Summary of the invention
CircRNA be after genetic transcription RNA montage process formed, thus cyclisation connecting portion structural dna sequence andComposition can't detect in genome.
First, we are by finding the possible DNA sequence of certain circular rna from gene bank, then by design a pair evenConnecing the reverse primer (Divergent primers) near part, we are named circ-XXX-F and circ-XXX-R.CarefullyThe RNA product (cDNA) of born of the same parents or tissue can detect that, and the PCR process with genomic DNA as template can't detect.PCRResult detects through agarose gel electrophoresis result, is positive for template with cDNA, and is negative with genomic DNA for template, i.e.It may be circular rna；Both are negative or the positive is not circular rna.
Secondly, circular rna is circulus because of it, by designing a pair reverse primer at its gene internal, after PCRExpanding out by whole circular RNA molecule, further order-checking just can obtain the gene order of circular rna.Accordingly, it is determined that the first stepAfter, we will design a pair primer (Convergent primers) in the same direction, and we are named Linear-XXX-F and Linear-XXX-R.Linear-XXX-F and circ-XXX-R of these primers, or circ-XXX-F and Linear-XXX-R needs part weightFolded.
Then carrying out PCR amplification with cDNA for template, PCR result detects through agarose gel electrophoresis result, to be predictedMolecular size range consistent after, PCR primer is checked order, finally determines circular RNA molecule sequence.
Technical solution of the present invention example:
From 12 circular rnas of circBase random choose, after finding their sequence, separately design primer with DNAstar software,Specific as follows:
BARD1 hsa_circ_0001098
circR-BARD1-F:CTGACTTTCTTACTTCGAGGGCTAAAC
circR-BARD1-R:GGTATCAAAATGACTCACCACTTCACG
PRKD3 hsa_circ_0000992
circR-PRKD3-as-F:CATTCTGGGGGGATGAACTTTTC
circR-PRKD3-as-R:GGAACTGTCTTTATCTGCTGTCAAGG
RSF1 hsa_circ_0000345
circ-RSF1-as-F:CACAGTAGTCTCTTCTAGAACATTGGC
circ-RSF1-as-R:CCTTCCAAACCATCCTGAGC
CBL hsa_circ_0000363
circ-CBL-F:CACAAATGGAGCCCAGACCAG
circ-CBL-R:CCAGAACTGATGGGATGCACTTC
APLF hsa_circ_0001023
circ-APLF-F:GTGGGCCAAGATGAGACTGATG
circ-APLF-R:GATTCGCAGCTGACCACCTG
MGAT5 hsa_circ_0001068
circ-MGAT5-F: GGTACATCAAGGCACTGGCAGAAG
circ-MGAT5-R: GAAAATGGTAGCAGGATCTTGAAGC
BTBD7 hsa_circ_0000563
circ-BTBD7-as-F:GCACCCATTTTCTTCAGTCACTCAG
circ-BTBD7-as-R:GGCAGAAGAAGCCATGGAACTTTAC
KPNB1 hsa_circ_0000780
circ-KPNB1-F:CCTGTTAAGGATGTTCCAAAGCAC
circ-KPNB1-R:CTTCACCAGATTCTGTAAAGCAGCC
CNTRL hsa_circ_0001884
circR-CNTRL-F:CTTCAGGTTGTTTTAAGGCAGATGTC
circR-CNTRL-R:CTCCGACTGCCGCTTGGATG
LYN hsa_circ_0001803
circ-LYN-F:GCCATGGGATAAAGATGCCTG
circ-LYN-R:CATCGTCACTCAAGCTGTCTTTCC
The extraction of total serum IgE (with reference to Total mRNA Extract kit (Omiga) workbook, specific as follows:
(1) 293T that 80%-90% merges, rinses gently with 1 × PBS twice, fully removes unnecessary liquid, add 1 mLRNA lysate (RNA-Solv), cell is blown down by room temperature；
(2) homogenate is transferred to 1.5 mL without in the EP of RNase and add the chloroform of 200 μ L, acutely after concussion 15 seconds on iceStand 10 min；
(3) after significantly layering occurs in homogenate, putting into 4 DEG C 12,000 g is centrifuged 15 min, and at this moment homogenate just can divideIt it is three layers；
(4) careful transfer upper strata aqueous phase (less than 80 % of upper strata cumulative volume) is in new EP without a RNase pipe, and addsThe dehydrated alcohol of 1/2 times of volume, fully mixing 15 seconds；
(5) being transferred to by mixing liquid in HiBind RNA centrifugal column, room temperature 10,000 g is centrifuged 1 min, abandons effluent；, (6)Being positioned in new collecting pipe by centrifugal column, and the RNA adding 300 μ L washes miscellaneous buffer I, 10,000 g are centrifuged 30 seconds, abandonRemove effluent；
(7) again adding 400 μ L RNA in centrifugal column and wash miscellaneous buffer I, 10,000 g are centrifuged 1 min, discard effluent；(8) washing miscellaneous buffer II toward the RNA adding 500 μ L dehydrated alcohol dilutions in centrifugal column, 10,000 g are centrifuged 1 min, abandonEffluent；
(9) after repeating (8) washing, 13,000 g are centrifuged 2 min and fully remove residual liquid；
(10) centrifugal column is placed in 1.5 new mL without in RNase centrifuge tube, centrifugal column adds 30 μ L DEPC water, room temperatureAfter standing 2 min, 13,000 g are centrifuged 1 min, again suck back in centrifugal column by eluate, and repeated centrifugation once, is finally collectedEluent ,-80 DEG C of preservations.
The qualification of RNA is with quantitative: take the RNA solution of 2 l gained, in 1 % agarose gel electrophoresis, at detection of nucleic acids instrumentLower observation RNA integrity, 28S, 18S band is complete, clear, and 28S brightness is 2 times of 18S, and preservation of taking a picture, and shows RNAComplete without degraded.Separately take 2 l RNA solution, carry out quantitatively at nucleic acid quantification instrument, obtained RNA concentration, OD 260/280 ratioIn the range of 1.8-2.0, show that the RNA purity of gained is higher, reach the requirement of next step experiment.
Reverse transcription reaction and process take the total serum IgE of the 5 above-mentioned gained of μ g, operate according to M-MLV First Strand KitHandbook reverse transcription synthesis cDNA, reaction system is 20 l.Specifically comprise the following steps that
(1) in 0.2 mL microcentrifugal tube of RNase, add the total serum IgE of 1 μ g, oligo (dT) 1 μ L and 10 mM dNTP1 μ L, then moisturizing is to 12 μ L, centrifugal mixes rear 65 DEG C of degeneration 5 min, is immediately placed in cooled on ice, of short duration centrifugal after respectivelyAdd 4 μ L 5 × the first chain synthesis buffer, 2 μ L 0.1 M DTT and 1 L RNaseOUT nucleic acid inhibitor；
(2) centrifugal mixing, is subsequently placed in PCR reaction instrument 37 DEG C and hatches 2 min, add 1 μ L M-MLV subsequently inverse under room temperatureTranscriptase (200 U/ μ L), centrifugal mixing；
(3) placing back in PCR instrument, pre-set programs 37 DEG C reaction 50 min, then 70 DEG C process 15 min to inactivate reverseRecord enzyme, last 4 DEG C of coolings.Preparation cDNA be saved in-20 DEG C standby.
PCR expands circular rna fragment concrete steps:
(1) on ice, primer, cDNA/gDNA template and 5 X PCR buffer are melted；
(2) 50 μ LPCR reaction systems (PrimeSTAR HS DNA Polymerase, Code are prepared by following descriptionNo.: R010A).(5 X PCR buffer, 10 μ L；Primerstar PCR enzyme, 0.5 μ L；DNTP Mix, 4 μL；Primers F, 2 μ L；Primers R, 2 μ L；GDNA template, 1 μ L (50 ng)；DdH2O, 30.5 μ L；) totalTotally 50 μ L；
(3) by the most centrifugal for mixed box liquid, reacting in PCR instrument, condition is as follows: (98 DEG C, 3 min；98 DEG C, 15 s；68 ℃, 45 s；68 DEG C, 2 min；Totally 34 cycles) last 4 DEG C of preservations；
(4) 5 μ L PCR product are taken in 2 % DNA agarose gel electrophoresiies, 120 V, 15 min, note of taking pictures under uviol lampRecord；
(5) selecting may be for the gene of circular rna, and standard is: be positive with cDNA for template, and with genomic DNA as templateIt is negative, it is possible to for circular rna；And person is negative or the positive is not circular rna.
According to the above results, separately design following primer with DNAstar software, wherein underscore for newly synthesized, do not haveHave underscore for original primer, specific as follows:
BARD1 hsa_circ_0001098
linearR-BARD1-F:GAAGTGGTGAGTCATTTTGATACCCG
circR-BARD1-R:GGTATCAAAATGACTCACCACTTCACG
PRKD3 hsa_circ_0000992
linearR-PRKD3-as-F:GCTTCAATGGTAACACTCTCCCG
circR-PRKD3-as-R:GGAACTGTCTTTATCTGCTGTCAAGG
RSF1 hsa_circ_0000345
circ-RSF1-as-F:CACAGTAGTCTCTTCTAGAACATTGGC
linearR-RSF1-as-R:CTTGTGAAACTGCCAGTCATAGTGAAGC
CBL hsa_circ_0000363
circ-CBL-F:CACAAATGGAGCCCAGACCAG
linearR-CBL-R:GCAATGAAAATGGAAGTGAGTGCC
APLF hsa_circ_0001023
linearR-APLF-F:CACACAAATCCATGTTTTTACCAGTC
circ-APLF-R:GATTCGCAGCTGACCACCTG
MGAT5 hsa_circ_0001068
circ-MGAT5-F: GGTACATCAAGGCACTGGCAGAAG
linearR-MGAT5-R: CTGCTTTCAGGCTGAGTTCGC
BTBD7 hsa_circ_0000563
linearR-BTBD7-as-F:CCTGGTCATGTTTGGCTTCC
circ-BTBD7-as-R:GGCAGAAGAAGCCATGGAACTTTAC
KPNB1 hsa_circ_0003650
circ-KPNB1-F:CCTGTTAAGGATGTTCCAAAGCAC
linearR-KPNB1-R:CTGAAGAGTTGCACAGAGTAAAGACTGAAG
CNTRL hsa_circ_0001884
linearR-CNTRL-F:GCGGGAAGAAAGGTGGATGAG
circR-CNTRL-R:CTCCGACTGCCGCTTGGATG
LYN hsa_circ_0001803
circ-LYN-F:GCCATGGGATAAAGATGCCTG
linearR-LYN-R:CCAATCTTCTGCACAAGCCATC。
PCR expands circular rna fragment concrete steps:
(1) on ice, primer, cDNA template and 5 X PCR buffer are melted；
(2) 50 μ LPCR reaction systems (PrimeSTAR HS DNA Polymerase, Code are prepared by following descriptionNo.: R010A).(5 X PCR buffer, 10 μ L；Primerstar PCR enzyme, 0.5 μ L；DNTP Mix, 4 μL；Primers F, 2 μ L；Primers R, 2 μ L；GDNA template, 1 μ L (50 ng)；DdH2O, 30.5 μ L；) totalTotally 50 μ L；
(3) by the most centrifugal for mixed box liquid, reacting in PCR instrument, condition is as follows: (98 DEG C, 3 min；98 DEG C, 15 s；68 ℃, 45 s；68 DEG C, 2 min；Totally 34 cycles) last 4 DEG C of preservations；
(4) 5 μ L PCR product are taken in 2 % DNA agarose gel electrophoresiies, 120 V, 15 min, note of taking pictures under uviol lampRecord；
(5) PCR result detects through agarose gel electrophoresis result, after band position is consistent with the molecular size range of prediction,PCR primer is checked order, finally determines circular RNA molecule sequence；
(6) PCR primer reclaims and sample presentation carries out gene sequencing.
Result and analysis:
Due to we design primer detection fragment all between 100-250 bp, agarose gel electrophoresis such as Fig. 1 institute after PCRShowing, in this regional extent, the sequence of circular rna junction can detect in c (cDNA) template group, and at g (genomic DNA)Template detection less than, can tentatively judge the existence of this circular rna, we have seen that from result, such as BARD1, PRKD3,RSF1, CBL, APLF, MGAT5, BTBD7, KPNB1, CNTRL have significantly band in region.And CBL is due to equal in c and g groupThere is band, it is likely that for non-specific, need next step PCR to determine.Next full genome is expanded by we with linear primer.
Result is as in figure 2 it is shown, PRKD3, and RSF1, CBL, APLF, MGAT5, BTBD7 all can detect relatively bright band,Compared with prediction stripe size, PRKD3 about 943 pb, RSF1 about 1982 bp, APLF about 1190 bp, BTBD7 about 1268 bp,And CBL and MGAT5 becomes clear, pillar location and prediction is inconsistent, is not likely to be circular rna to be detected.CNTRL, MGAT5 are thenMore non-specific band occurs, is also regarded as can not be detected.We carry out gene survey after the recovery of bright band for next stepSequence, in order to finally determine whether the circular rna detected by us.
Through gene sequencing result, sequence is consistent with the circular rna sequence of prediction, such as Fig. 3, shown in 4,5,6.
In sum, the present invention is a kind of method that batch quickly detects circular rna, first, to circular rna interestedFirst synthesizing the primer circ-XXX-F/R of its junction, whether PCR detection junction exists in batches, if there is after, then setCount one group of linear Yu circ-XXX-F/R and there is partly overlapping primer, through PCR, whole circular rna is expanded, after through surveyingSequence determines its sequence accuracy.
Fig. 1 circular rna junction primer circ-XXX-F/R is through PCR batch Preliminary detection, and the region between dotted line is 100-The fragment of 250 bp sizes, detects with c (cDNA) template group in this region, and in g (genomic DNA) template detectionLess than band may be i.e. circular rna；
The full-length gene PCR result of Fig. 2 circular rna, screens the gene of obvious band from Fig. 1, design one group of linear withThere is partly overlapping primer in circ-XXX-F/R, it is ring-type that what PCR result was similar with the size that predicts the outcome is i.e. likely to thisRNA；
Fig. 3 circular rna PRKD3(hsa_circ_0000992) full length fragment sequencing result；
Fig. 4 circular rna RSF1(hsa_circ_0000345) full length fragment sequencing result；
Fig. 5 circular rna APLF(hsa_circ_0001023) full length fragment sequencing result；
Fig. 6 circular rna BTBD7(hsa_circ_0000563) full length fragment sequencing result.