CN113122533A - Method for removing ribosome RNA in total RNA of animals - Google Patents
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Abstract
The invention discloses a method for removing ribosome RNA in total RNA of animals. The invention provides a method for removing rRNA in an animal total RNA sample, which sequentially comprises the following steps: (1) taking an animal total RNA sample, carrying out denaturation, and then carrying out reverse transcription by adopting a specific primer group; the specific primer group consists of a plurality of rRNA specific reverse transcription primers; (2) treating with RNase H enzyme; (3) performing DNase I treatment; (4) recovering the RNA. The method provided by the invention has the following advantages: (1) specifically removing rRNA from the total RNA sample of the animal, reserving other target RNA such as mRNA, LncRNA and the like, and being suitable for LncRNA sequencing; (2) the species tolerance is high, and the method is widely suitable for removing rRNA in total RNA samples of mammals, birds, reptiles, amphibians, fishes, insects and the like; (3) the operation is simple, the cost is low, and the method is suitable for the operation of batch automatic workstations.
Description
Technical Field
The invention relates to a method for removing ribosomal RNA (rRNA) in total RNA of animals.
Background
Transcriptome sequencing or RNA sequencing is a powerful tool that has wide application in life science research today. However, current RNA sequencing presents significant challenges. Ribosomal RNA, rRNA, the most abundant RNA in cells, accounts for over 80% of the total RNA, and its presence makes the study of other target RNAs (such as mRNA or LncRNA) in the total RNA difficult, and consumes a lot of valuable sequencing resources if not eliminated. Therefore, in general, when RNA sequencing is performed, rRNA is removed from total RNA, and sequencing and analysis are performed.
At present, the methods for removing rRNA in total RNA samples of animals mainly comprise: (1) poly (A) extraction method, wherein mRNA in eukaryote has poly (A) tail structure, and enrichment of mRNA in eukaryote by using poly (T) can indirectly remove rRNA; (2) rRNA is removed from the total RNA by binding the rRNA-DNA probe (biotin group-labeled) hybridization product with magnetic beads having streptavidin groups; (3) the RNase H enzyme is used to specifically degrade rRNA in the double strands hybridized by the rRNA-DNA probe.
The method (1) is most widely used, and various commercial kits with reliable performance, such as polyA Spin, have been introduced in the marketTMmRNA Isolation Kit (NEB), etc. However, this method can only enrich mRNA, and cannot enrich and transfer other target RNAs such as LncRNA.
The method (2) has stable performance, and Ribo-Zero Human/Mouse/rat (Illumina), Ribo-Minus are available on the marketTM(Thermo Fisher Scientific) and the like. However, this method is expensive and complicated to operate.
The method (3) has high cost performance and good rRNA removing effect. However, because the probe needs to fully cover the whole rRNA sequence, species compatibility is limited, and a good rRNA removal effect cannot be achieved in other species except for human/mouse.
Disclosure of Invention
The invention aims to provide a method for removing ribosomal RNA in total RNA of animals.
The invention provides a method for removing rRNA in an animal total RNA sample, which sequentially comprises the following steps:
(1) taking an animal total RNA sample, carrying out denaturation, and then carrying out reverse transcription by adopting a specific primer group (aiming at forming all rRNA into a DNA-RNA hybrid double chain); the specific primer group consists of a plurality of rRNA specific reverse transcription primers;
(2) treatment with RNase H (in order to specifically digest the RNA strand in the double strand DNA-RNA hybrid, i.e., to specifically digest the rRNA);
(3) treatment with DNase I (for the purpose of digesting DNA, including cDNA strands and reverse transcription primers);
(4) recovering the RNA.
The specific primer group consists of 40 primers which are sequentially shown as a sequence 1 in a sequence table to a sequence 40 in the sequence table.
The denaturation method is specifically as follows: 5min at 65 ℃.
In step (1), denaturation is performed, and then the mixture is placed on an ice box for 1min, and then the reverse transcription is performed.
The RNA concentration of the total RNA sample of the animal can be 1 mug/10 muL.
The reaction system (20. mu.L) of the reverse transcription is specifically as follows:
the reaction conditions of the reverse transcription can be specifically as follows: 10min at 25 ℃, 40min at 42 ℃ and 15min at 70 ℃.
The reaction system (30. mu.L) treated with RNase H enzyme was as follows:
the reaction conditions for the treatment with the RNase H enzyme may specifically be: 30min at 37 ℃.
The reaction system (50. mu.L) treated with DNase I was as follows:
the reaction conditions for the treatment with DNase I may specifically be: 30min at 37 ℃.
RNA recovery was specifically performed using RNAclean XP magnetic beads.
The invention also protects the application of any one of the methods in the preparation of an RNA library. The RNA is RNA of animals.
The invention also protects the application of any one of the methods in RNA sequencing. The RNA is RNA of animals.
The invention also protects a specific primer group which consists of 40 primers and is sequentially shown as a sequence 1 in a sequence table to a sequence 40 in the sequence table.
The invention also protects the application of the specific primer group in removing rRNA in total RNA samples of animals.
The invention also protects the application of the specific primer group in the preparation of an RNA library. The RNA is RNA of animals.
The invention also protects the application of the specific primer group in RNA sequencing. The RNA is RNA of animals.
The invention also provides a kit for removing rRNA in an animal total RNA sample, which comprises a component A, a component B and a component C;
the element A is used for specific reverse transcription of rRNA;
the element B is used for specifically digesting an RNA strand in a DNA-RNA hybrid double strand;
the element is used for digesting DNA.
The element A comprises an element A-1 and an element A-2; the element A-1 is a specific primer group, and the specific primer group consists of a plurality of rRNA specific reverse transcription primers; the element A-2 comprises a kit or a reagent or a combination of reagents for carrying out reverse transcription.
The specific primer group consists of 40 primers which are sequentially shown as a sequence 1 in a sequence table to a sequence 40 in the sequence table.
The element A-2 comprises SuperScriptII reverse transcriptase.
The element a-2 comprises: 5 XSuperScript II First-Strand Buffer, DTT, SuperScriptII reverse transcriptase, dNTP solution, RNase inhibitor. The specific ratios of the components are shown in table 1.
The element B comprises a kit or a reagent combination for specifically digesting an RNA strand in a DNA-RNA hybrid double strand.
The element b comprises RNAse H.
The element B comprises: 10 XRNAse H Buffer, RNAse H. The specific ratios of the components are shown in Table 2.
The element also includes a kit or reagent or combination of reagents for digesting DNA.
The element c comprises DNase I.
The element C comprises: 10 XDNase I Buffer, DNase I. The specific ratios of the components are shown in Table 3.
Any of the animals described above is a non-human animal or a human.
Such non-human animals include, but are not limited to: insect, fish, amphibian, or mammal.
The insect may be an entomoid. The Insecta may specifically be a Diptera. The dipteran can be specifically Drosophila, specifically Drosophila.
The fish may be a chondrocytid or teleostomid. The Osteichthyes animals can be Osteichthyes animals. The animal of the order amethyidae may be a medaka, and may be a medaka.
The amphibian may be an amphibian. The amphibian may specifically be a non-urodele. The animals of the order Gaultheria may be Bufo siccus, specifically Bufo siccus of the family Bufo siccus.
The mammal may be a mammal of the class mammalia. The mammal may be a primate. The primate can be a monkey. The monkey animal can be an animal of genus Macaca, specifically rhesus monkey.
The invention provides a brand-new method for removing rRNA from an animal total RNA sample based on an rRNA specificity reverse transcription method, which can efficiently and conveniently remove the rRNA and is suitable for mammals, birds, reptiles, amphibians, fishes, insects and other organisms.
The method has the following advantages: (1) specifically removing rRNA from the total RNA sample of the animal, reserving other target RNA such as mRNA, LncRNA and the like, and being suitable for LncRNA sequencing; (2) the species tolerance is high, and the method is widely suitable for removing rRNA in total RNA samples of mammals, birds, reptiles, amphibians, fishes, insects and the like; (3) the operation is simple, the cost is low, and the method is suitable for the operation of batch automatic workstations.
Drawings
Fig. 1 shows the results of the test using the Ailgent 2100 in step 1 of example 3.
Fig. 2 shows the results of the detection using the airfent 2100 in step 8 of example 3.
Fig. 3 shows the results of the test using the airfent 2100 in the comparative example.
In FIGS. 1 to 3, A corresponds to Drosophila, B corresponds to medaka, C corresponds to Xenopus laevis, and D corresponds to rhesus monkey.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
SuperScriptII reverse transcriptase trans, 5 XSuperScript II First-Strand Buffer and DTT, are components of the same kit. The name of the kit is SuperScriptTMII Reverse Transcriptase, #18064014, Thermo Scientific, USA. RNAse inhibitor: # Y9240L, QIAGEN, Germany. 10 XRNAse H Buffer: # Y9220L, QIAGEN, germany; RNAse H: # Y9220L, QIAGEN, Germany. 10 XDNAse I Buffer: # M0303S, NEB, usa; DNAse I: # M0303S, NEB, USA. dNTP Solution is Deoxynucleotide (dNTP) Solution Mix, # N0447S, NEB, USA. RNAclean XP magnetic beads
XP, Beckman Coulter, Germany.
Example 1 design and preparation of primer set
And screening to obtain a primer group (rRNA RT primer group) through a large number of preliminary experiments and effect verification. The primer set consisted of 40 primers.
The characteristics of the primer group are as follows: based on rRNA conserved sequence design, the method has species compatibility.
The sequences of the 40 primers were as follows (all in the 5 '→ 3' direction):
ANRT18-38 (SEQ ID NO: 1): TGGCTTAATCTTTGAGACAAGCATATGCTACTGGCAGGATCAACCAGGTA, respectively;
ANRT18-33 (SEQ ID NO: 2): GCCCCCCGGCCGGGGCCGGAGAGGGGCTGACCGGGTTGGTTTTGATCTGA, respectively;
ANRT18-28 (SEQ ID NO: 3): TTTCGTCACTACCTCCCCGGGTCGGGAGTGGGTAATTTGCGCGCCTGCTG, respectively;
ANRT18-23 (SEQ ID NO: 4): TAAGAGCATCGAGGGGGCGCCGAGAGGCAAGGGGCGGGGACGGGCGGTGG, respectively;
ANRT18-18 (SEQ ID NO: 5): ATGAAAACATTCTTGGCAAATGCTTTCGCTCTGGTCCGTCTTGCGCCGGT, respectively;
ANRT18-13 (SEQ ID NO: 6): GCCGGGTGAGGTTTCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACTC, respectively;
ANRT18-8 (SEQ ID NO: 7): GCAGCCCCGGACATCTAAGGGCATCACAGACCTGTTATTGCTCAATCTCG, respectively;
ANRT18-3 (SEQ ID NO: 8): GTGGGCCGACCCCGGCGGGGCCGATCCGAGGGCCTCACTAAACCATCCAA, respectively;
ANRT28-101 (SEQ ID NO: 9): GACTAATATGCTTAAATTCAGCGGGTCGCCACGTCTGATCTGAGGTCGCG, respectively;
ANRT28-96 (SEQ ID NO: 10): CCCAAGCAACCCGACTCCGGGAAGACCCGGGCGCGCGCCGGCCGCTACCG, respectively;
ANRT28-91 (SEQ ID NO: 11): GTGGAGGGGTCGGGAGGAACGGGGGGCGGGAAAGATCCGCCGGGCCGCCG, respectively;
ANRT28-86 (SEQ ID NO: 12): CGGGGGAGGAGGAGGACGGACGGACGGACGGGGCCCCCCGAGCCACCTTC, respectively;
ANRT28-81 (SEQ ID NO: 13): CGCGCCCCCGCGGGGGAGACCCCCCTCGCGGGGGATTCCCCGCGGGGGTG, respectively;
ANRT28-76 (SEQ ID NO: 14): GACGTCGCCGCCGACCCCGTGCGCTCGCTCCGCCGTCCCCCTCTTCGGG, respectively;
ANRT28-71 (SEQ ID NO: 15): CTGCCCAGGCATAGTTCACCATCTTTCGGGTCCTAACACGTGCGCTCGTG, respectively;
ANRT28-66 (SEQ ID NO: 16): CCATTTAAAGTTTGAGAATAGGTTGAGATCGTTTCGGCCCCAAGACCTCT, respectively;
ANRT28-61 (SEQ ID NO: 17): ATCCATTTTCAGGGCTAGTTGATTCGGCAGGTGAGTTGTTACACACTCCT, respectively;
ANRT28-56 (SEQ ID NO: 18): ACCGCAGCGGCCCTCCTACTCGTCGCGGCGTAGCGTCCGCGGGGCTCCGG, respectively;
ANRT28-51 (SEQ ID NO: 19): GCCACTCCGGATTCGGGGATCTGAACCCGACTCCCTTTCGATCGGCCGAG, respectively;
ANRT28-46 (SEQ ID NO: 20): TGCTGCGGATATGGGTACGGCCCGGCGCGAGATTTACACCCTCTCCCCCG, respectively;
ANRT28-41 (SEQ ID NO: 21): GGGCGCGGGGTGGGGAGGGAGCGAGCGGCGCGCGCGGGTGGGGCGGGGGA, respectively;
ANRT28-36 (SEQ ID NO: 22): CGGGGGCGGCCGCGACGCCCGCCGCAGCTGGGGCGATCCACGGGAAGGGC, respectively;
ANRT28-31 (SEQ ID NO: 23): CCGGGGGCGGACCCGGCGGGIGGGACCGGCCCGCGGCCCCTCCGCCGCCT, respectively;
ANRT28-26 (SEQ ID NO: 24): CACTAATTAGATGACGAGGCATTTGGCTACCTTAAGAGAGTCATAGTTAC, respectively;
ANRT28-21 (SEQ ID NO: 25): GTAGTGGTATTTCACCGGCGGCCCGCAGGGCCGCGGACCCCGCCCCGGGC, respectively;
ANRT28-16 (SEQ ID NO: 26): TGTATTCGTACTGAAAATCAAGATCAAGCGAGCTTTTGCCCTTCTGCTCC, respectively;
ANRT28-11 (SEQ ID NO: 27): GTAGGGTAAAACTAACCTGTCTCACGACGGTCTAAACCCAGCTCACGTTC, respectively;
ANRT28-6 (SEQ ID NO: 28): GGGCGCGTGGAGGGGIGGGCGGCCCGCCGGCGGGGACAGGCGGGGGACCG, respectively;
ANRT5.8 (sequence 29): GCAGCTAGCTGCGTTCTTCATCGACGCACGAGCCGAGTGATCCACCGCTA, respectively;
ANRT16-31 (SEQ ID NO: 30): AATGGTTTGGCTAAGGTTGTCTGGTAGTAAGGTGGAGTGGGTTTGGGGCT, respectively;
ANRT16-26 (SEQ ID NO: 31): ATAAATCTTCCCACTATTTTGCTACATAGACGGGTGTGCTCTTTTAGCTG, respectively;
ANRT16-21 (SEQ ID NO: 32): TTTAGGTAGTGGGTGTTGAGCTTGAACGCTTTCTTAATTGGTGGCTGCTT, respectively;
ANRT16-16 (SEQ ID NO: 33): GCCGAGTTCCTTTTACTTTTTTTAACCTTTCCTTATGAGCATGCCTGTGT, respectively;
ANRT16-11 (SEQ ID NO: 34): AGGGTCTTCTCGTCTTGCTGTGTTATGCCCGCCTCTTCACGGGCAGGTCA, respectively;
ANRT16-6 (SEQ ID NO: 35): AACCCTATTGTTGATATGGACTCTAGAATAGGATTGCGCTGTTATCCCTA, respectively;
ANRT12-19 (SEQ ID NO: 36): CTTGCATGTGTAATCTTACTAAGAGCTAATAGAAAGGCTAGGACCAAACC, respectively;
ANRT12-14 (SEQ ID NO: 37): CTAAAACACTCTTTACGCCGGCTTCTATTGACTTGGGTTAATCGTGTGAC, respectively;
ANRT12-9 (SEQ ID NO: 38): TCCTCTAGAGGGATATGAAGCACCGCCAGGTCCTTTGAGTTTTAAGCTGT, respectively;
ANRT12-4 (SEQ ID NO: 39): AGCACTCTACTCTTAGTTTACTGCTAAATCCACCTTCGACCCTTAAGTTT, respectively;
ANRT5 (sequence 40): TTCCGAGATCAGACGAGATCGGGCGCGTTCAGGGTGGTATGGCCGTAGAC are provided.
In the above sequences, I represents Inosine (Inosine). Inosine is also known as inosine, inosine. In the sequence listing, I is represented by N.
Example 2 establishment of the method
1. Total RNA of the biological material was extracted and the mass, concentration and integrity of the total RNA was tested using 2100.
RNA meeting the following quality control standards is subjected to the following steps: no DNA pollution peak; RIN > 7; concentrations >100 ng/. mu.L.
2. And (3) taking 1 mu g of RNA obtained in the step (1), and supplementing water to 10 mu L to obtain the sample to be detected.
3. Taking a sample to be detected, carrying out denaturation at 65 ℃ for 5min, and then placing on an ice box for 1min to obtain a product solution.
4. And carrying out reverse transcription.
The method comprises the following specific steps: the reaction system was prepared according to table 1, and then reacted to obtain a product solution.
TABLE 1
| Components | Amount of addition |
| The product solution obtained in step 3 | 10μL |
| 5×SuperScript II First-Strand Buffer | 4μL |
| DTT(0.1M) | 0.5μL |
| SuperScriptII reverse transcriptase(200U/μl) | 0.5μL |
| Primer Mix | 0.5μL |
| dNTP solution | 0.2μL |
| RNase inhibitor(40U/μl) | 0.5μL |
| DEPC-H2O | 3.8μL |
| Total volume | 20μL |
The dNTP solution provides effective components of dATP, dCTP, dGTP and dTTP. In the dNTP solution, the concentrations of dATP, dCTP, dGTP and dTTP were all 10 mM.
The effective components provided by the Primer Mix are the 40 primers of example 1. In the Primer Mix, the concentration of each Primer was 10. mu. mol/L.
Reaction procedure: storing at 25 deg.C for 10min, 42 deg.C for 40min, 70 deg.C for 15min, and 4 deg.C.
5. The RNA template (rRNA) bound to the first strand cDNA was digested with RNase H enzyme.
The method comprises the following specific steps: the reaction system was prepared according to table 2, and then reacted to obtain a product solution.
TABLE 2
| Composition of | Amount of addition |
| The product solution obtained in |
|
| 10×RNAse H Buffer | 3μL |
| RNAse H(5U/μL) | 4μL |
| DEPC-H2O | 3μL |
| Total volume | 30μL |
Reaction procedure: storing at 37 deg.C for 30min and 4 deg.C.
6. The DNA (including the remaining cDNA strand and primers) was digested with DNase I.
The method comprises the following specific steps: the reaction system was prepared according to table 3, and then reacted to obtain a product solution.
TABLE 3
| Composition of | Amount of addition |
| The product solution obtained in step 5 | |
| 10×DNase I Buffer | 5μL |
| DNase I(2U/μL) | 2μL |
| DEPC-H2O | 13μL |
| Total volume | 50μL |
Reaction procedure: storing at 37 deg.C for 30min and 4 deg.C.
7. And (5) sample purification.
And (4) taking the product solution obtained in the step (6), and purifying by using 100 mu L of RNAclean XP magnetic beads to obtain the product solution. The RNA contained in the product solution is rRNA-remove RNA.
8. And (4) taking the product solution obtained in the step (7), and detecting by using Ailgent 2100.
Example 3 application of the method
The biological materials are respectively: drosophila (intact individual), medaka (intact individual), xenopus laevis (larval intact individual), rhesus monkey (blood).
The assay was performed according to the method established in example 2.
The results of step 1 are shown in FIG. 1.
The results of step 8 are shown in FIG. 2. The result shows that the main peaks of the 28S and 18S bands in the total RNA sample are basically eliminated, which indicates that the method for removing rRNA in the animal total RNA sample provided by the invention has good effect.
Comparative example (c),
Samples from step 2 of example 3 were taken and run as described using the Ribo-off rRNA deletion Kit (Human/Mouse/Rat) and the protocol, and product solutions were taken and tested using the Ailgent 2100.
Ribo-off rRNA deletion Kit (Human/Mouse/Rat), Novozan, China. Suitable subjects for the Ribo-off rRNA deletion Kit (Human/Mouse/Rat) are Human, Mouse and Rat. The detection principle of Ribo-off rRNA deletion Kit (Human/Mouse/Rat) is the method (3) in the background art.
The results are shown in FIG. 3. The result shows that obvious residual rRNA miscellaneous peaks exist in the total RNA sample, which indicates that the rRNA removing scheme provided by the method (3) has poor rRNA removing effect on the total RNA sample of other animals and has limited species adaptability.
SEQUENCE LISTING
<110> Shenzhen Hua Dagen shares GmbH
<120> a method for removing ribosomal RNA from total RNA of animals
<130> GNCYX200257
<160> 40
<170> PatentIn version 3.5
<210> 1
<211> 50
<212> DNA
<213> Artificial sequence
<400> 1
tggcttaatc tttgagacaa gcatatgcta ctggcaggat caaccaggta 50
<210> 2
<211> 50
<212> DNA
<213> Artificial sequence
<400> 2
gccccccggc cggggccgga gaggggctga ccgggttggt tttgatctga 50
<210> 3
<211> 50
<212> DNA
<213> Artificial sequence
<400> 3
tttcgtcact acctccccgg gtcgggagtg ggtaatttgc gcgcctgctg 50
<210> 4
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<212> DNA
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<400> 4
taagagcatc gagggggcgc cgagaggcaa ggggcgggga cgggcggtgg 50
<210> 5
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<213> Artificial sequence
<400> 5
atgaaaacat tcttggcaaa tgctttcgct ctggtccgtc ttgcgccggt 50
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gccgggtgag gtttcccgtg ttgagtcaaa ttaagccgca ggctccactc 50
<210> 7
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<212> DNA
<213> Artificial sequence
<400> 7
gcagccccgg acatctaagg gcatcacaga cctgttattg ctcaatctcg 50
<210> 8
<211> 50
<212> DNA
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<400> 8
gtgggccgac cccggcgggg ccgatccgag ggcctcacta aaccatccaa 50
<210> 9
<211> 50
<212> DNA
<213> Artificial sequence
<400> 9
gactaatatg cttaaattca gcgggtcgcc acgtctgatc tgaggtcgcg 50
<210> 10
<211> 50
<212> DNA
<213> Artificial sequence
<400> 10
cccaagcaac ccgactccgg gaagacccgg gcgcgcgccg gccgctaccg 50
<210> 11
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<212> DNA
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gtggaggggt cgggaggaac ggggggcggg aaagatccgc cgggccgccg 50
<210> 12
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<212> DNA
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cgggggagga ggaggacgga cggacggacg gggccccccg agccaccttc 50
<210> 13
<211> 50
<212> DNA
<213> Artificial sequence
<400> 13
cgcgcccccg cgggggagac ccccctcgcg ggggattccc cgcgggggtg 50
<210> 14
<211> 49
<212> DNA
<213> Artificial sequence
<400> 14
gacgtcgccg ccgaccccgt gcgctcgctc cgccgtcccc ctcttcggg 49
<210> 15
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<212> DNA
<213> Artificial sequence
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ctgcccaggc atagttcacc atctttcggg tcctaacacg tgcgctcgtg 50
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<212> DNA
<213> Artificial sequence
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ccatttaaag tttgagaata ggttgagatc gtttcggccc caagacctct 50
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<211> 50
<212> DNA
<213> Artificial sequence
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atccattttc agggctagtt gattcggcag gtgagttgtt acacactcct 50
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<400> 18
accgcagcgg ccctcctact cgtcgcggcg tagcgtccgc ggggctccgg 50
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tgctgcggat atgggtacgg cccggcgcga gatttacacc ctctcccccg 50
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cgggggcggc cgcgacgccc gccgcagctg gggcgatcca cgggaagggc 50
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<212> DNA
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ccgggggcgg acccggcggg ngggaccggc ccgcggcccc tccgccgcct 50
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cactaattag atgacgaggc atttggctac cttaagagag tcatagttac 50
<210> 25
<211> 50
<212> DNA
<213> Artificial sequence
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gtagtggtat ttcaccggcg gcccgcaggg ccgcggaccc cgccccgggc 50
<210> 26
<211> 50
<212> DNA
<213> Artificial sequence
<400> 26
tgtattcgta ctgaaaatca agatcaagcg agcttttgcc cttctgctcc 50
<210> 27
<211> 50
<212> DNA
<213> Artificial sequence
<400> 27
gtagggtaaa actaacctgt ctcacgacgg tctaaaccca gctcacgttc 50
<210> 28
<211> 50
<212> DNA
<213> Artificial sequence
<400> 28
gggcgcgtgg aggggngggc ggcccgccgg cggggacagg cgggggaccg 50
<210> 29
<211> 50
<212> DNA
<213> Artificial sequence
<400> 29
gcagctagct gcgttcttca tcgacgcacg agccgagtga tccaccgcta 50
<210> 30
<211> 50
<212> DNA
<213> Artificial sequence
<400> 30
aatggtttgg ctaaggttgt ctggtagtaa ggtggagtgg gtttggggct 50
<210> 31
<211> 50
<212> DNA
<213> Artificial sequence
<400> 31
ataaatcttc ccactatttt gctacataga cgggtgtgct cttttagctg 50
<210> 32
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<400> 32
tttaggtagt gggtgttgag cttgaacgct ttcttaattg gtggctgctt 50
<210> 33
<211> 50
<212> DNA
<213> Artificial sequence
<400> 33
gccgagttcc ttttactttt tttaaccttt ccttatgagc atgcctgtgt 50
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<211> 50
<212> DNA
<213> Artificial sequence
<400> 34
agggtcttct cgtcttgctg tgttatgccc gcctcttcac gggcaggtca 50
<210> 35
<211> 50
<212> DNA
<213> Artificial sequence
<400> 35
aaccctattg ttgatatgga ctctagaata ggattgcgct gttatcccta 50
<210> 36
<211> 50
<212> DNA
<213> Artificial sequence
<400> 36
cttgcatgtg taatcttact aagagctaat agaaaggcta ggaccaaacc 50
<210> 37
<211> 50
<212> DNA
<213> Artificial sequence
<400> 37
ctaaaacact ctttacgccg gcttctattg acttgggtta atcgtgtgac 50
<210> 38
<211> 50
<212> DNA
<213> Artificial sequence
<400> 38
tcctctagag ggatatgaag caccgccagg tcctttgagt tttaagctgt 50
<210> 39
<211> 50
<212> DNA
<213> Artificial sequence
<400> 39
agcactctac tcttagttta ctgctaaatc caccttcgac ccttaagttt 50
<210> 40
<211> 50
<212> DNA
<213> Artificial sequence
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ttccgagatc agacgagatc gggcgcgttc agggtggtat ggccgtagac 50
Claims (10)
1. A method for removing rRNA in an animal total RNA sample sequentially comprises the following steps:
(1) taking an animal total RNA sample, carrying out denaturation, and then carrying out reverse transcription by adopting a specific primer group; the specific primer group consists of a plurality of rRNA specific reverse transcription primers;
(2) treating with RNase H enzyme;
(3) performing DNase I treatment;
(4) recovering the RNA.
2. The method of claim 1, wherein: the specific primer group consists of 40 primers which are sequentially shown as a sequence 1 in a sequence table to a sequence 40 in the sequence table.
3. Use of the method of claim 1 or 2 for the preparation of an RNA library.
4. Use of the method of claim 1 or 2 for RNA sequencing.
5. The specific primer group consists of 40 primers which are sequentially shown as a sequence 1 in a sequence table to a sequence 40 in the sequence table.
6. Use of the specific primer set of claim 5 for removing rRNA from total RNA samples of animals.
7. Use of the specific primer set of claim 5 for preparing an RNA library.
8. The use of the specific primer set of claim 5 for RNA sequencing.
9. A kit for removing rRNA in an animal total RNA sample comprises a component A, a component B and a component C;
the element A is used for specific reverse transcription of rRNA;
the element B is used for specifically digesting an RNA strand in a DNA-RNA hybrid double strand;
the element is used for digesting DNA.
10. The kit of claim 9, wherein:
the element A comprises an element A-1 and an element A-2; the element A-1 is a specific primer group, and the specific primer group consists of a plurality of rRNA specific reverse transcription primers; the element A-2 comprises a kit or a reagent or a combination of reagents for performing reverse transcription;
the element B comprises a kit or a reagent combination for specifically digesting an RNA strand in a DNA-RNA hybrid double strand;
the element also includes a kit or reagent or combination of reagents for digesting DNA.
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| CN113652469A (en) * | 2021-08-17 | 2021-11-16 | 上海市肺科医院 | Method and kit for removing ribosomal RNA and/or mitochondrial RNA in sample |
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