Deoxyribose nucleic acid (DNA) and ribonucleic acid (RNA) methylation analysis method for single cell or few cells
Single-cell DNA and RNA methylation analysis was resolved by directly extracting DNA and RNA from single cells, eliminating cell lysis and DNA/RNA extraction steps, and using methylation-sensitive endonuclease and bisulfite treatments The problems of DNA sample loss and complex operation steps in the process have achieved high sensitivity and stable analysis results.
Patent Information
- Application Number
- CN202310992803.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-08
- Publication Date
- 2025-02-18
AI Technical Summary
The prior art has problems with DNA sample loss and complex operating steps in single-cell DNA and RNA methylation analysis, resulting in unstable analysis results and low detection sensitivity.
By extracting DNA and RNA directly from individual cells, eliminating cell lysis and DNA/RNA extraction steps, treating broken cells with temperature changes or protease digestion, and treating with methylation-sensitive endonuclease and bisulfite, reducing Background interference, achieving simultaneous analysis of DNA and RNA.
It significantly reduces the loss of DNA samples, improves the stability and detection sensitivity of the analysis results, simplifies the operation steps, and enhances the quality and quantity of DNA methylation analysis in individual cells.
Smart Images
Figure CN119464449A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine and digital medicine, and more specifically relates to a method for analyzing DNA and RNA methylation of a single cell or a small number of cells. Background Art
[0002] More than 90% of CpG sites in the human genome are methylated, but the methylation level of CpG islands is usually low, which generally does not significantly affect the protein binding to the promoter region of DNA to initiate transcription, thereby affecting gene expression. However, if the CpG island of this DNA is significantly methylated, the promoter protein cannot bind to the promoter region of DNA, which will silence transcription and cause the gene to not express RNA and protein.
[0003] CpG islands generally refer to DNA sequences with a length greater than 200bp where the G+C content is greater than 50%. There are approximately 25,000 CpG islands in the human genome, of which 75% are less than 850bp long. Approximately 50% of CpG islands are located in gene promoter regions, while the other 25% are located within genes, usually acting as alternative promoters. Approximately 60-70% of genes have CpG islands in their promoter regions. Most CpG islands are structurally unmethylated, and in somatic tissues, only 10% of CpG islands are methylated, most of which are located in intergenic and intragenic regions.
[0004] The functions of methylation mainly include gene regulation: methylation of the promoter region will silence gene expression; developmental regulation: it can be inherited during cell division to establish differences in cell and tissue differentiation; DNA replication initiation and error correction positioning; changing the conformation of the DNA region, affecting the interaction between protein and DNA, and inhibiting the binding efficiency of transcription factors and promoter region DNA. Hypermethylation includes continuous methylation leading to the inactivation of one X chromosome in women; chromosome imprinting: gene expression activity is limited to the gene version from one of the parents; inhibition of transposons: in CpG-dense regions, DNA methylation is a strong transcriptional repressor. Although DNA methylation does not have the flexibility required for fine-tuning gene regulation, its stability is very suitable for ensuring the permanent silencing of transposable elements. This methylation will keep some genes permanently silent in almost all tissues. DNA methylation is easily affected by the environment. The degree of DNA methylation in tissues directly affects the expression level of related genes, and the significance of DNA methylation in individual cells may be more significant.
[0005] The main process of single-cell DNA methylation analysis includes single cell lysis, DNA extraction and purification, DNA conversion and amplification analysis, and sequencing to obtain the DNA methylation status in a single cell. DNA analysis of a single cell will lose a lot of DNA samples after cell lysis, DNA extraction and purification, and conversion. Even if DNA is not lost, the amount of DNA in a single cell is very small (on average, a cell has only 6pg of DNA and 10pg of RNA), so the amount of DNA is the main factor limiting the effective analysis of DNA. Summary of the invention
[0006] In view of the above-mentioned problems, the purpose of the present invention is to provide a method for DNA and RNA methylation analysis of a single cell or a small number of cells, which reduces the operating steps of single-cell DNA and RNA analysis, significantly reduces the loss of DNA, and effectively utilizes the DNA and RNA in a single cell. The method is easy to implement, the results are stable, the detection sensitivity is high, the information is more complete, the operation is simple, and the limitation of the single-cell DNA amount in DNA methylation analysis is greatly improved.
[0007] In order to achieve the above-mentioned purpose, the present invention adopts the following technical measures:
[0008] The overall idea of the present invention is as follows: when analyzing DNA methylation of a single cell, the inventors found that the analysis of DNA of a single cell can avoid the steps of cell lysis, DNA separation and extraction, and purification. The cells can be directly treated by temperature changes to break the cells, so that cellular components such as DNA / RNA are effectively released for single-cell multi-omics analysis, which greatly reduces the loss of samples such as DNA of a single cell; methylation-sensitive endonucleases are used to cut DNA molecules, which can effectively reduce the background level of DNA methylation analysis when analyzing DNA methylation. At the same time, the DNA and RNA of the same single cell are analyzed together, which is equivalent to increasing the sample size several times, making the subsequent analysis reaction more effective, accurate and reproducible.
[0009] A single cell or a small number of cells DNA and RNA methylation analysis method, starting directly from a single cell or a small number of target cells, the steps are:
[0010] A. Directly use a single or a small number of mammalian cells;
[0011] B. Use high temperature or protease digestion to break up target cells; it has the advantages of breaking up cells without changing the sample volume or with very little change in volume;
[0012] C. Treat the DNA in the sample of step B above with a methylation-sensitive endonuclease; this helps reduce background interference;
[0013] E. Treating the sample in step C above with bisulfite converts the unmethylated cytosine (C) in its DNA and RNA into uracil (U), which becomes thymine (T) in the subsequent DNA replication, while the methylated cytosine (C), adenine (A), guanine (G) and uracil (U) remain unchanged; reducing the steps of DNA separation, extraction and purification from a single cell or a small number of cells, greatly reducing the possibility of loss of single cell components including DNA, RNA and other component samples;
[0014] F. Using the sample from step E above, synthesize cDNA from its RNA portion;
[0015] G. Analyze the methylation results of DNA and RNA from single cells in step G above using gene amplification / library construction / Southern Blot analysis and gene sequencing.
[0016] Optionally, the single or small amount of mammalian cells in step A include fresh, living or reagent-fixed, dead target cells.
[0017] Optionally, the high temperature or protease digestion treatment in step B includes high temperature treatment at 50°C-100°C, or various protein digestion enzymes including Proteinase K, so as to effectively break up the target cells while minimizing the damage or loss of their DNA and RNA.
[0018] Optionally, the use of a methylation-sensitive endonuclease to treat the DNA in the sample in step C includes using one endonuclease (such as: the product on the market: Not I recognizes GC↓GGCCGC) or multiple endonucleases (two or more such as Not I (GC↓GGCCGC), SalI (G↓TCGAC), Nae I (GCC↓GGC)), which can cut unmethylated DNA sequences but not methylated sequences.
[0019] Optionally, the bisulfite conversion in step E includes converting unmethylated cytosine (C) in DNA and RNA into uracil (U), which becomes thymine (T) in subsequent DNA replication.
[0020] Optionally, the cDNA synthesis in step F includes synthesizing cDNA from the RNA sample that has been subjected to bisulfite conversion treatment in the sample of step E, which provides materials for subsequent RNA methylation analysis, and simultaneously uses the DNA and RNA in a single cell, which is beneficial for obtaining more DNA and RNA templates in a single cell, thereby improving the sample nucleic acid quantity and analysis effect.
[0021] Optionally, the DNA and RNA methylation interpretation method in step G includes using the converted DNA and RNA templates to perform Southern Blot methylation-sensitive enzyme digestion nucleic acid analysis, amplification, library construction and sequencing analysis of various methylation methods.
[0022] Through the above technical measures: the following technical problems have been solved:
[0023] The present invention directly starts from a single cell or a small number of cells, omitting the steps of cell lysis, DNA / RNA extraction and purification, reducing the loss of DNA samples, analyzing the methylation of RNA and DNA simultaneously, automatically increasing the amount of sample nucleic acid components, confirming the RNA source through the label of the special sequence of the cDNA synthesis primer, making the analysis step more stable and highly sensitive, using DNA methylation sensitive endonuclease treatment to reduce the background interference of non-methylated DNA, greatly improving the quality and quantity of DNA methylation analysis of a single cell or a small number of cells, simplifying the operation procedure, and being practical.
[0024] The technical advancement of the present invention is that the fidelity and integrity of DNA samples of single cells and a small number of cells are maintained, so that the detection and analysis results are closer to the authenticity, accuracy and high detection sensitivity of complete single cells.
[0025] The main difference between the technical solution of the present invention and the prior art is that the operation steps such as cell lysis, DNA / RNA extraction and purification are reduced, which is equivalent to reducing the DNA loss of a single cell, increasing the double analysis of DNA and RNA, and reducing the interference of non-methylated DNA background.
[0026] Compared with the prior art, the present invention has the following advantages and effects:
[0027] 1. The cell lysis step of a single cell is omitted, which not only reduces the volume of the single-cell reaction, but also reduces the loss of DNA samples.
[0028] 2. It eliminates the need for separation and extraction of DNA samples, thus improving the fidelity of single-cell DNA analysis.
[0029] 3. Add DNA methylation-sensitive endonuclease treatment to reduce the background interference of non-methylated DNA in the analysis.
[0030] 4. The DNA and RNA of a single cell or a small number of cells are analyzed simultaneously, which increases the stability of the sample amount and the stability of the analysis results during the detection and analysis, and improves the sensitivity and information content of the detection (dual results of DNA methylation and RNA methylation). BRIEF DESCRIPTION OF THE DRAWINGS
[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.
[0032] Figure 1 It is the methylation sequencing map of the whole DNA and whole RNA of a single cell;
[0033] Figure 2 Schematic diagram for construction of single cell DNA / RNA amplification library;
[0034] Figure 3 Schematic diagram of single-cell methylation detection of RASSF1A and SHOX2 genes;
[0035] Figure 4 Schematic diagram of methylation detection of known genes in single-cell DNA;
[0036] Figure 5 Schematic diagram of methylation detection of known genes in single-cell RNA. DETAILED DESCRIPTION
[0037] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.
[0038] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.
[0039] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.
[0040] The DNA and RNA methylation analysis method and operation of a single cell or a small number of cells of the present application will be described in detail below in combination with examples, control examples and experimental data.
[0041] Example 1: Methylation sequencing analysis of whole DNA and whole RNA of a single cell, such as Figure 1 As shown:
[0042] 1. Enrichment and isolation of circulating tumor cells from the peripheral blood of breast cancer patients: Use a magnetic capture device to separate and extract circulating tumor cells from 5 ml of blood samples.
[0043] 2. Use markers such as DAPI (4',6-diamidino-2-phenylindole), CK / EpCAM (keratin / epithelial cell adhesion molecule), CD45 (leukocyte common antigen), Vimentin / Twist1 (vimentin / twist-associated protein 1), PD-L1 (programmed death ligand-1) to identify and analyze the above tumor cells, and obtain single, identified, analyzed and confirmed target cells.
[0044] 3. Place the single cells identified in step 2 above directly into the PCR reaction tubes.
[0045] 4. Add proteinase K and treat at 65°C for 10 minutes.
[0046] 5. Treat at 75°C for 10 minutes to inactivate the above-mentioned protease.
[0047] 6. Treat the sample from step 5 above with bisulfite at 70°C for 10 minutes and 60°C for 60 minutes, repeat twice.
[0048] 7. Perform conventional RT-PCR reaction using a PCR instrument.
[0049] 8. Single cell gene library construction and sequencing, such as Figure 2 shown.
[0050] 9. Analyze gene sequences and obtain DNA and RNA methylation results of single cells.
[0051] like Figure 3 As shown, the results of DNA / RNA broad-spectrum methylation analysis of single cells were successfully obtained, and methylation of genes such as RASSF1A and SHOX2 was detected.
[0052] Example 2: Single-cell single-gene DNA methylation detection
[0053] 1. Enrichment and isolation of circulating tumor cells from the peripheral blood of breast cancer patients: Use a magnetic capture device to separate and extract circulating tumor cells from 5 ml of blood samples.
[0054] 2. The above tumor cells are identified and analyzed using tumor cell protein markers (such as DAPI, CK, EpCAM, CD45, etc.) to obtain single, identified, analyzed and confirmed circulating tumor cells.
[0055] 3. Place the single cells from step 2 above directly into the PCR reaction tubes.
[0056] 4. Add proteinase K and treat at 65°C for 10 minutes.
[0057] 5. Treat at 75°C for 10 minutes to inactivate the above-mentioned protease.
[0058] 6. Directly treat the single cell lysate with NotI DNA methylation sensitive endonuclease for 30 minutes.
[0059] 7. Treat at 75°C for 10 minutes to inactivate the endonuclease.
[0060] 8. Using a PCR instrument, directly perform conventional PCR pre-amplification for 30 cycles.
[0061] 9. Perform a second PCR amplification reaction.
[0062] 10. Detect the length of PCR product. Figure 4 As shown, the methylated ones are longer and the unmethylated ones are shorter.
[0063] In short, DNA methylation-sensitive endonucleases can be used to directly detect DNA methylation at consistent sites. Methylation-sensitive enzyme treatment can also be performed in advance in single-cell whole DNA methylation sequencing, which can reduce background interference from unmethylated DNA and improve the sensitivity of methylation detection.
[0064] Example 3: RNA methylation sequencing of known genes in single cells, such as Figure 5 As shown:
[0065] 1. Enrichment and isolation of circulating tumor cells from the peripheral blood of breast cancer patients: Use a magnetic capture device to separate and extract circulating tumor cells from 5 ml of blood samples.
[0066] 2. The above tumor cells are identified and analyzed using tumor cell protein markers (such as DAPI, CK, EpCAM, CD45, etc.) to obtain single, identified, analyzed and confirmed circulating tumor cells.
[0067] 3. Place the single cells identified in step 2 above directly into the PCR reaction tubes.
[0068] 4. Add proteinase K and treat at 65°C for 10 minutes.
[0069] 5. Treat at 75°C for 10 minutes to inactivate the above-mentioned protease.
[0070] 6. Treat the sample from step 5 with bisulfite at 70°C for 10 minutes, 60°C for 60 minutes, and repeat 3 times.
[0071] 7. Use a conventional PCR instrument to perform RT-PCR reaction.
[0072] The PCR products were sequenced and analyzed to detect the methylation composition, thus achieving the successful detection of complete single-cell RNA methylation.
Claims
1. A method for analyzing DNA and RNA methylation in a single cell or a small number of cells, characterized in that: The following steps are involved: A. Directly use a single or a small number of mammalian cells; B. Use high temperature or protease digestion to break up target cells; C. Treat the DNA in the sample of step B above with a methylation-sensitive endonuclease; E. Treat the sample in step C above with bisulfite to convert the unmethylated cytosine (C) in its DNA and RNA into uracil (U), which becomes thymine (T) in the subsequent DNA replication, while the methylated cytosine (C), adenine (A), guanine (G) and uracil (U) are not changed; F. Using the RNA portion of the sample from step E above to synthesize cDNA; G. Analyze the methylation results of DNA and RNA from single cells in step G above using gene amplification / library construction / Southern Blot analysis and gene sequencing.
2. The method for analyzing DNA and RNA methylation of a single cell or a small number of cells according to claim 1, characterized in that: The single or small amount of mammalian cells in step A include fresh, living or reagent-fixed, dead target cells.
3. The method for analyzing DNA and RNA methylation of a single cell or a small number of cells according to claim 1, characterized in that: The high temperature or protease digestion treatment in step B includes high temperature treatment at 50° C.-100° C., or various protein digestion enzymes including Proteinase K.
4. The method for analyzing DNA and RNA methylation in a single cell or a small number of cells according to claim 1, characterized in that: The use of a methylation-sensitive endonuclease to treat the DNA in the sample in step C includes using one endonuclease or multiple endonucleases, wherein the one endonuclease includes Not I that recognizes GC↓GGCCGC, and the multiple endonucleases include two or three of Not I (GC↓GGCCGC), Sal I (G↓TCGAC), and Nae I (GCC↓GGC), which can cut unmethylated DNA sequences but cannot cut methylated sequences.
5. The method for analyzing DNA and RNA methylation of a single cell or a small number of cells according to claim 1, characterized in that: The conversion of bisulfite in step E includes the conversion of unmethylated cytosine (C) in DNA and RNA into uracil (U), which becomes thymine (T) in the subsequent DNA replication.
6. The method for analyzing DNA and RNA methylation of a single cell or a small number of cells according to claim 1, characterized in that: The cDNA synthesis in step F includes synthesizing cDNA from the RNA sample that has been subjected to bisulfite conversion treatment in the sample in step E.
7. The method for analyzing DNA and RNA methylation in a single cell or a small number of cells according to claim 1, characterized in that: The DNA and RNA methylation interpretation method in step G includes using the converted DNA and RNA templates to perform Southern Blot methylation-sensitive enzyme digestion nucleic acid analysis, amplification, library construction and sequencing analysis of various methylation methods.