阅读说明：本技术 一种去除样本中核糖体rna和/或线粒体rna的方法和试剂盒 (Method and kit for removing ribosomal RNA and/or mitochondrial RNA in sample ) 是由 倪健 杨玉霞 陈曦 罗俊峰 于 2021-08-17 设计创作，主要内容包括：本发明公开了一种去除样本中核糖体RNA和/或线粒体RNA的方法和试剂盒,属于基因检测技术领域。本发明提供由异尾双链探针杂交捕获人类、小鼠和大鼠核糖体RNA和线粒体RNA,达到分离去除核糖体RNA和线粒体RNA的目的。本发明具有操作简单,流程简短,成本低廉,去除效率高,兼容多种商用RNA二代测序建库试剂盒的特点。(The invention discloses a method and a kit for removing ribosome RNA and/or mitochondrial RNA in a sample, belonging to the technical field of gene detection. The invention provides a method for capturing human, mouse and rat ribosomal RNA and mitochondrial RNA by hybridization of a heterotail double-stranded probe, thereby achieving the purpose of separating and removing the ribosomal RNA and the mitochondrial RNA. The method has the characteristics of simple operation, short flow, low cost, high removal efficiency and compatibility with various commercial RNA second-generation sequencing library building kits.)

1. A method for removing ribosomal RNA and/or mitochondrial RNA in a sample is characterized in that a plurality of heterotail double-stranded probes are adopted to capture ribosomal RNA and/or mitochondrial RNA in the sample to form a DNA-RNA hybrid complex, the hybrid complex is separated, and the ribosomal RNA and/or mitochondrial RNA in the sample is removed; wherein the heterotail double-stranded probe consists of a probe sequence with a label and a protection sequence; the probe sequence is formed by sequentially connecting a TH region, an overlap region and an NH region, wherein the TH region and the overlap region are designed according to a subunit fragment of RNA to be removed in a sample and are complementary with part of the sequence of the subunit fragment of the RNA to be removed, and the NH sequence is not complementary with the sequence of the subunit fragment of the RNA to be removed; the protective sequence is complementary with an overlap region and an NH region in the probe sequence.

2. The method of claim 1, wherein the subunit fragments of RNA to be removed are one or more of ribosomal RNA 45S subunit, ribosomal RNA18S subunit, ribosomal RNA5.8S subunit, ribosomal RNA5S subunit, mitochondrial RNA 12S subunit, mitochondrial RNA 16S subunit.

3. The method of claim 1, wherein the probe sequence comprises one or more of the following characteristics at a concentration of 0.3M NaCl at 60 ℃:

(1) the Gibbs free energy of a TH region of the probe sequence is (-4.00 to-16.00) kcal/mol; gibbs free energy at the NH terminus is (-1.00 to-12.00) kcal/mol;

(2) gibbs free energy difference Δ Δ G (Δ G) between TH terminal and NH terminal of probe sequence_TH-ΔG_NH) Between (4.00 and-15.00) kcal/mol;

(3) the Gibbs free energy of the overlap region of the probe sequence is (-5.00 to-40.00) kcal/mol.

4. The method of claim 1, wherein the probe sequence is 20-80bp in length and the protection sequence is 10-70bp in length.

5. The method of claim 1, wherein the sample is a human, mouse or rat RNA sample.

6. The method of claim 1, wherein the probe sequences and protection sequences are as shown in the following table:

7. the method of claim 1, wherein the label is a biotin label or an inter-biotin arm label, and the label is at the 5 'end or 3' end of the probe sequence.

8. The method of claim 1, wherein the capturing comprises:

(1) performing warm bath treatment at 70-95 ℃ for 2-10 minutes to denature RNA;

(2) carrying out hybridization treatment by adopting warm bath treatment at 40-80 ℃ for 10-60 minutes;

(3) and performing bath treatment for 10-60 minutes at 40-80 ℃ by using streptavidin magnetic beads to capture the hybrid complex.

9. A kit for removing ribosomal RNA and/or mitochondrial RNA in a sample is characterized by comprising an heterotail double-stranded probe, wherein the heterotail double-stranded probe consists of a probe sequence with a label and a protection sequence; the probe sequence is formed by sequentially connecting a TH region, an overlap region and an NH region, wherein the TH region and the overlap region are designed according to a subunit fragment of RNA to be removed in a sample and are complementary with part of the sequence of the subunit fragment of the RNA to be removed, and the NH sequence is not complementary with the sequence of the subunit fragment of the RNA to be removed; the protective sequence is complementary with an overlap region and an NH region in the probe sequence.

10. The kit of claim 9, further comprising 2x B & W buffer, 1x B & W buffer, streptavidin magnetic beads, beads wash, positive control, and negative control.

Technical Field

The invention relates to a method and a kit for removing ribosome RNA and/or mitochondrial RNA in a sample, belonging to the technical field of gene detection.

Background

Cancer is one of malignant diseases affecting human health, in order to research the occurrence and development mechanism of cancer, scientists cannot leave the help of model organisms, scientists use mice and rats to establish various cancer models, and only can research the change of various technologies and mechanisms, try the change of the drug effect and the metabolic mechanism of various drugs and the like by using the biological models.

However, in human, mouse and rat samples, ribosomal RNA is the most abundant RNA species in total RNA, accounting for approximately 90% of total RNA, and ribosomal RNA provides little useful transcript information. In the second generation sequencing application, two approaches are available in order to obtain the most useful transcript information possible. Firstly, oligo-dT is used for enriching poly-tail A (polyA) of mRNA, however, the method can only enrich mRNA with polyA, and some eukaryotic mRNA does not have polyA, and other RNA species such as snRNA, snorRNA, miRNA and the like in non-coding RNA can be lost when the method is used for capturing; in addition, because high quality intact RNA is required for capture, the enrichment of mRNA with low incomplete quality and low yield may not be as good. The other method is a method for removing ribosome RNA, and the method is more suitable for RNA with low quality or low yield, simultaneously reserves more RNA species diversity and improves the information content of transcripts.

Mitochondrial rRNA comprises two subunits, namely 12S subunit and 16S subunit, the expression of the two subunits is greatly changed in different cells and development stages, and the data needs to be filtered out in the quality control process of second-generation high-throughput sequencing RNA-seq. These two subunits typically account for 10% of the RNA-seq data.

The existing method for removing ribosomal RNA mainly uses probe capture, and specific probe types comprise RNA probe, DNA probe, locked nucleic acid probe and the like, but all have certain defects and shortcomings. For example, the reported RiboZero kit RNA probe of Epicentre company needs to be stored at the ultralow temperature of-70 ℃, is not easy to operate, can capture non-target RNA and causes the loss of transcript information; the ribosome RNA removal efficiency of Invitrogen RiboMinus is only 80%, and the ribosome RNA still occupies a large amount of data; in addition, trials on the marketThe cartridge also has problems of an excessively long operation time and an excessively high cost. E.g. of NEB corporationrRNA deletion Kit and Ribo-off rRNA deletion Kit from Vazyme Inc. all over the experimental procedure for more than 2 hours. It can be known from the patent document CN104685071A that the probe of the qiagen RNA-eliminating composition needs to form a complex double-stranded hybrid with the target region, and then bind with the magnetic bead with antibody to achieve the purpose of removing ribosomal RNA or mitochondrial RNA, and the cost of such a complex design is difficult to reduce. Furthermore, some kits do not provide for ribosomal RNA 45s subunit and mitochondrial rRNA removal, and ultimately affect the effective data size of the RNA-seq.

Disclosure of Invention

Aiming at the defects of the existing method for removing ribosome RNA and mitochondrial RNA and better meeting the requirements in cancer research, the invention discloses a method and a kit for removing ribosome RNA and mitochondrial RNA.

The first purpose of the invention is to provide a method for removing ribosomal RNA and/or mitochondrial RNA in a sample, which comprises the steps of capturing ribosomal RNA and/or mitochondrial RNA in the sample by using a plurality of heterotail double-stranded probes to form a DNA-RNA hybrid complex, separating the hybrid complex, and removing the ribosomal RNA and/or the mitochondrial RNA in the sample; wherein the heterotail double-stranded probe consists of probe sequences (probes) with labels and protection sequences (protectors); the probe sequence is formed by sequentially connecting a TH region, an overlap region and an NH region, wherein the TH region and the overlap region are designed according to a subunit fragment of RNA to be removed in a sample and are complementary with part of the sequence of the subunit fragment of the RNA to be removed, and the NH sequence is not complementary with the sequence of the subunit fragment of the RNA to be removed; the protective sequence is complementary with an overlap region and an NH region in the probe sequence.

Further, the subunit fragment of the RNA to be removed is one or more of ribosomal RNA 45S subunit, ribosomal RNA18S subunit, ribosomal RNA5.8S subunit, ribosomal RNA5S subunit, mitochondrial RNA 12S subunit and mitochondrial RNA 16S subunit.

Further, the probe sequence comprises one or more of the following characteristics at a concentration of 0.3M NaCl and 60 ℃:

(1) the Gibbs free energy of a TH region of the probe sequence is (-4.00 to-16.00) kcal/mol; gibbs free energy at the NH terminus is (-1.00 to-12.00) kcal/mol;

(2) gibbs free energy difference Δ Δ G (Δ G) between TH terminal and NH terminal of probe sequence_TH-ΔG_NH) Between (4.00 and-15.00) kcal/mol;

(3) the Gibbs free energy of the overlap region of the probe sequence is (-5.00 to-40.00) kcal/mol

Furthermore, the length of the probe sequence is 20-80bp, and the length of the protective sequence is 10-70 bp.

Further, the sample is a human, mouse or rat RNA sample.

Further, based on the design of ribosomal RNA and mitochondrial RNA subunits in human, mouse and rat, the probe sequences and protection sequences are shown in the following table and can be used for the removal of ribosomal RNA and mitochondrial RNA in human, mouse and rat samples:

further, the label is a biotin label or a biotin-spacer label, and is labeled at the 5 'end or the 3' end of the probe sequence.

The second purpose of the invention is to provide a kit for removing ribosomal RNA and/or mitochondrial RNA in a sample, wherein the kit comprises a heterotail double-stranded probe, and the heterotail double-stranded probe consists of a probe sequence with a label and a protection sequence; the probe sequence is formed by sequentially connecting a TH region, an overlap region and an NH region, wherein the TH region and the overlap region are designed according to a subunit fragment of RNA to be removed in a sample and are complementary with part of the sequence of the subunit fragment of the RNA to be removed, and the NH sequence is not complementary with the sequence of the subunit fragment of the RNA to be removed; the protective sequence is complementary with an overlap region and an NH region in the probe sequence.

Further, the kit also comprises 2x B & W buffer solution (1xTE,1M NaCl, 0.1% Tween-20), 1x B & W buffer solution (0.5xTE,0.5M NaCl, 0.05% Tween-20), streptavidin magnetic beads, beads cleaning solution (0.1M NaCl +0.1M NaOH), positive control and negative control.

The invention has the beneficial effects that:

the invention provides a method for capturing human, mouse and rat ribosomal RNA and mitochondrial RNA by hybridization of a heterotail double-stranded probe, thereby achieving the purpose of separating and removing the ribosomal RNA and the mitochondrial RNA. The method has the characteristics of simple operation, short flow, low cost, high removal efficiency and compatibility with various commercial RNA second-generation sequencing library building kits.

Drawings

FIG. 1 is a structural design diagram of a heterotail double-stranded probe;

FIG. 2 is a sample capture hybridization experiment procedure;

FIG. 3 is an integrated genome browser map (IGV).

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1: human ribosomal RNA heterotail double-stranded probe design

1) The sequences of the human/rat/mouse ribosomal RNA 45S, 18S, 5.8S, 5S subunits were downloaded from the NCBI database, and the post-data download sequence alignments found the positions of consensus sequences and intraspecies conserved sequences of these subunits for different species, and examined for predicted RNA secondary structures (http:// www.unafold.org/mfold/applications/RNA-folding-form. php).

2) Probes are respectively designed aiming at a consensus sequence and a conserved sequence of human ribosomal RNA, the position of the probe is prevented from being designed in a double-stranded region of a secondary structure as much as possible, the restriction condition of Gibbs free energy is considered, and the specific principle of the energy of each part of the probe is that the following conditions are simultaneously met under the conditions of 0.3M NaCl concentration and 60 ℃:

(1) the Gibbs free energy of a TH region of the probe sequence is (-4.00 to-16.00) kcal/mol; gibbs free energy at the NH terminus is (-1.00 to-12.00) kcal/mol;

(2) gibbs free energy difference Δ Δ G (Δ G) between TH terminal and NH terminal of probe sequence_TH-ΔG_NH) Between (4.00 and-15.00) kcal/mol;

(3) the Gibbs free energy of the overlap region of the probe sequence is (-5.00 to-40.00) kcal/mol.

TABLE 1

Ribosome bindingSubunit type	Number of probes (strip)
		28s	51
18s	19
		5.8s	3
5s	2
		45s	5

The number of co-designed human ribosomal RNA probes is shown in Table 1.

After the probe design is completed, a probe with biotin or an arm between biotin is synthesized, and the storage concentration of the probe is 100 mu M. The amount of probe used was 15pmol for 100ng-1ug of total RNA in a 100ul system. Compared with the prior art, the cost of hybridization capture is saved at the concentration because other modifications such as antibodies, RNA probes or locked nucleic acids are not used in the synthesis.

According to the principle of energy in each part, the probe sequences and protection sequences designed according to the human ribosomal RNA and mitochondrial RNA subunits are shown in Table 2 (sequence numbers 1-80 are probe sequences, and sequence numbers 81-160 are protection sequences):

TABLE 2

Example 2: human ribosomal RNA and mitochondrial RNA ablation

1) Fresh 500ul of fresh human whole blood was taken and total RNA extraction was performed using a whole blood total RNA extraction kit (Hangzhou New Zealand Biopsis). 1ml of buffer L9 was added to a 2ml centrifuge tube, 500. mu.l of whole blood was added, and the mixture was vortexed and shaken for 30 seconds to mix well. 13000rpm for 10 minutes, sucking 700 mul of centrifugal supernatant and transferring to a new 1.5ml centrifuge tube, adding 500 mul of absolute ethyl alcohol; transferring 600. mu.l of the above-mentioned mixed solution to a nucleic acid purification column, placing the purification column in a 2ml collection tube, centrifuging at 12000rpm for 30 seconds, discarding the filtrate in the 2ml collection tube, returning the nucleic acid purification column to the 2ml collection tube, pouring the whole of the remaining liquid in the 1.5ml centrifuge tube into the nucleic acid purification column, and centrifuging at 12000rpm for 30 seconds. The filtrate in the collection tube WAs discarded, and the tube WAs returned to the nucleic acid purification cartridge, and 500. mu.l of buffer WA WAs added to the cartridge and centrifuged at 12000rpm for 30 seconds, and the filtrate WAs discarded, and the tube WAs returned to the nucleic acid purification cartridge, and 700. mu.l of buffer WBR WAs added to the cartridge and centrifuged at 12000rpm for 30 seconds. The filtrate was discarded, and the column was returned to the nucleic acid purification column and centrifuged at 14000rpm for 1 minute. The collection tube was discarded, and the nucleic acid purification column was placed in a 1.5ml RNase-free centrifuge tube, 50. mu.l RNase-free water was added to the center of the purification column, allowed to stand at room temperature for 1 minute, and centrifuged at 12000rpm for 30 seconds. The purification column was discarded for subsequent ribosomal RNA and mitochondrial RNA removal experiments.

2) NanoDrop quantitates RNA, 9 copies of 100ng total human RNA are taken for use, 3 copies are captured by Probes + Protectors, 3 copies are captured by Probes, and 3 total are used as controls to directly construct the library.

3) Heterotailed double-stranded probe mix preparation included 1.5. mu.l of 100. mu.M probes, 3. mu.l of 100. mu.M protectors, 4.5. mu.l of 2x B & W buffer, and 91. mu.l of 1x B & W buffer. After the mixture of Probes and detectors is shaken and centrifuged, the mixture is placed on a PCR instrument, and a touchdown program is set to 94-25 ℃ for 70 cycles, wherein each cycle is reduced by 1 ℃.

The second set of probe mixtures (Probes) specifically included 1.5. mu.l of 100. mu.M probe, 1.5. mu.l of 2x B & W buffer, and 97. mu.l of 1x B & W buffer.

4) And (3) hybridization and capture:

100ng of RNA was placed in the PCR tube, the same volume of 2x B & W buffer was added, and 1x B & W buffer was added to 90. mu.l. Placing on a PCR instrument for 75 ℃ warm bath. After centrifugation, 10. mu.l of the mixture of Probes + Protectors Probes or 10. mu.l of the mixture of Probes was added, and the sample was placed on a PCR instrument after being turned upside down and mixed, and then incubated at 60 ℃ for 30 minutes.

Wash 75. mu.l streptavidin magnetic beads (Dynabeads)^TM MyOne^TMStreptavidin T1), collecting magnetic Beads, placing on magnetic rack for clarification, pouring out supernatant, adding 100 μ l of 1 xBeads cleaning solution, waiting for clarification, removing supernatant, and adding 200 μ l of 1x B&The W buffer re-suspends the magnetic beads, leaves the magnetic rack for 1 minute, then returns the magnetic rack, waits for clarification, removes the supernatant, and repeats the above steps for 2 times. Using 20. mu.l of 1x B&Suspending in W buffer solution, performing warm bath in a dry thermostat at 60 ℃ for 10 minutes, centrifuging, placing in a magnetic rack, and removing supernatant. Adding magnetic beads into the probe mixed solution of RNA, uniformly mixing, carrying out warm bath at 60 ℃ for 15 minutes, putting back a magnetic frame, taking the supernatant as a sample for removing ribosome RNA, and purifying the RNA sample by using RNA clean beads.

5) Synthesizing and purifying a first strand and a second strand, synthesizing cDNA by using Illumina TruSeq RNA sample Prep kit V2, and specifically configuring the following reaction system:

PCR instrument program: hold at 25 ℃ for 10min,42 ℃ for 50min,70 ℃ for 15min and 4 ℃.

After the reaction was complete, the second strand master mix was added to the single stranded cDNA, shaken well and separated out briefly, and placed on a PCR instrument for 1 hour. After completion the product was purified as indicated by AMPure XP beads.

6) Tip repair

The Library was constructed using VAHTS Universal DNA Library Prep Kit for Illumina V3, the End Prep Mix 4 was thawed and mixed by inversion and placed in a 0.2mL PCR tube for the following reactions:

components	Volume of
		Fragmenting DNA	25μL
End Prep Mix 4	7.5μL

Gently blowing and beating the mixture by using a pipettor, and centrifuging the mixture for a short time to collect reaction liquid to the bottom of the tube; the PCR tube was placed in a PCR machine and the following reactions were performed (105 ℃ hot lid):

temperature of	Time
		20℃	15min
65℃	15min
		4℃	Hold

7) Joint connection

Thawing Rapid Ligation buffer 2, inverting and mixing, and configuring the following reactions in an End precipitation step PCR tube:

components	Volume of
		End Preparation product	32.5μL
Rapid Ligation buffer 2	12.5μL
		Rapid DNA Ligase	2.5μL
DNA Adapter X	2.5μL

Gently blowing and beating the mixture by using a pipettor, and centrifuging the mixture for a short time to collect reaction liquid to the bottom of the tube; the PCR tube was placed in a PCR machine and the following reactions were performed (105 ℃ hot lid):

temperature of	Time
		20℃	15min
4℃	Hold

After completion the product was purified as indicated by AMPure XP beads.

8) After thawing, the PCR Primer Mix3 and the HiFi Amplification Mix were mixed by inversion, and the following reactions were performed in a PCR tube:

the PCR cycle was set as follows:

9) display of ribosomal RNA removal Effect

Total in the table refers to three samples which are not processed by the probe, Probes + detectors refer to three samples which are removed by capturing with the heterotail double-stranded probe, and Probes refer to samples which are removed by capturing with the single-stranded probe. As can be seen from the table, the average removal efficiency of Probes + protectors can reach 98.04%, and the removal efficiency of Probes alone can also reach 98.79%. Although it is better to remove the probe sequence alone from the results, the probe sequence used in the present invention is designed based on the principle that the energy of the regions of both the probe sequence and the protection sequence are satisfied, and therefore, the protection sequence is also important.

10) Effect of removing mitochondrial RNA 16S

In the IGV diagram, CG240-C1 and CG240-C2 represent the capture removal effect of probes; CG240-CP1 and CG240-CP2 represent the capture removal effect of probes + protectors probes; CG240-T1 and CG240-T2 represent samples that were not probe treated. It was calculated that up to 96% of mitochondrial RNA 16S was removed.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.