CN114410744A - Method for processing sample, nucleic acid extraction method and library thereof - Google Patents
Method for processing sample, nucleic acid extraction method and library thereof Download PDFInfo
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- CN114410744A CN114410744A CN202210101853.9A CN202210101853A CN114410744A CN 114410744 A CN114410744 A CN 114410744A CN 202210101853 A CN202210101853 A CN 202210101853A CN 114410744 A CN114410744 A CN 114410744A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
Abstract
A method for processing a sample and a nucleic acid extraction method and a library thereof are provided, wherein the method for processing the sample comprises the following steps: a centrifugation step, which comprises the steps of centrifuging a sample to be detected, and then removing supernatant to obtain a centrifuged precipitate; a rinsing step comprising resuspending the pellet with a buffer and then removing the supernatant to obtain a rinsed pellet; and a filtering step, which comprises diluting the precipitate with a buffer solution, filtering, and taking the liquid passing through the filter membrane, namely the filtered sample. The processing method remarkably improves the impurity removal efficiency of the sample, remarkably reduces the influence of impurities on subsequent sequencing, and effectively improves the quality of nucleic acid. In addition, a nucleic acid protective agent can be added to obtain a sample which can be transported and stored under the refrigeration condition of 2-8 ℃.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for processing a sample, a nucleic acid extraction method and a library thereof.
Background
Urine samples are complex in composition, with low cellular content and are not easy to store, making it difficult to obtain acceptable RNA for downstream molecular biological assays. However, more and more studies have shown that efficient analysis of RNA in urine epithelial cells can help in the diagnosis of genetic diseases. The clinical routine urine sample collection mode is not beneficial to subsequent nucleic acid extraction, RNA is extremely easy to degrade, and the sample transportation requirement is high; however, the collection and storage method of urine samples is the first problem to be solved in order to obtain RNA satisfying the analysis conditions.
In the prior art, substances such as exosomes and urine proteins are mainly enriched from a body fluid sample such as urine, and the like, and the prior art does not relate to the treatment of cells and the preservation of the sample.
Disclosure of Invention
According to a first aspect, in an embodiment, there is provided a method of processing a sample, comprising:
a centrifugation step, which comprises the steps of centrifuging a sample to be detected, and then removing supernatant to obtain a centrifuged precipitate;
a rinsing step comprising resuspending the pellet with a buffer and then removing the supernatant to obtain a rinsed pellet;
and a filtering step, which comprises diluting the precipitate with a buffer solution, filtering, and taking the liquid passing through the filter membrane, namely the filtered sample.
According to a second aspect, in an embodiment, there is provided the method of the first aspect, processing the obtained sample.
According to a third aspect, in an embodiment, there is provided a nucleic acid extraction method comprising:
extracting nucleic acid from the sample according to the second aspect.
According to the fourth aspect, in one embodiment, the nucleic acid extracted by the nucleic acid extraction method of the third aspect is provided.
According to a fifth aspect, in an embodiment, there is provided a sequencing library constructed from the nucleic acid of the fourth aspect.
According to the method for processing the sample, the nucleic acid extraction method and the library, the impurity removal efficiency of the sample is obviously improved, the influence of impurities on subsequent sequencing is obviously reduced, and the quality of the nucleic acid is effectively improved.
In one embodiment, a nucleic acid protective agent can be added to obtain a sample which can be transported and stored under the refrigeration condition of 2-8 ℃.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments. Wherein like elements in different embodiments are numbered with like associated elements. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances. In some instances, certain operations related to the present application have not been shown or described in detail in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that they may be fully understood from the description in the specification and the general knowledge in the art.
Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the method descriptions may be transposed or transposed in order, as will be apparent to one of ordinary skill in the art. Thus, the various sequences in the specification are for the purpose of clearly describing one embodiment only and are not meant to be necessarily order unless otherwise indicated where a certain order must be followed.
The numbering of the components as such, e.g., "first", "second", etc., is used herein only to distinguish the objects as described, and does not have any sequential or technical meaning.
Urine samples are complex in composition, with low cellular content and are not easy to store, making it difficult to obtain acceptable RNA for downstream molecular biological assays. However, more and more studies have shown that efficient analysis of RNA in urine cells can help in the diagnosis of genetic diseases. The clinical routine urine sample collection mode is not beneficial to subsequent nucleic acid extraction, RNA is extremely easy to degrade, and the sample transportation requirement is high; however, the collection and storage method of urine samples is the first problem to be solved in order to obtain RNA satisfying the analysis conditions.
At present, a better method is not available for extracting nucleic acid in a urine sample in high quality, a clinical laboratory generally extracts RNA by using a conventional Trizol method after the urine is simply centrifuged, the obtained RNA concentration can meet the requirement generally, but a stable RT-qPCR result cannot be obtained, the fragmentation degree of the RNA is too high, and RNAseq sequencing cannot be carried out.
According to a first aspect, in an embodiment, there is provided a method of processing a sample, comprising:
a centrifugation step, which comprises the steps of centrifuging a sample to be detected, and then removing supernatant to obtain a centrifuged precipitate;
a rinsing step comprising resuspending the pellet with a buffer and then removing the supernatant to obtain a rinsed pellet;
and a filtering step, which comprises diluting the precipitate with a buffer solution, filtering, and taking the liquid passing through the filter membrane, namely the filtered sample.
In one embodiment, the treated object of the present invention is mainly exfoliated epithelial cells in urine (including other body fluid samples such as amniotic fluid), and has an intact cell structure, which is different from the substances extracted in the prior art such as exosome and urine protein. Therefore, the enrichment target of the present invention is different, and the specific treatment method such as centrifugation is also different.
In one embodiment, the sample to be tested comprises a body fluid sample.
In one embodiment, the body fluid sample includes, but is not limited to, at least one of urine, amniotic fluid.
In one embodiment, the temperature during centrifugation is 2-8 ℃, and low-temperature centrifugation reduces temperature rise in the cavity caused by too long operation time of the centrifuge, so that cells are protected. Temperatures include, but are not limited to, 2 deg.C, 3 deg.C, 4 deg.C, 5 deg.C, 6 deg.C, 7 deg.C, 8 deg.C.
In one embodiment, the centrifugation is carried out at 1600rpm for 10 min.
In one embodiment, the buffer includes, but is not limited to, a Duchen phosphate buffer (also known as a DPBS solution).
In one embodiment, the pore size of the filter membrane may be 40 μm during filtration. The purpose of using a 40 μm filter is to allow cells to pass through the filter while removing macromolecular substances or impurities.
In one embodiment, the method further comprises centrifuging the filtered sample again, and obtaining the sediment as the centrifuged sample.
In one embodiment, the speed of re-centrifugation is 1600rpm for 10 min.
In one embodiment, after centrifugation again, the resulting pellet is mixed with a nucleic acid protecting agent to obtain a sample. The sample is a sample for nucleic acid extraction.
In one embodiment, the nucleic acid protecting agent includes, but is not limited to, RNAlater et al nucleic acid protecting agents.
In one embodiment, the sample to be tested comprises at least one of DNA and RNA. The enriched cells of the invention can be used for DNA extraction and RNA extraction.
According to a second aspect, in an embodiment, there is provided the method of the first aspect, processing the obtained sample.
According to a third aspect, in an embodiment, there is provided a nucleic acid extraction method comprising:
extracting nucleic acid from the sample according to the second aspect.
In one embodiment, the kit for nucleic acid extraction is commercially available, and the extraction method is performed with reference to the kit instructions.
In one embodiment, the nucleic acid comprises at least one of DNA, RNA.
In one embodiment, the nucleic acid is RNA.
According to the fourth aspect, in one embodiment, the nucleic acid extracted by the nucleic acid extraction method of the third aspect is provided.
According to a fifth aspect, in an embodiment, there is provided a sequencing library constructed from the nucleic acid of the fourth aspect.
In one embodiment, the kit required for library construction is commercially available and the construction method is performed with reference to kit instructions.
In one embodiment, the method provided by the present invention is as follows:
the urine of about 100mL is collected by using sterile consumables, and collection after a large amount of drinking water is avoided. Subpackaging into 2 sterile 50mL centrifuge tubes. The samples were immediately centrifuged at 4 ℃ and 1600rpm for 10 minutes. The whole procedure was performed on ice, the supernatant was removed with a pipette, a volume of about 1mL was left at the bottom of the tube, 10mL of 1 XDPBS was added to resuspend the pellet, and this was repeated once; adding 1mL of DPBS, gently pipetting the precipitate with a pipette and transferring the suspension to a new centrifuge tube through a 40 μm cell filter; centrifugation was carried out at 1600rpm for 10 minutes, the supernatant was removed, 1mL of RNAlater nucleic acid protectant was added, and the new suspension was transferred to a sterile enzyme-free cryopreservation tube.
The samples can be stored at 4 ℃ for 2 days and at-20 ℃ for a long time. The quality of the extracted nucleic acid has no obvious change when the Trizol method is adopted to extract the RNA.
Urine has complex components, and when the impurities are more, the extraction efficiency of nucleic acid is greatly influenced, and in one embodiment, the invention can obtain epithelial cells in urine with less impurities after the treatment of steps such as centrifugation, rinsing, filtration and the like.
In one embodiment, the RNAlater protective agent is added into the cell sediment, and the agent can effectively protect urine epithelial cells to maintain complete morphology at 4 ℃, reduce RNA degradation caused by RNase release, and is free from ultralow temperature transportation and storage, so that the method is more convenient for clinical adoption.
Example 1:
reagent: DPBS solution (i.e. duller's phosphate buffer and no calcium magnesium), RNAlater protectant.
Consumable material: sterile urine collection cups, sterile enzyme-free 50mL conical centrifuge tubes, 40 μm cell filters.
The instrument comprises the following steps: centrifuge, pipettor.
The experimental procedure was as follows:
1. the sterile consumables are used to collect about 100mL of fresh urine, avoiding collection after a large amount of drinking water.
2. Urine was aliquoted into 2 sterile 50mL centrifuge tubes.
3. The samples were immediately centrifuged at 4 ℃ and 1600rpm for 10 minutes. The low temperature is to protect the cells and the centrifugation is to enrich the cells.
4. The following operations were performed on ice: the supernatant was removed by pipette, leaving a volume of about 1mL at the bottom of the tube, 10mL of 1 XDPBS was added to resuspend the pellet, the supernatant was discarded, and the process repeated once. This step is intended to wash the cells.
5. Add 1mL of pre-cooled DPBS, gently pipette the pellet and transfer the suspension in 2 tubes through a 40 μm cell filter to 1 new tube, and remove the filtered liquid. The addition of pre-chilled DPBS serves to wash, dilute, and facilitate the next step of filtration. The filtration functions to remove macromolecules and impurities.
6. Centrifugation is carried out at 1600rpm for 10 minutes (the centrifugation is used for enriching cells), the supernatant is removed, 1mL of RNAlater nucleic acid protectant is added, and the new suspension is transferred to a sterile enzyme cryopreservation tube.
7. RNA from the treated sample was extracted by the Trizol method and purified using the RNA purification kit from Zymo, and the RNA purity was measured as a260/280 ═ 1.969, a260/230 ═ 2.064, RNA concentration was 20.64 ng/. mu.l, and RNA integrity index RIN was 5.8 using Nanodrop. RNA library construction can be performed using an RNA library construction kit for enucleated ribosomal RNA depending on the degree of integrity of the RNA and can be used for downstream RNAseq.
Example 2
Reagents, consumables, and instruments were the same as in example 1. Approximately 300mL of fresh urine was collected and aliquoted into 6 portions. The experimental procedure was as follows:
1. the sterile consumables are used to collect about 300mL of fresh urine, avoiding collection after a large amount of drinking water.
2. Urine was aliquoted into 6 sterile 50mL centrifuge tubes.
3. The samples were immediately centrifuged at 4 ℃ and 1600rpm for 10 minutes.
4. The following operations were performed on ice: the supernatant was removed by pipette, leaving a volume of about 1mL at the bottom of the tube, 10mL of 1 XDPBS was added to resuspend the pellet, the supernatant was discarded, and the process repeated once.
5. 1mL of pre-cooled DPBS was added, the pellet was gently pipetted and the suspension in the centrifuge tube was transferred through a 40 μm cell filter into 1 new centrifuge tube.
6. And (3) centrifuging at 1600rpm for 10 minutes, removing the supernatant, adding 1mL of RNAlater nucleic acid protective agent, and transferring the new suspension into a sterile enzyme cryopreservation tube, wherein the number is 1-6.
7. The storage conditions of samples 1-6 were as follows:
TABLE 1
| Sample numbering | Storage temperature (. degree.C.) | Storage time |
| 1 | 4 | Extracted immediately on the day of collection |
| 2 | 4 | 1 day |
| 3 | 4 | 2 days |
| 4 | -20 | 1 month |
| 5 | -20 | 3 months old |
| 6 | -20 | 6 months old |
8. Extracting RNA of the treated sample by using a Trizol method and purifying the RNA by using an RNA purification kit of Zymo, wherein the RNA quality test result is as follows:
TABLE 2
A260/280 is preferably between 1.7 and 2.0, A260/230 is preferably about 2.0, and A260/280 and A260/230 are used for representing the purity of the extracted RNA; as can be seen from Table 2, RNA extracted from the urine cells enriched in this example meets the aforementioned requirements. Furthermore, a higher RIN value indicates a better integrity of the extracted RNA. If urine is collected without the method of the embodiment, the RIN value of RNA is about 1-3 mostly.
As can be seen from table 2, the freshly collected urine samples are enriched in cells by the method of the present embodiment, and the samples are stored at 4 ℃ for 2 days, so that the quality of the extracted RNA is not significantly reduced, i.e., the purity and concentration of the RNA are basically unchanged from the samples extracted on the same day, and the RIN value is slightly reduced, but the requirements of downstream library construction are still met; the sample is stored for 6 months at the temperature of minus 20 ℃, the quality of the extracted RNA is not obviously reduced, namely the purity and the concentration of the RNA are not changed much compared with the sample extracted on the same day, the RIN value is reduced, and the requirement of downstream bank establishment is still met.
Comparative example 1
The method of this comparative example is as follows: urine samples from the same sources as in example 2 were collected, centrifuged at 1600rpm for 10 minutes, the supernatant was removed, 1mL of Trizol was added directly to the pellet and the pellet was gently blown and stored at-80 ℃ for 2 days for transportation under simulated dry ice conditions for 2 days, and RNA was obtained by the same nucleic acid extraction method as in example 1. RNA concentration was measured to be 7.96 ng/. mu.l using Nanodrop and RIN ═ 1.3. The RIN value is too low, which indicates that the RNA degradation is very serious and the downstream library building cannot be carried out.
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.
Claims (10)
1. A method of processing a sample, comprising:
a centrifugation step, which comprises the steps of centrifuging a sample to be detected, and then removing supernatant to obtain a centrifuged precipitate;
a rinsing step comprising resuspending the pellet with a buffer and then removing the supernatant to obtain a rinsed pellet;
and a filtering step, which comprises diluting the precipitate with a buffer solution, filtering, and taking the liquid passing through the filter membrane, namely the filtered sample.
2. The method of claim 1, wherein the test sample comprises a bodily fluid sample.
3. The method of claim 2, wherein the body fluid sample comprises at least one of urine and amniotic fluid.
4. The method of claim 1, wherein the temperature during centrifugation is 2 to 8 ℃;
preferably, the rotation speed is 1600rpm and the time is 10min during centrifugation;
preferably, the buffer comprises a Duchenne phosphate buffer;
preferably, the filter membrane has a pore size of 40 μm upon filtration.
5. The method of claim 1, further comprising re-centrifuging the filtered sample to obtain a pellet as the centrifuged sample;
preferably, the rotation speed of the re-centrifugation is 1600rpm, and the time is 10 min;
preferably, after centrifuging again, mixing the obtained precipitate with a nucleic acid protective agent to obtain a sample;
preferably, the nucleic acid protecting agent comprises RNAlater;
preferably, the sample to be tested contains at least one of DNA and RNA.
6. A sample treated according to any one of claims 1 to 5.
7. A method for extracting nucleic acid, comprising: nucleic acid extracted from the sample of claim 6.
8. The method for extracting nucleic acid according to claim 7, wherein the nucleic acid comprises at least one of DNA and RNA.
9. The nucleic acid extracted by the method according to any one of claims 7 to 8.
10. A sequencing library constructed from the nucleic acid of claim 9.
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