rotating speed of 20000 × g to remove small fragments, sucking the supernatant, transferring the supernatant into a clean sterile EP tube, collecting the supernatant, and then dropwise adding the supernatant into a pretreated molecular sieve centrifugal column to obtain the treated molecular sieve centrifugal column;
step three, centrifuging the treated molecular sieve centrifugal column at the temperature of 3 ℃, wherein the centrifugation speed is 600 × g, centrifuging for 2min, and collecting filtrate to obtain a primary extract RNC;
step four, adding nuclease into the primary extract RNC for enzyme digestion, wherein the enzyme digestion temperature is 25 ℃, and the reaction time is 20 min; the addition volume of the nuclease V = (A260)RNC－A260buffer）×VE×800／VE，A260bufferAnd A260RNCRespectively measuring the absorbance value V at 260nm of the enzymolysis buffer solution of the nuclease obtained by using a spectrophotometer and the primary extract RNC obtained in the step twoEVolume of primary carrier RNC (m L), CEConcentration of nuclease (U/ml);
and step five, adding an enzyme digestion reaction stopping reagent (EGTA stop solution with the final concentration of 45 mM) into the product obtained in the step four, and then extracting RNA in the solution by using Trizol (Invitrogen company) according to an instruction manual to obtain the RFPs.
Example 8 measurement and verification of RFPs
The RFPs prepared in examples 5-7 and the RFPs obtained by the comparative example are subjected to identification and sequencing in examples 3 and 4 respectively, and the results show that the sizes of the detected fragments of the RFPs prepared in examples 5-7 are all between 20 and 35bp, the main fragments are concentrated at about 28 and 29bp, the size characteristics of the RFPs are met, and the correct extraction is proved. The sequencing experiment results show that the ribosome occupation ratio of the obtained RFPs is obviously reduced, the CDS region mapping rate is obviously higher than that of a control group, the number of identified genes is also larger than that of the control group, and the reaction time and the loss cost are obviously reduced.
In some embodiments of the present invention, the inventor finds that the method of the present invention can be used for not more than 3 hours only by extracting the RNC. The original ultra-high speed centrifugation method is adopted, including the ultra-high speed centrifugation method, the experiment time is about 5-12 hours, impurities are easily mixed, and the quality of the product obtained by the experiment result is influenced.
Example 9A method of obtaining animal tissue RFPs Using a molecular Sieve spin column
This example describes mainly a method of obtaining animal tissue RFPs using a molecular sieve column centrifuge, which differs from example 5 in that the animal tissue used is heart tissue of a 4-week-old Balb/c mouse.
Example 10A method of obtaining animal tissue RFPs Using a molecular Sieve spin column
This example describes mainly a method of obtaining animal tissue RFPs using a molecular sieve column centrifuge, which differs from example 6 in that the animal tissue used is heart tissue of a 4-week-old Balb/c mouse.
Example 11A method for obtaining animal tissue RFPs Using a molecular Sieve spin column
This example describes mainly a method of obtaining animal tissue RFPs using a molecular sieve column centrifuge, which differs from example 7 in that the animal tissue used is heart tissue of a 4-week-old Balb/c mouse.
Example 12A method of obtaining animal tissue RFPs Using a molecular Sieve spin column
This example describes mainly a method for obtaining animal tissue RFPs using a molecular sieve column centrifuge, which differs from example 2 in that the animal tissue used was heart tissue of 4-week-old Balb/c mice, and the extraction of RNC from the experimental group of the method described in example 1 was performed.
The inventor finds out in the research process that: in the process of extracting the RFPs from the animal heart tissue by the method of the invention described in the aforementioned embodiments 1-12, the components and volume of the added cell lysate play an important role in the finally obtained effective data. The limitations of the composition and volume ranges of cell lysates can result in technical effects including, but not limited to: effectively reduces the degradation of target RNA products and ensures the quality of final extracted products.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (10)

1. A method for obtaining animal tissue RFPs using a molecular sieve column centrifuge, the method comprising:
step one, animal tissue pretreatment: cleaning collected animal tissues with blood, freezing the animal tissues in liquid nitrogen for at least 2-5 min, grinding the frozen animal tissues into powder with the granularity of 5-10 meshes, adding cell lysate, and cracking the animal tissues at the temperature of 10-35 ℃ for 20-30 min to obtain a pretreated substance;
step two, centrifuging the pretreated substance obtained in the step one at a high speed, collecting supernatant, then dropwise adding the supernatant into a pretreated molecular sieve centrifugal column to obtain a treated molecular sieve centrifugal column, centrifuging the treated molecular sieve centrifugal column at a low temperature and a low speed, and collecting filtrate to obtain a primary extract RNC;
adding nuclease into the primary extract RNC for enzyme digestion, wherein the enzyme digestion temperature is 20-30 ℃, and the reaction time is 10-60 min; the addition volume of the nuclease V = (A260)RNC－A260buffer）×VE×800／VE，A260bufferAnd A260RNCAre respectively made asMeasuring the enzymolysis buffer solution of the nuclease obtained by the step two and the light absorption value V of the primary extract RNC obtained by the step two at 260nm by using a spectrophotometerEVolume of primary carrier RNC (m L), CEConcentration of nuclease (U/ml);
and step four, adding an RNA extraction reagent into the product obtained in the step three to obtain RFPs.
2. The method according to claim 1, wherein the volume of the cell lysate added in step one is 3 to 20 times the volume of the frozen animal tissue, and the cell lysate comprises 20mM potassium dihydrogen phosphate (pH7.4) and 15mM MgCl2200mM KCl, 2mM DTT, 100ug/m L cycloheximide and 1% (v/v) Triton X-100.
3. The method of claim 1,
the temperature of the high-speed centrifugation in the second step is 0-5 ℃, the speed of the high-speed centrifugation in the second step is 13000-24000 g, and the time of the high-speed centrifugation in the second step is 3-30 min;
and the temperature of the low-temperature low-speed centrifugation in the second step is 0-5 ℃, the speed of the low-temperature low-speed centrifugation in the second step is 300-800 g, and the time of the low-temperature low-speed centrifugation in the second step is 1-3 min.
4. The method of claim 1, wherein said pretreated molecular sieve spin column of step two comprises a pretreated MicroSpin S400 HR-GE molecular sieve spin column; the fourth step also comprises: and (4) adding an enzyme digestion reaction stopping reagent into the product obtained in the step three.
5. The method of claim 1, wherein the Nuclease comprises Micrococcus Nuclease (Micrococcus Nuclease) and the RNA extraction reagent comprises Trizol.
6. The method of claim 1, wherein the pretreated molecular sieve column is prepared by adjusting the temperature of the molecular sieve column to 10-35 ℃, adding 2.5-6 m L RNase-free molecular sieve column balance buffer solution to perform column balance, standing at 2-6 ℃ for 12-18h, centrifuging at 300-800 g for 1-5 min, and removing the liquid in the column to obtain the pretreated molecular sieve column.
7. The method of claim 6, wherein said high speed centrifugation of step two is performed at least twice, and said molecular sieve spin column equilibration buffer comprises: 20mM monopotassium phosphate (pH7.4), 15mM MgCl2200mM KCl, sterile water without RNase and sterile instruments are not needed when the molecular sieve centrifugal column balance buffer solution is prepared, and the molecular sieve centrifugal column balance buffer solution is prepared without sterile treatment.
8. The method of claim 1, wherein the sequence length of said RFPs is no more than 40 bp.
9. A nucleic acid prepared according to the method of any one of claims 1 to 8.
10. Use of a nucleic acid according to claim 9 in the biological, medical or chemical field.
CN202010256695.5A 2020-04-02 2020-04-02 Method for obtaining animal tissue RFPs by using molecular sieve centrifugal column Pending CN111440787A (en)

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