CN110904192A - Ultra-micro RNA methylation m6A detection method and application thereof - Google Patents
Ultra-micro RNA methylation m6A detection method and application thereof Download PDFInfo
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Abstract
The invention provides a construction method and application of an ultramicro RNA methylation m6A detection library. Compared with the existing report, the invention is a method specially aiming at detecting m6A by using RNA with lower initial quantity; the method has the advantages of simple experimental process, complete RNA in the whole process, high library purity and good quality, is particularly suitable for the condition of small initial sample amount, realizes high-efficiency connection, finally obtains a library with high enough concentration, completes on-machine sequencing and subsequent sequencing related application, and effectively realizes the MeRIP-seq of single cells, exosomes or precious clinical samples to be applied to the fields of diagnosis, treatment, research and development and the like.
Description
The application claims priority of Chinese patent application with application number of 201811621335.X and title of "a detection method for ultra-trace RNA methylation m6A and application thereof" filed in Chinese patent office in 2018, 12, 28, and the entire content of the application is incorporated by reference.
Technical Field
The invention relates to the field of high-throughput sequencing and library construction, in particular to a detection method for ultramicro RNA methylation m6A and application thereof.
Background
Recent studies have found that Methylation at the 6 th nitrogen atom of the RNA base, also known as m6A RNA modification, is highly conserved, widely occurs in most eukaryotic (e.g., yeast, plant, fruit fly, mammalian, etc.) and viral RNAs, and plays a key regulatory role in post-transcriptional RNA processing and metabolism [ Methylation modifications in eukaryotic gene rna.j t genes.2014; 41:21-3.].
At present, the technology for detecting the modification of RNA m6A is mainly m6A RNA immunoprecipitation combined with high-throughput sequencing (MeRIP-seq). The main process of the technology is as follows:
1) isolation and purification of total RNA: generally, a kit for isolating and purifying total RNA of a specimen, such as a kit for RNA extraction and purification by QIAGEN, Takara, etc., is used, which is based on a column method. The method has the advantages of simple operation, high purity of the obtained RNA, good integrity and high yield; because EDTA and salts influence subsequent interruption and DNA influences the specific recognition of the m6A antibody on RNA, the total RNA should avoid the pollution of EDTA, salts and DNA;
2) disruption of RNA: total RNA was broken into 100nt fragments by chemical means (metal ion induction) and briefly, EDTA was added immediately after incubation at 94 ℃ for 8-12min and transferred to ice. Incubation time and temperature, residual EDTA and salts, and RNA concentration are key influencing factors of RNA interruption efficiency;
3) and (3) immunoprecipitation: performing an immunoprecipitation reaction with the m6A binding site of RNA using the m6A antibody;
4) eluting RNA;
5) and (3) quality detection: qPCR detects the reliability of the immunoprecipitation experiment;
6) preparing a library; preparing a library by using mRNA-seq or TruSeq;
7) sequencing;
8) and (4) bioinformatics analysis.
However, the MeRIP-seq technology still requires 300-500. mu.g of total RNA. This greatly limits the application of m6A modification detection technology to clinical and basic research. Since in most cases it is very difficult to obtain hundreds of micrograms of total RNA from a patient sample, and some samples are very precious. Although m6A is the most abundant modification in mammalian mRNA and plays a key regulatory role in RNA metabolism, the role of m6A hinders the profound understanding of human diseases, particularly the changes and dynamics of cancer, due to the lack of efficient analytical methods. Therefore, a m6A assay applicable to microrna samples would provide a solid foundation for understanding the m6A modification mechanism in human diseases, especially cancer.
Therefore, there is a need to provide a MeRIP-seq method suitable for use with ultra-low initial, i.e., trace, RNA samples.
Disclosure of Invention
The invention provides a detection method of ultramicro RNA methylation m6A and application thereof.
The above purpose of the invention is realized by the following technical scheme:
in a first aspect, the invention provides a method for constructing a detection library of ultramicro RNA methylation m6A, which comprises the following steps:
1) obtaining total RNA in a sample, breaking and purifying to obtain a product after fragmentation treatment;
2) adding the product obtained in the step 1) into a precipitation buffer solution containing an anti-m 6A-antibody, a protein A-magnetic bead and a protein G-magnetic bead, uniformly mixing and incubating overnight; magnetic separation, removing supernatant, adding 5 Xprecipitation buffer solution and RNase inhibitor, reacting for 1-3 hours, and washing for 2-3 times by using low-salt precipitation buffer solution; washing with high salt buffer solution for 2-3 times; eluting RNA by using an eluent to obtain a purified product;
3) adding the product obtained in the step 2) into a cDNA first strand synthesis system, synthesizing a first strand cDNA product, and performing PCR amplification to obtain an ultramicro RNA methylation m6A detection library.
Preferably, in the step 2), the components of the precipitation buffer include:
preferably, in the step 2), the low salt buffer comprises the following components:
preferably, in the step 2), the high salt buffer comprises the following components:
preferably, in the step 3), the first strand cDNA synthesis system comprises a reverse transcription primer and a TSO primer; wherein:
the reverse transcription primer can form a stem-loop structure, and the sequence of the reverse transcription primer comprises:
stem-loop-stem-poly (N) region, N being selected from any one of ATCGs;
the sequence of the TSO primer comprises:
a first linker sequence-a second linker sequence-a poly (G) region;
the TSO primer is connected to the 5 'end of the first strand cDNA, and the reverse transcription primer is connected to the 3' end of the first strand cDNA.
Preferably, in the step 3), the PCR amplification includes a first round of PCR amplification and a second round of PCR amplification, wherein amplification primers used in the first round of PCR system include the following primer sets:
a first primer group: the FR primer, the P7 RF primer,
wherein, the sequence of the FR comprises: a first linker sequence of a TSO primer; the sequence of P7 RF includes: reverse transcription of the loop-stem sequence of the primer;
in the second round of PCR amplification, the amplification primers adopted comprise the following primer groups:
and (2) primer group II: p5 FR primer, P7 RF primer,
wherein, the sequence of P5 FR includes: a first linker sequence and a second linker sequence of the TSO primer.
As would be known to those skilled in the art, the poly (g) region of the TSO primer includes: there are two ribo-guanosines (rG) and one LNA-modified guanosine (+ G).
Preferably, the sequences of the reverse transcription primer, the TSO primer, the FR primer, the P7 RF primer and the P5 FR primer are shown in Table 1.
In a second aspect, the invention provides a detection method for ultramicro RNA methylation m6A, which comprises the step of using the ultramicro RNA methylation m6A detection library obtained in the first aspect for high-throughput sequencing to obtain a detection result of ultramicro RNA methylation m 6A.
In a third aspect, the invention provides a kit for constructing a detection library of ultramicro RNA methylation m6A, which comprises the primers in Table 1.
Preferably, the kit for constructing the m6A test library including ultra-trace RNA methylation further includes a precipitation buffer, a low-salt buffer and a high-salt buffer described in the first aspect.
In a fourth aspect, the present invention provides a method for constructing a supermicro RNA methylation m6A test library according to the first aspect, a method for detecting supermicro RNA methylation m6A according to the second aspect, or a kit for constructing a supermicro RNA methylation m6A test library according to the third aspect, for detecting supermicro RNA methylation m 6A.
The invention has the following beneficial effects:
in conclusion, compared with the existing MeRIP-seq technology, the method is a method specially for detecting m6A by aiming at the RNA with lower initial quantity, has simple experimental process, complete RNA in the whole process, high library purity and good quality, is particularly suitable for the condition of small initial sample quantity, realizes high-efficiency connection, finally obtains a library with high enough concentration, completes the application related to on-machine sequencing and subsequent sequencing, and effectively realizes the MeRIP-seq of single cells, exosomes or precious clinical samples to be applied to the fields of diagnosis, treatment, research and development and the like.
Drawings
FIG. 1 is a schematic diagram of a detection process of ultra-trace RNA methylation m6A according to an embodiment of the present invention;
FIG. 2 is a distribution diagram of the methylation of ultramicro RNA m6A Peak on RNA structure provided by the embodiment of the invention;
FIG. 3 is a graph showing the results of analysis of motif modified with ultra-trace RNA methylation m6A according to an embodiment of the present invention.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available. The experimental method not specified for the specific conditions is usually carried out under the conventional conditions or the conditions recommended by the manufacturer.
In an embodiment of the present invention, the present invention provides a detection method of ultramicro RNA methylation m6A and applications thereof, in combination with the schematic diagram of the detection process of ultramicro RNA methylation m6A shown in fig. 1.
The invention provides a detection method of ultramicro RNA methylation m6A, which adopts the detection library construction kit of the ultramicro RNA methylation m6A provided by the invention, and the method comprises but is not limited to one or more of the following steps:
1. trizol method for extracting total RNA of test cell sample
RNA disruption and purification recovery
2.1 starting with 20. mu.g total RNA, the following system was used:
2.2 the reaction was carried out according to the following procedure, hot lid 105 ℃:
2.3 purification of the cleavage product with RNA Clean & Concentrator-5 kit (ZymoResearch, Cat No: R1015)
3. Immunoprecipitation
3.1 the following buffers were prepared:
1) precipitation buffer
2) Low salt precipitation buffer
3) High salt precipitation buffer
3.2 preparation of antibody + magnetic bead mixture: placing 10-30 μ L of protein A magnetic beads and 10-30 μ L of Lof protein G magnetic beads on a magnetic frame, discarding the supernatant, adding 500 μ L of precipitation buffer to wash the magnetic beads, repeating twice, re-suspending the magnetic beads with 500 μ L of precipitation buffer, adding 1-5 μ G of anti-m6A anti, mixing well, rotating at 4 deg.C overnight
3.3 step 3.2 antibody + magnetic bead mixture on magnetic frame, remove supernatant and wash twice with 500. mu.L precipitation buffer, add 100. mu.L 5 Xprecipitation buffer, 5. mu.L 20U/. mu.L RNase inhibitor, break purified total RNA, add water to 500. mu.L, rotate 2 hours at 4 deg.C
3.4 after the reaction, 1000. mu.L of low-salt precipitation buffer was added, mixed well and rotated at 4 ℃ for 10 minutes, and this was repeated once.
3.5 washing twice with low-salt precipitation buffer, adding 1000. mu.L of high-salt precipitation buffer, mixing, rotating at 4 ℃ for 10 min, and repeating once.
Extraction of RNA: RNA extraction by phenol chloroform method
5. Denaturation of template
5.1 preparation of the reaction System according to the following Table
5.2 mixing the tube walls, centrifuging for a short time, and placing on ice
5.3 the reaction was carried out according to the following procedure, hot lid 105 ℃:
6. first Strand cDNA Synthesis
6.1 the reaction system is formulated as follows:
6.2 after the preparation, flicking the tube wall, mixing uniformly, and centrifuging for a short time to collect the sample.
6.3 the reaction was carried out according to the following procedure:
7. first round PCR amplification
7.1 the reaction system is configured according to the following table:
7.2 after the preparation, shaking and mixing uniformly, and centrifuging for a short time to collect samples.
7.3 the reaction was carried out according to the following procedure:
7.4 first round PCR products were purified with 1 Xmagnetic beads and eluted with 12. mu.L water.
8. Second round of PCR amplification
8.1 the reaction system is configured according to the following table:
8.2 after the preparation, shaking and mixing uniformly, and centrifuging for a short time to collect samples.
8.3 the reaction was carried out according to the following procedure:
8.4 second round PCR products were purified with 1 Xmagnetic beads and eluted with 23. mu.L water.
Qsep100 full-automatic nucleic acid protein analysis system for detecting size of main peak of library
9. The sequences of the primers used in the above steps are shown in Table 1:
10. the index sequence used in the technical scheme provided by the invention is shown in table 2:
concrete experimental results
1. Genome alignment
After the quality control of the sequence data obtained by sequencing, HISAT2 software is adopted[1]Comparing with the genome of a human species, wherein the genome comparison rate is a standard for measuring the data qualityMethod initial amount (20ug) (TEST _ IP) genome alignment>90% unique position alignment ratio>80%, the alignment data reached a level consistent with the conventional method (control _ IP) of high initial volume (300 ug).
TABLE 1 genome alignment data statistics
AllReads: the total number of sequences involved in genome alignment, i.e.the number of sequencing sequences after quality control
Unmap: number of sequences not aligned with genome
Applying: the number of sequencing sequences which can be aligned to the genome in the total reference alignment sequence
Mapping Rate: genome alignment, i.e., the ratio of the sequenced sequences to the total sequenced filtered sequences
Unique Mapped: the number of sequences in the total of the sequences involved in the alignment that can have only one unique alignment position on the genome
Unique Mapped Rate: unique position alignment ratio, i.e., the ratio of the unique position aligned sequence to the sequence filtered from sequencing
Control-IP data is from the conventional m6A library building method reference: a549 MeRIP-seq data from published paper: Lin S, Choe J, Du P, Triboulet R, growth RI. the m (6) amino transferase METTL3 proteins transformation in Human Cancer cells. MolCell.2016; 62:335-345.
M6A methylation-rich region search (Peak Calling)
The task after alignment of Reads obtained from MeRIP-seq to the genome is to find the enrichment of these short sequences in the genome, also known as Peak Calling. For Peak Calling, the software used for this analysis is exoMePeak [2 ]. Peak Calling is an important step in analyzing the level of m6A and is another criterion for verifying the quality of data.
A meta gene plot was generated from the identified m6A Peak, which reflects the distribution of Peak on the RNA structure, and the results are shown in FIG. 2, which shows that m6A modifications are mainly distributed near the stop-coding region, and the plot conforms to the structural distribution characteristics of m6A modifications.
The number of peaks identified by the method is greater than 10000, some samples even exceed 20000, and m6A modification of mRNA is mainly distributed at the coding termination sites, and the results are consistent with the reports of mainstream literature and even better.
Motif analysis
The specific base sequence for binding to proteins is motif, we use HOMER
(http:// homer. ucsd. edu/homer/ngs/peakmotifs. html.) software mol tif analysis was performed on Peaks. A typical motif modified with m6A is "GGAC".
The Motif analysis result is shown in fig. 3, the Motif analysis result shows that the sample has obvious 'GGAC' enrichment, the graph conforms to the Motif characteristic modified by m6A, and the specificity of capturing m6A modification by the method is strong.
The embodiment provided by the technical scheme of the invention has the following beneficial effects:
1) lower initial amount of sample. The conventional MeRIP-seq requires a minimum of 300. mu.g of total RNA, and the initial amount of the sample can be reduced to 500 ng using the present invention, which is equivalent to one six hundredths of the conventional initial amount. Particularly provides a high-efficiency RNA m6A analysis method for samples which are difficult to obtain clinically or extremely precious trace samples.
2) The modified MeRIP experiment, a special library building method and a bioinformatics analysis method are used for enabling the MeRIP peak to be detected in the trace RNA sample to be the most and the FDR to be less than 0.05.
Student's analysis reference:
[1]Kim D,Langmead B,Salzberg S L.HISAT:a fast spliced aligner with lowmemory requirements[J].Nature methods,2015,12(4):357.
[2]Meng J,Lu Z,Liu H,et al.A protocol for RNA methylation differentialanalysis with MeRIP-Seq data and exomePeak R/Bioconductor package[J].Methods,2014,69(3):274-281.
[3]Heinz S,Benner C,Spann N,et al.Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required formacrophage and B cell identities[J].Molecular cell,2010,38(4):576-5
in conclusion, the technical scheme provided by the invention can realize the method for detecting m6A by using ultralow initial amount of RNA, has the advantages of simple experimental process, complete RNA in the whole process, high library purity and good quality, is particularly suitable for the condition of small initial sample amount, realizes high-efficiency connection, finally obtains a library with high enough concentration, completes the related application of on-machine sequencing and subsequent sequencing, effectively realizes the application of MeRIP-seq of single cells, exosomes or precious clinical samples in the fields of diagnosis, treatment, research and development and the like, and is an ideal choice for detecting the ultramicro RNA methylation m 6A.
It should be noted that, for those skilled in the art, without departing from the principle of the embodiments of the present invention, several improvements and modifications can be made, and these improvements and modifications are also considered to be within the scope of the embodiments of the present invention.
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Claims (10)
1. A construction method of a detection library of ultramicro RNA methylation m6A comprises the following steps:
1) obtaining total RNA in a sample, breaking and purifying to obtain a product after fragmentation treatment;
2) adding the product obtained in the step 1) into a precipitation buffer solution containing an anti-m 6A-antibody, a protein A-magnetic bead and a protein G-magnetic bead, uniformly mixing and incubating overnight; magnetic separation, removing supernatant, adding 5 Xprecipitation buffer solution and RNase inhibitor, reacting for 1-3 hours, and washing for 2-3 times by using low-salt precipitation buffer solution; washing with high salt buffer solution for 2-3 times; extracting RNA by using phenol chloroform lysate to obtain a purified product;
3) adding the product obtained in the step 2) into a cDNA first strand synthesis system, synthesizing a first strand cDNA product, and performing PCR amplification to obtain an ultramicro RNA methylation m6A detection library.
2. The method for constructing a library for the detection of ultramicro RNA methylation m6A according to claim 1, wherein the components of the precipitation buffer in the step 2) comprise:
3. the method for constructing a library for the detection of ultramicro RNA methylation m6A according to claim 1, wherein in the step 2), the components of the low-salt buffer comprise:
4. the method for constructing a library for the detection of ultramicro RNA methylation m6A according to claim 1, wherein in the step 2), the components of the high-salt buffer comprise:
5. the method for constructing a detection library of ultra-trace RNA methylation m6A according to claim 1, wherein the first strand cDNA synthesis system in step 3) comprises a reverse transcription primer and a TSO primer; wherein:
the reverse transcription primer can form a stem-loop structure, and the sequence of the reverse transcription primer comprises:
stem-loop-stem-poly (N) region, N being selected from any one of ATCGs;
the sequence of the TSO primer comprises:
a first linker sequence-a second linker sequence-a poly (G) region;
the TSO primer is connected to the 5 'end of the first strand cDNA, and the reverse transcription primer is connected to the 3' end of the first strand cDNA.
6. The method for constructing the ultramicro RNA methylation m6A detection library according to claim 1, wherein the PCR amplification comprises a first round of PCR amplification and a second round of PCR amplification, wherein the amplification primers used in the first round of PCR system comprise the following primer sets:
a first primer group: the FR primer, the P7 RF primer,
wherein, the sequence of the FR comprises: a first linker sequence of a TSO primer; the sequence of P7 RF includes: reverse transcription of the loop-stem sequence of the primer;
in the second round of PCR amplification, the amplification primers adopted comprise the following primer groups:
and (2) primer group II: p5 FR primer, P7 RF primer,
wherein, the sequence of P5 FR includes: a first linker sequence and a second linker sequence of the TSO primer.
7. The method for detecting the ultramicro RNA methylation m6A, which is characterized in that the ultramicro RNA methylation m6A detection library of claim 1 is used for high-throughput sequencing to obtain the detection result of the ultramicro RNA methylation m 6A.
8. A kit for constructing a detection library of ultramicro RNA methylation m6A, which is characterized by comprising primers in a table 1.
9. The kit for constructing a ultramicro RNA methylation m6A assay library according to claim 8, wherein the kit for constructing a ultramicro RNA methylation m6A assay library further comprises the precipitation buffer according to claim 2, the low salt buffer according to claim 3 and the high salt buffer according to claim 4.
10. Use of the method for constructing a ultramicro RNA methylation m6A assay library according to claim 1, the method for detecting ultramicro RNA methylation m6A according to claim 7 or the kit for constructing an ultramicro RNA methylation m6A assay library according to claim 7 in the detection of ultramicro RNA methylation m 6A.
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| CN111560423A (en) * | 2020-06-05 | 2020-08-21 | 中山大学孙逸仙纪念医院 | Method for detecting RNA m6A with high-throughput and high-sensitivity single-base resolution and application thereof |
| CN111560423B (en) * | 2020-06-05 | 2023-11-24 | 中山大学孙逸仙纪念医院 | Method for detecting RNA m6A with high flux and high sensitivity and single base resolution and application thereof |
| CN112662749A (en) * | 2020-09-27 | 2021-04-16 | 中山大学 | Nucleic acid modification detection method with single base resolution |
| CN113061648A (en) * | 2021-03-24 | 2021-07-02 | 中山大学 | Method for constructing micro sample m6A modification detection library by aid of Tn5 transposase and application of method |
| CN113308514A (en) * | 2021-05-19 | 2021-08-27 | 武汉大学 | Construction method and kit for detection library of trace m6A and high-throughput detection method |
| CN113774121A (en) * | 2021-09-13 | 2021-12-10 | 武汉大学 | Low sample size m based on RNA connection label6A high throughput sequencing method |
| CN113774121B (en) * | 2021-09-13 | 2024-02-20 | 武汉大学 | Low sample size m based on RNA (ribonucleic acid) connection tag 6 A high throughput sequencing method |
| CN114250267A (en) * | 2021-12-13 | 2022-03-29 | 南京诺唯赞生物科技股份有限公司 | Method for constructing sequencing library of RNA (ribonucleic acid) containing modification sites |
| CN114292914A (en) * | 2021-12-28 | 2022-04-08 | 中山大学 | Visual RNA methylation detection method and application thereof |
| CN114292914B (en) * | 2021-12-28 | 2024-04-30 | 中山大学 | Visual RNA methylation detection method and application thereof |
| CN113999898A (en) * | 2021-12-31 | 2022-02-01 | 北京恩泽康泰生物科技有限公司 | method for detecting methylation sites of m6A RNA |
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