CN105803101A - Probe, gene chip and method for detecting expression abundance of circular RNA - Google Patents
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Abstract
The invention discloses a probe for detecting expression abundance of circular RNA. The probe includes backsplicing sites at the 5' terminal of a donor exon and at the 3' terminal of an accepter exon of the circular RNA. The invention also discloses a gene chip containing the probe, and a method for detecting the expression abundance of the circular RNA by virtue of the gene chip, wherein the method mainly comprises the following steps: 1) extracting and purifying total RNA of a to-be-detected sample; 2) removing ribosome RNA; 3) enriching circular RNA; 4) conducting linear amplification to the enriched circular RNA and labeling fluorescence; 5) hybridizing a fluorescence-labeled product with a chip probe; and 6) washing and scanning the chip, and analyzing data. By designing the probe and the gene chip which are capable of achieving specific detection of the expression abundance of the circular RNA, the rapid and high-throughput detection of the expression abundance of the circular RNA can be achieved, so that shortcomings in an existing circular RNA detection technology are overcome.
Description
Technical field
The present invention relates to biological field, particularly relate to the detection of circular rna gene expression abundance.
Background technology
CircularRNA (circRNA) be a class with covalency closed-loop for feature, be widely present in the novel RNA molecule in eukaryote.CircRNA derives from exon or the intron region of gene, exists in a large number in mammalian cell.The formation of circRNAs is different from the standard scissors pattern of linear rna, is by joining end to end for 3 ' ends of the 5 ' of Exon ends with receptor exon, forming reverse splicing site (backsplicing) mode and shear.Existing circRNA forms model mainly following several (shown in Figure 1):
(1) " lariat-drivencircularization (lock set driving cyclization) " or " exonskipping (exon skipping cyclization) ", as shown in Figure 1A；
(2) " intron-pairing-drivencircularization (pairing intron drives cyclization) " or " directbacksplicing (is directly opposite splicing cyclization) ", as shown in Figure 1B；
(3) ring-type introne RNA (ciRNAs) rock mechanism, as shown in Figure 1 C；
(4) RBPs cyclization patterns is depended on, as shown in figure ip；
(5) the variable cyclization patterns of variable sheer it is similar to, as referring to figure 1e.
Now there are some researches show that most circRNA is conservative between different plant species.Simultaneously as its circulus can resist the degraded of RNaseR and more stable.The complexity of the circRNA specificity due to its expression and regulation and control, and the important function in disease occurs, be increasingly subject to everybody attention.Just as miRNA is the same with long-chain non-coding RNA, circRNA has become as a new study hotspot in RNA field.The technological means of circular rna gene expression abundance detection conventional at present is real-time PCR, but the method has the shortcoming that research flux is low, and the research of circRNA is at the early-stage, is badly in need of a high-throughout reliable detection technique and meets research needs.
Chip gene expression profile technology refer to by microarray technology by high-density DNA fragment by high speed robot or fabricated in situ mode sequentially or arrangement mode make it be attached to such as slide, the solid phase surfaces such as silicon chip, with fluorescence or biotin labeled purpose fragment, by base complementrity Hybridization principle, carry out the revolutionary technology of substantial amounts of gene expression research.Biochip technology is accompanied by the enforcement of the Human Genome Project and forward position biotechnology in the life science that grows up.At present, the classification of disease and the level of diagnosis have been had further raising by people, and the Feature Selection based on gene chip serves critical effect wherein.Through the development of more than ten years, biochip technology is also constantly perfect, ripe, and is widely used in the every field of life sciences.
Summary of the invention
One of the technical problem to be solved in the present invention is to provide a kind of probe detecting circular rna gene expression abundance, it can specificity, high flux, detect the gene expression abundance of circular rna rapidly.
For solving above-mentioned technical problem, the probe of the detection circular rna gene expression abundance of the present invention, include 3 ' the reverse splicing sites held sub with receptor exon for 5 ' ends of Exon of described circular rna.
It is also preferred that the left the sequence length of described probe is 60nt.
It is also preferred that the left described probe has the sequence as shown in SEQIDNO:3 or its complementary series.
The two of the technical problem to be solved in the present invention are to provide the gene chip of a kind of detection circular rna gene expression abundance including above-mentioned probe, and this chip can provide reliable and stable and high-throughout circular rna gene expression abundance testing result.
The three of the technical problem to be solved in the present invention are to provide the method for the above-mentioned genechip detection circular rna gene expression abundance of a kind of use, and the method comprises the steps:
1) also purification sample to be tested total serum IgE is extracted；
2) ribosomal RNA is removed；
3) enrichment circular rna；
4) circular rna that enrichment is obtained carries out linear amplification mark fluorescent；
5) by step 4) fluorescent mark product that obtains hybridizes with the probe on described gene chip；
6) washing gene chip, scanning, data analysis.
Described step 3), adopt RNaseR to digest linear rna, thus being enriched with circular rna.
The present invention is by being designed to probe and the gene chip of specific detection circular rna gene expression abundance, achieve quick, high throughput testing to circular rna gene expression abundance, nearly 90,000 circular rnas can be detected simultaneously, so that biochip technology is better applied to circular rna expression pattern analysis, compensate for the deficiencies in the prior art.
Accompanying drawing explanation
Fig. 1 is that existing 5 kinds of circRNA form model.Wherein, A figure is that lock set drives cyclization or exon skipping to become ring model；B figure is that pairing intron drives cyclization or is directly opposite and is spliced into ring model；C figure is that ciRNAs forms model；D figure is for depending on RBPs cyclisation model；E figure is variable cyclisation model.
Fig. 2 is the probe design schematic diagram of the gene chip of the detection circular rna gene expression abundance of the embodiment of the present invention.
Fig. 3 is the method flow schematic diagram with the genechip detection circular rna gene expression abundance of the embodiment of the present invention.
Fig. 4 is the DNA Microarray Scanning figure of the detection circular rna gene expression abundance of the embodiment of the present invention.
Detailed description of the invention
Understanding more specifically for the technology contents of the present invention, feature and effect are had, in conjunction with accompanying drawing, the present invention detects the concrete grammar of circular rna gene expression abundance, and details are as follows:
One, design chips probe sequence
According to the splice mode that circular rna is special, there are the special 5 ' ends for Exon and hold end to end reverse splicing site (backsplicing with the 3 ' of receptor exon, linear rna does not have this site), circular rna probe design is being positioned at the 60nt chip probe sequence in reverse splicing site, and this probe sequence can circular rna in specific detection sample.
The present embodiment detection circular rna be hsa_circ_0034688, it be the gene by an OIP5 by name the 4th exon to the 5th exon head and the tail be reversely spliced.According to this circular rna 5 ' terminal sequence:
CTATCTCTTAAAAACAAAAGCCATAGTAAATGCATCAGAGATGGATATTCAAAATG TTCCTCTATCAGAAAAGATTG (SEQIDNO:1) and 3 ' terminal sequences:
The feature of ACTAATACATCACTTTTTAACTTCTGTAAAATACAGATCATAATATTCTATAGGTA ATGTTTAATAAATTGCCTGAATAATATACAAT (SEQIDNO:2), we design the specific probe sequence containing this splicing site:
TAATATACAACTATCTCTTAAAAACAAAAGCCATAGTAAATGCATCAGAGATGGAT ATTC (SEQIDNO:3), this probe can specific detection this circular rna hsa_circ_0034688.
Two, gene chip is prepared
Utilize the SurePrint technology of Agilent company, in the way of inkjet printing (ink-print) on slide the oligonucleotide probe on fabricated in situ chip, preparation is for detecting the gene chip of above-mentioned circular rna gene expression abundance.
Three, with the experiment flow of above-mentioned genechip detection circular rna gene expression abundance
1. extraction, purification sample total serum IgE
Trizol extracting sample total serum IgE, then use QIAGENKit (article No. 74106) purification total serum IgE, detailed method of operation following (referring to RNeasyMiniProtocol):
1) take in the total serum IgE≤100 μ g water being dissolved in 100 μ lRNasefree (without RNase), add 350 μ l buffer RLT and fully mix.
2) add 250 μ l dehydrated alcohol, fully mix with Tip head (the rifle head of sample loading gun).
3) proceed in the RNeasy pillar being enclosed within 2ml centrifuge tube by amounting to the 700 μ l solution containing total serum IgE, centrifugal 15 seconds of 13200rpm, discard filtered solution.
4) draw in 350 μ l buffer RW1 to RNeasymini pillars, centrifugal 15 seconds of 13200rpm, discard filtered solution.
5) DNase I of 10 μ l is joined in the buffer RDD of 70 μ l, mixing, join in pillar, room temperature places 15min.
6) draw in 350 μ l buffer RW1 to RNeasymini pillars, centrifugal 15 seconds of 13200rpm, discard filtered solution.
7) draw in 500 μ l buffer RPE to RNeasymini pillars, centrifugal 15 seconds of 13200rpm, discard filtered solution, repeat this step once.
8) sleeve pipe renewed, 13200rpm, 2min.And pillar is transferred in eluting pipe.
9) RNeasymini pillar is proceeded in collecting pipe.
10) draw 30 μ lRNasefree water, stand the centrifugal 1min of 1min, 13200rpm.
11) again the 30ul sample in eluting pipe is gone back to pillar, stand the centrifugal 1min of 1min, 13200rpm.
12) NanoDrop (NanoDropND-1000UV-VISspectrophotometer) records RNA concentration and 260/280.
2.rRNA removes
RRNA (ribosomal RNA) occupies the ratio of about 95% in total serum IgE, and rRNA is relatively stable in intracellular expression.The removal step of rRNA is as follows:
1) the RNA sample moisturizing after being purified by 1-3 μ g with the water of nuclease-free (without nuclease) is to 10 μ l.
2) rRNABindingBuffer (binding buffer liquid) of 5 μ l is added.
3) add the rRNARemovalMix (removal mixed liquor) of 5 μ l, blow and beat gently with pipettor 10 times, mix homogeneously.
4) sample is placed in PCR instrument, 65 DEG C, 5min.
5), after hatching end, sample room temperature is placed 1min.
6) rRNARemovalBeads (removal magnetic bead) is placed on spiral instrument, makes magnetic bead even suspension.
7) add the rRNARemovalBeads of 35 μ l in sample, and blow and beat 20 times with pipettor, it is ensured that sample is mixed homogeneously with magnetic bead.
8) mixed magnetic bead and sample room temperature are placed 1min, then room temperature is placed on magnetic frame 1min, after solution is clarified, pipette supernatant to new centrifuge tube, then it is placed on magnetic frame by centrifuge tube 1min, after solution is clarified, then pipettes supernatant to new centrifuge tube.
9) RNACleanXPbeads is placed on spiral instrument, makes magnetic bead even suspension.
10) taking 90 μ lRNACleanXPbeads and join in sample, fully blow and beat with pipettor, it is ensured that sample is mixed homogeneously with magnetic bead, room temperature places 15min, then room temperature is placed on magnetic frame 5min, after treating sample clarification, removes supernatant.
11) sample is placed on magnetic frame, adds the 200 μ l70% ethanol newly prepared, place 30s, remove supernatant, repeat this step once.
12) sample is placed dry 15min at room temperature, then sample is taken off from magnetic frame.
13) room temperature is vibrated elution buffer slightly, adds 11 μ l elution buffers in sample, is suspended in full and uniform for magnetic bead in elution buffer.
14) sample added with elution buffer is placed 2 minutes in room temperature, then, sample is placed on magnetic frame upper 5 minute, after sample is clarified, shifts the supernatant of 8.5 μ l in the centrifuge tube of 0.3ml.
15) add 8.5 μ lElute, Prime, FragmentHighMix in sample, and be sufficiently mixed uniformly.
3. linear rna is removed
Utilize RNaseR can digest linear rna, but the principle of the circular rna with closed hoop structure can not be digested, adopt RNaseR to digest linear rna, reach the purpose of enrichment circular rna.Specifically comprise the following steps that
1) add 2 μ l (40U/ μ l) RNaseR to remove in the sample after rRNA to what above-mentioned steps 2 obtained.
2) in the water of 100 μ lRNase-free, add 350 μ l buffer RLT and fully mix.
3) adding 250 μ l dehydrated alcohol, Tip head fully mixes.
4) proceed in the RNeasymini pillar being enclosed within 2ml centrifuge tube by amounting to the 700 μ l solution containing total serum IgE, centrifugal 15～30 seconds of 8000g, discard filtered solution.
5) 350 μ l buffer RW1 are added in centrifugal column.Lid upper tube cap, 8000g is centrifuged 15s, discards filtered solution.
6) being directly added into 80 μ lDNaseI mixed liquors to centrifugal column film, room temperature is placed 15 minutes.
7) 350 μ l buffer RW1 are added in centrifugal column.Lid upper tube cap, 8000g is centrifuged 15s, discards filtered solution.
8) draw in 500 μ l buffer PRE to RNeasymini pillars, 8000g centrifuge washing 15 seconds, discards filtered solution, then with 500 μ l buffer PRE 8000g centrifuge washing 2 minutes, discard filtered solution and 2ml sleeve pipe, RNeasymini pillar is proceeded in a new 1.5mlEppendorf pipe.
9) water of 40 μ lRNasefree is drawn, the centrifugal eluting of >=8000g 1 minute.
4. the circular rna of pair enrichment carries out linear amplification mark fluorescent cy3
1) single target spike-in (RNASpike-InKit, One-Color, Agilent5188-5282) is prepared.
According to different RNA initial amounts, dilute spike-in by dilution buffer, as shown in table 1:
Table 1RNAspike-in
2) reverse transcription.
The reaction solution that preparation composition is as shown in table 2:
Table 2 reverse transcription reaction solution composition
PCR instrument (MJPTC-100) upper 65 DEG C of insulations 10min, ice bath 5min.At 80 DEG C, 5Xfirststrandbuffer being preheated 3min, room temperature is standby simultaneously.Configuration reverse transcription mixed solution, particular make-up is as shown in table 3:
Table 3 reverse transcription mixed solution forms
| 5X First Strand Buffer | 2.0μl |
| 0.1M DTT | 1.0μl |
| 10mM dNTP mix | 0.5μl |
| AffinityScript RNase Block Mix | 1.2μl |
| Cumulative volume | 4.7μl |
Above-mentioned 4.7 μ l reverse transcription mixed solutions are added in the RNA of the ice bath after degeneration, mixing, centrifugal, carry out PCR reaction.PCR reaction condition: 40 DEG C are reacted 2 hours；70 DEG C inactivate 15 minutes；4 DEG C are reacted 5 minutes.
3) fluorescent labeling
Preparation fluorescent labeling mixed solution (LowInputQuickAmpLabelingKit, One-Color, Agilent5190-2305), particular make-up is as shown in table 4:
Table 4 fluorescent labeling mixed solution forms
| Nuclease free water | 0.75μl 5 --> |
| 5* transcription buffer | 3.2μl |
| 0.1M DTT | 0.6μl |
| NTP mix | 1.0μl |
| T7 RNA polymerase mixture | 0.21μl |
| Cy3-CTP | 0.24μl |
| Cumulative volume | 6.0μl |
Add above-mentioned 6.0 μ l fluorescent labeling mixed solutions, mixing, centrifugal, carry out PCR reaction, obtain fluorescent mark product.PCR reaction condition: 40 DEG C are reacted 2 hours；4 DEG C are reacted 5 minutes.
4) fluorescent mark product purification
A) adding the nuclease free water of 84 μ l, cumulative volume is to 100 μ l.
B) RLT of 350 μ l is added, mixing.
C) dehydrated alcohol of 250 μ l is added, mixing, it is not centrifuged.
D) by the mix of 700 μ l, forward on pillar.13000rpm, 4 DEG C of centrifugal 30sec.Abandon and flow through liquid.
E) RPE, 13000rpm, 4 DEG C of 500 μ l is added centrifugal 30 seconds.Abandon and flow through liquid.
F) RPE, 13000rpm, 4 DEG C of 500 μ l is separately added centrifugal 60 seconds.Abandon and flow through liquid.
G) sleeve pipe renewed, 13000rpm, 4 DEG C are dallied 30 seconds, and are transferred to by pillar in eluting pipe.
H) add the nuclease free water of 30 μ l, stand 1min, 13000rpm, 4 DEG C centrifugal 30 seconds.
I) again 30 μ l samples in eluting pipe are gone back to pillar, stand 1min, 13000rpm, 4 DEG C centrifugal 30 seconds.
J) RNA concentration is recorded with NanoDrop, Cy3 concentration, 260/280.
Used by fluorescent mark product purification, the requirement of probe amount is as shown in table 5:
Probe amount used by table 5 fluorescent mark product purification
| 1 × chip | cRNA>5μg | Cy3>6pmol/μg |
| 2 × chip | cRNA>3.75μg | Cy3>6pmol/μg |
| 4 × chip | cRNA>1.65μg | Cy3>6pmol/μg |
| 5 × chip | cRNA>0.825μg | Cy3>6pmol/μg |
5. chip hybridization
Fluorescent mark product after purification utilizes base complementrity Hybridization principle to hybridize with the probe on circular rna chip.Hybridization kit adopts GeneExpressionHybridizationKit (Agilent5188-5242).Specifically comprise the following steps that
1) fragmentation mixed solution is joined by table 6:
Table 6 fragmentation mixed solution forms
| Constituent | 1x | 2x | 4x | 8x |
| Cy3-cRNA | 5μg | 3.75μg | 1.65μg | 600ng |
| 10X blocker | 50μL | 25μL | 11μL | 5μL |
| Nuclease free water | Mend to 240 μ L | Mend to 120 μ L | Mend to 52.8 μ L | Mend to 24 μ L |
| 25X fragmentation buffer | 10μL | 5μL | 2.2μL | 1μL |
| Cumulative volume | 250μL | 125μL | 55μL | 25μL |
2) 60 DEG C of insulations 30min, then ice bath 1min, of short duration centrifugal.
3) isopyknic 2xGEx hybridization buffer HI-RPM is added, as shown in table 7, mixing.
Table 7 hybrid mixed solution composition
4) 13000rpm, centrifugal 1min, be subsequently placed on ice.
5) hybridization storehouse (AgilentG2534A) is placed in horizontal table top, puts belt washer coverslip, add sample by volume shown in table 8:
Table 8 hybridizes injection volume
| Constituent | 1x | 2x | 4x | 8x |
| Prepare volume | 500μL | 250μL | 110μL | 50μL |
| Hybridization volume | 490μL | 240μL | 100μL | 40μL |
6) facing down with " Agilent " on chip, lid, to coverslip, assembles rapidly hybridization storehouse, in hybrid heater (AgilentG2545A), and 65 DEG C, 10rpm, hybridize 17h.
6. chip washing, scanning
1) washing liquid 1 and washing liquid 2 add the 10%TritonX-102 of 2ml, and washing liquid 2 needs 37 DEG C of preheatings overnight.
2) chip being complete hybridization is taken out from hybrid heater, takes hybridization storehouse apart, wash chip by the step 1-3 in table 9:
Table 9 chip washing step
| Operating procedure | Washing liquid composition | Temperature | Wash time |
| Tear sheet open | GE washing liquid 1 | Room temperature | |
| Washing liquid 1 is washed | GE washing liquid 1 | Room temperature | 1min |
| Washing liquid 2 is washed | GE washing liquid 2 | 37℃ | 1min |
GE washing liquid 1 in table 9, GE washing liquid 2 are from GeneExpressionWashBufferKit (gene expression washing buffer test kit, brand: Agilent, article No.: 5188-5327)
3) washed chip is loaded in sheet folder, put into scanner (AgilentMicroarrayScannerG2565CA) and be scanned.Sweep parameter is as shown in table 10:
Table 10 chip scanning parameter
7. data analysis
Initial data is with, after limma bag normalization in R software, adopting Fold-change (differential expression multiple) and T to check (Student'st-test) statistical method that differential gene is screened.For actual a pair experiment and check sample, after cDNA microarray and data analysis, screening obtains the circRNAs that experimental group raises relative to matched group difference 2 times 1163, and the circRNAs of difference 2 times downward has 1334, altogether screens 2 times of differential expression cyclic group because of 2497.
Claims (6)
1. detect the probe of circular rna gene expression abundance, it is characterised in that described probe includes 3 ' the reverse splicing sites held sub with receptor exon for 5 ' ends of Exon of described circular rna.
2. probe according to claim 1, it is characterised in that the sequence length of described probe is 60nt.
3. probe according to claim 1 and 2, it is characterised in that described probe has the sequence as shown in SEQIDNO:3 or its complementary series.
4. detect the gene chip of circular rna gene expression abundance, it is characterised in that include the probe described in any one of claims 1 to 3.
5. by the method for genechip detection circular rna gene expression abundance described in claim 4, it is characterised in that step includes:
1) also purification sample to be tested total serum IgE is extracted；
2) ribosomal RNA is removed；
3) enrichment circular rna；
4) circular rna that enrichment is obtained carries out linear amplification mark fluorescent；
5) by step 4) fluorescent mark product that obtains hybridizes with the probe on described gene chip；
6) washing gene chip, scanning, data analysis.
6. method according to claim 5, it is characterised in that described step 3), adopt RNaseR to digest linear rna, be enriched with circular rna.
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| CN106939306A (en) * | 2017-04-11 | 2017-07-11 | 江苏吉诺思美精准医学科技有限公司 | It is a kind of based on PSP suitable for high-flux sequence elRNA library constructing methods |
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| CN111607588A (en) * | 2019-02-25 | 2020-09-01 | 上海印序生物科技有限公司 | Annular RNA rapid enrichment kit and method |
| CN110628884A (en) * | 2019-09-19 | 2019-12-31 | 中国人民解放军陆军军医大学 | Rapid identification method for circular RNA |
| CN111696627A (en) * | 2020-03-26 | 2020-09-22 | 上海生物芯片有限公司 | Design method of long-chain RNA specific probe |
| CN111696627B (en) * | 2020-03-26 | 2024-02-23 | 上海生物芯片有限公司 | Design method of long-chain RNA specific probe |
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