CN104328112B - A kind of method of the endogenous circular rna of quantitative detection for non-disease diagnostic purpose - Google Patents
A kind of method of the endogenous circular rna of quantitative detection for non-disease diagnostic purpose Download PDFInfo
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Abstract
The invention discloses a kind of method for preparing external source circular rna and the method for quantitatively detecting endogenous circular rna.The present invention artificial synthesized DNA genes are cloned, breed, transcription DNA gene be linear rna afterwards, by RNA it is end modified after, head and the tail connection, be prepared into external source circular RNA molecule.External source circular RNA molecule is added in total serum IgE in specific proportions, reference gene during as endogenous circular rna Real_time quantitative detection, realizes the Real_time quantitative detection to endogenous circular rna first.
Description
Technical field
The present invention relates to biology field, more particularly to a kind of quantitative detection for non-disease diagnostic purpose are endogenous
The method of circular rna.
Background technology
Most RNA (Ribonucleic acid, ribonucleic acid) is linear in organism, but has small part RNA first
Tail connects to form ring-type, referred to as circular rna.In recent years, circular rna rapidly becomes the focus of RNA world research, because
Circular rna is found closely related with important biomolecule function and disease etc..In addition, high throughput sequencing technologies are applied to detect
Circular rna, greatly accelerate the discovery to circular rna and the analysis to circular rna function.
For RNA, its expression quantity is a highly important attribute.Whether RNA expresses and the number of expression quantity,
All decide RNA biological function.For example, the generation of many diseases is exactly because caused by the exception of rna expression amount.Weigh
Measure RNA expression quantity, it is necessary to which the RNA of an expression quantity relative constancy under various regimes is as reference gene.By a large amount of
Research find, the linear isogenic expression quantity relative constancy of actin (Actin), often as linear rna expression ginseng
Examine gene.
During the present invention is realized, inventor has found that prior art at least has problems with：
Known constant expressing gene is linear rna molecule, it is impossible to the reference gene as circular RNA molecule so that
The circular rna expression quantity of different sample rooms compares shortage normative reference, thus lacks effective circular rna detection technique, greatly
Ground limits the research and application of circular rna.
The content of the invention
In order to solve the problems, such as to lack the reference gene of circular rna in the prior art, the embodiments of the invention provide one kind
The method for preparing the method for external source circular rna and quantitatively detecting endogenous circular rna.The technical scheme is as follows：
On the one hand, the embodiments of the invention provide a kind of method for preparing external source circular rna, methods described to include：
Select external source linear rna gene；
By the external source linear rna gene cloning into prokaryotic expression carrier, the external source linear rna gene of clone is obtained；
The external source linear rna gene of the clone is transcribed in vitro, synthesizes external source linear rna；
The triphosphoric acid structure at 5 ' ends of the external source linear rna is removed, and in 5 ' end synthesis hydroxyls of the external source linear rna
Base；
Phosphorylation modification, the institute of the phosphorylated modification in the end of synthesis 5 ' are carried out for the external source linear rna of hydroxyl to 5 ' ends
State external source linear rna；
5 ' ends of the external source linear rna of described 5 ' the phosphorylated modifications in end and 3 ' ends are connected, synthesize external source circular rna；
Cut away and be not cyclized the successful external source linear rna, obtain external source circular rna.
Specifically, DNA sequence dna such as sequence table SEQ ID NO corresponding to the external source linear rna gene:Shown in 1.
Specifically, cut away using RNase R and be not cyclized the successful external source linear rna.
On the other hand, the invention provides a kind of method for quantitatively detecting endogenous circular rna, methods described to include：
Adopt and prepare external source circular rna with the aforedescribed process；
Quality control is carried out to the external source circular rna；
The external source circular rna, the total serum IgE and the external source are added into the total serum IgE of sample to be checked and check sample
The mass ratio of circular rna is 2000:1, obtain the mixture of the external source circular rna and the total serum IgE；
Detect the external source circular rna and the target in the mixture of the total serum IgE respectively using real time quantitative PCR method
The content of circular rna and the external source circular rna；
The expression of target circular rna described in the sample to be checked is determined using the external source circular rna as reference gene
Amount.
Specifically, it is described that external source circular rna progress quality control is included：
Reverse transcription, synthesis external source cDNA are carried out to the external source circular rna of synthesis using random primer；
Using the first primer and the second primer, cDNA linear to the external source and external source ring-type cDNA is carried out in fact respectively
When quantitative pcr amplification, obtain C respectivelyT1 value and CT2 values, first primer are included such as SEQ ID NO in sequence table:Shown in 2
SEQ ID NO in first forward primer and such as sequence table:The first reverse primer shown in 3, second primer include such as sequence
SEQ ID NO in table:SEQ ID NO in the second forward primer and such as sequence table shown in 4:The second reverse primer shown in 5；
Pass through CT1 value and CT2 values and formulaCalculate the ring of the external source circular rna of synthesis
Change ratio, and select the external source circular rna of the cyclisation ratio more than 90%.
Specifically, it is described that the external source circular rna, the total serum IgE and the external source are added into the total serum IgE of sample to be checked
The mass ratio of circular rna is 2000:1, the mixture of the external source circular rna and the total serum IgE is obtained, including：
Extract and purify the total serum IgE of the sample to be checked and the check sample；
The mass concentration of the total serum IgE of measurement after purification；
According to the mass concentration of the sample to be checked and the total serum IgE of the check sample according to the total serum IgE with it is described outer
The mass ratio of source circular rna is 2000:1 ratio mixes with the external source circular rna, obtains the external source circular rna and institute
State the mixture of total serum IgE.
Further, the quality using sample to be checked and the total serum IgE of the check sample described in spectrophotometer measurement is dense
Degree.
Specifically, it is described to detect the mixed of the external source circular rna and the total serum IgE respectively using real time quantitative PCR method
The content of target circular rna and the external source circular rna in compound, including：
Reverse transcription is carried out to the total serum IgE for adding the external source circular rna using the random primer, reversed
Product is recorded, the reverse transcription product includes the target ring-type cDNA and external source ring-type cDNA；
The target ring-type cDNA of the sample to be checked and the check sample is determined respectively using three-primer
PCR detections are measured, obtain C after amplification respectivelyT3 values and CT4 value, using second primer respectively to the sample to be checked with it is described
The external source ring-type cDNA of check sample carries out quantitative PCR detection, obtains CT after amplification respectively5Value and CT6Value；
The amplified production of the three-primer and second primer spans the cyclisation node of circular rna.
Further, according to formulaCalculate target ring-type described in the sample to be checked
RNA ratio.
Further, the amplification program of the real-time quantitative PCR is：95 DEG C 20 seconds；95 DEG C 3 seconds, 60 DEG C 20 seconds, totally 40
Circulation.
The beneficial effect that technical scheme provided in an embodiment of the present invention is brought is：The embodiments of the invention provide one kind to prepare
The method of external source circular rna, its external source circular rna obtained can solve endogenous ring as the reference gene of endogenous circular rna
The defects of shape RNA can not carry out real-time quantitative PCR detection due to shortage reference gene, contribute to the research of circular rna with answering
With.The method provided in an embodiment of the present invention for quantitatively detecting endogenous circular rna, the external source circular rna of preparation is added and treats sample
This is with the total serum IgE of check sample, and together being expanded and being detected with the total serum IgE of sample to be checked and check sample.Thus, outside
Source circular rna has substantially been equal to the endogenous circular rna of stable expression, can be used as different samples between detect endogenous ring
The reference gene of shape rna expression amount, solves the problem of no endogenous circular rna reference gene, it is achieved thereby that to endogenous ring
The purpose of shape rna expression amount detection.
Brief description of the drawings
Technical scheme in order to illustrate the embodiments of the present invention more clearly, make required in being described below to embodiment
Accompanying drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the present invention, for
For those of ordinary skill in the art, on the premise of not paying creative work, other can also be obtained according to these accompanying drawings
Accompanying drawing.
Fig. 1 is the method flow diagram for the preparation external source circular rna that the embodiment of the present invention one provides；
Fig. 2 is the method flow diagram for the preparation external source circular rna that the embodiment of the present invention two provides.
Embodiment
To make the object, technical solutions and advantages of the present invention clearer, embodiment of the present invention will be made into one below
It is described in detail on step ground.
Embodiment one
The embodiments of the invention provide a kind of method for preparing external source circular rna, as shown in figure 1, this method includes：
Step 101：Select external source linear rna gene；
Step 102：By external source linear rna gene cloning into prokaryotic expression carrier, the external source linear rna base of clone is obtained
Cause；
Step 103：By the external source linear rna gene of clone reverse transcription in vitro, external source linear rna is synthesized；
Step 104：The triphosphoric acid structure at 5 ' ends of external source linear rna is removed, and in 5 ' end synthesis hydroxyls of external source linear rna
Base；
Step 105：Phosphorylation modification, the phosphorylated modification in the end of synthesis 5 ' are carried out for the external source linear rna of hydroxyl to 5 ' ends
External source linear rna；
Step 106：5 ' ends of the external source linear rna of 5 ' the phosphorylated modifications in end and 3 ' ends are connected, synthesize external source ring-type
RNA；
Step 107：Cut away and be not cyclized the successful external source linear rna, obtain external source circular rna；
Step 108：Purify external source circular rna.
Specifically, sample to be checked provided in an embodiment of the present invention is Chlamydomonas reinhardtii, and the strain of the Chlamydomonas reinhardtii is CC503
(chlamydomonas resource center is purchased from, network address is：http://chlamycollection.org/).
Step 101：Select external source linear rna gene, DNA sequence dna such as sequence table SEQ corresponding to the external source linear rna gene
ID NO:Shown in 1.Above-mentioned SEQ ID NO:1 and its complementary strand synthesized by Sangon Biotech (Shanghai) Co., Ltd..It is logical
NCBI blast program retrieval is crossed, does not find the high gene of the homology that external source linear rna gene in biology be present.Selection
External source linear rna gene of length no more than 1000bp.It with the addition of respectively at 5 ' ends of external source linear rna gene and 3 ' ends
Ctcgag and aagctt sequences, ctcgag the and aagctt sequences are respectively restriction enzyme XhoI and HindIII digestion
Site, it is easy to external source linear rna gene cloning entering prokaryotic expression carrier, is connected at 5 ' ends of external source linear rna gene
Taatacgactcactata sequences are the promoter sequence of T7 transcriptases, and remaining sequence is transcribed sequence, and
Ggg sequences are connected with after taatacgactcactata sequences, ggg sequences can increase the transcriptional efficiency of T7 transcriptases.
Step 102：By external source linear rna gene cloning into prokaryotic expression carrier, the external source linear rna base of clone is obtained
Cause, including：
Selected cloning vector is that (cloning vector wins profit biotechnology from Changsha to be had pBluescript II SK (+)
Limit company buys, article No. VKS0288), using restriction endonuclease XhoI (buying from NEB companies, article No. R0146) and restriction endonuclease
HindIII (buying from NEB companies, article No. R0104) carries out double digestion to pBluescript II SK (+) carrier.Specifically
Ground, in 20 μ l reaction system, include 10 μ g pBluescript II SK (+) carriers, 20U restriction endonuclease HindIII,
20U restriction endonuclease XhoI and 2 × NEBuffer 2.1 (being provided by NEB companies).Above-mentioned reaction system is well mixed, through short
Temporarily centrifugation, after 37 DEG C of insulations 1 hour, through 80 DEG C 20 minutes by enzyme-deactivating, and obtain linear pBluescript II SK (+)
Carrier, the concentration of linear pBluescript II SK (+) carrier is 10/20=0.5 μ g/ μ l.
By DNA sequence dna (such as sequence table SEQ ID NO of external source linear rna gene:Shown in 1) and its complementary strand sterilized water
After dissolving, the solution that concentration is 0.5 μ g/ μ l is configured to respectively, by volume 1：1 is well mixed, undergoes 94 successively in PCR instrument
DEG C 10 minutes；65 DEG C 10 minutes；37 DEG C 10 minutes, formed external source double-stranded DNA gene.
Include linear μ l of pBluescript II SK (+) carrier 10, external source double-stranded DNA in 20 μ l reaction system
μ l of gene 1,400U T4DNA ligases (buying from NEB companies, article No. M0202) and 1 × T4DNA ligase buffer solutions (with
T4DNA ligases are bought from NEB companies together), after 16 DEG C are incubated overnight, 65 DEG C 10 minutes, T4DNA ligases through heat inactivation,
Obtain pBluescript II SK (+) plasmid vector containing external source linear rna gene.
PBluescript II SK (+) plasmid vector containing external source linear rna gene is converted using heat shock method and entered
Escherichia coli, specific method are as follows：50 μ l competent cells are taken (to be produced by Beijing Quanshijin Biotechnology Co., Ltd, catalogue
Number it is CD501) naturally to thaw in ice bath, add pBluescript II SK (+) plasmid containing external source linear rna gene
Carrier, gently mix, be incubated 30 minutes on ice bath, 42 DEG C of heat shocks 30 seconds, fast transfer ice bath 3 minutes into ice bath, the mistake
Centrifuge tube is not shaken in journey, then add 300 μ l without antibiotic sterile LB fluid nutrient mediums (LB fluid nutrient mediums into
Part：Beef extract 0.5g, peptone 1.0g, sodium chloride 0.5g, distilled water 100mL, pH7.2~7.5), in 37 DEG C 200 revs/min,
Recovery 1 hour, the μ l of bacterium solution 200 after recovery are taken to be spread evenly across LB solid mediums (the LB solids containing amicillin resistance
Culture medium includes：Tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L and agar powder 15g/L) on, treat that bacterium solution is complete
It is inverted in after hypersorption in 37 DEG C of constant incubators, light culture is overnight.The monoclonal to be grown on picking LB solid mediums is extremely
In LB fluid nutrient mediums of the 5ml containing ampicillin, through 37 DEG C 200 revs/min, bacterium amplification breeding 6-8 hours are shaken, are prepared into sense
By state cell.
Using TIANGEN Biotech (Beijing) Co., Ltd. production the small extraction reagent kit (article No. DP105) of rapid plasmid from
In above-mentioned competent cell, extract and purify pBluescript II SK (+) plasmid vector containing external source linear rna gene
DNA, the method for extraction and purification carries out according to the specification provided with kit.Utilize spectrophotometer (U.S. Quawell
Company produces, model Q5000) in double-stranded DNA program, detect the matter of pBluescript II SK (+) plasmid after purification
Measure concentration.Expanded outside from pBluescript II SK (+) plasmid after purification using the PCR primer of external source linear rna gene
Source linear rna gene.Specifically, 20 μ l amplification system includes：PBluescript II SK (+) plasmid 50ng after purification,
10 μ l Q5 high-fidelity amplifications mixtures (being purchased from NEB companies, article No. M0492) and 10 μM of external source linear rna gene
PCR primer (PCR primer is the equimolar mixture of forward primer and reverse primer).Amplification program is as follows：98 DEG C 30 seconds；
98 DEG C 8 seconds, 56 DEG C 20 seconds, 72 DEG C 20 seconds, totally 30 circulation.PCR primer is purified using ethanol precipitation, purified product is molten
Solution without in enzyme water, obtains the PCR primer of external source linear rna gene in 10 μ l, using Q5000 (Quawell companies of the U.S. produce,
Model Q5000) in double long, rectangular bag DNA quant programs quantitative detection is carried out to PCR primer, testing result shows：This amplification obtains
The PCR primer concentration of the external source linear rna gene obtained is 560ng/ μ l.The PCR primer of external source linear rna gene is delivered into Wuhan
Qing Ke Bioisystech Co., Ltd is sequenced, and the correctness of the external source linear rna gene of clone is confirmed, so as to obtain with correct gram
PBluescript II SK (+) plasmid of grand external source linear rna gene.
Wherein, SEQ ID NO in the forward primer of the PCR primer of external source linear rna gene such as sequence table:Shown in 9, external source
SEQ ID NO in the reverse primer of the PCR primer of linear rna gene such as sequence table:Shown in 10.SEQ ID NO in sequence table:9
In poly T-sequence be in order to transcription when obtain poly A, for simulating biological RNA poly A tract bar, there is poly A tract bar
RNA can be by Poly (T) primer reverse transcriptions into cDNA.Meanwhile poly A tract bar can also be used for purifying RNA.
Step 103：The external source linear rna gene of clone is transcribed in vitro, synthesizes external source linear rna, including：
Using T7RNA rapidly and efficiently synthetic agent box (being purchased from NEB companies, article No. E2050) transcription synthesis external source lines
Property RNA.Composition in the kit includes：, should without enzyme water, the buffer solution mixture containing NTP and t7 rna polymerase mixture
Reaction system includes：The PCR primer of the above-mentioned external source linear rna genes of 1 μ g, 10 μ l contain NTP buffer solution mixture and 2 μ l
T7 rna polymerase mixture, add water to supply 20 μ l and be well mixed, and of short duration centrifugation, after 37 DEG C are incubated 2 hours, add 30 μ l
Without enzyme water and 2 μ l DNase I (being purchased from NEB companies, article No. M0303), after being well mixed, 15 minutes are incubated through 37 DEG C, is gone
Except DNA profiling.Dynabeads mRNA purification kits (being produced by ABI companies, article No. 61006) are recycled to be purified,
And purified product is dissolved without enzyme water with 50 μ l, obtain the external source linear rna of purifying.
Step 104：The triphosphoric acid structure at 5 ' ends of external source linear rna is removed, and in 5 ' end synthesis hydroxyls of external source linear rna
Base.Because 5 ' ends of the external source linear rna synthesized in vitro are triphosphoric acid structure so that 5 ' ends of external source linear rna can not be with 3 ' ends
Connection forms ring-type, so must go to except the triphosphoric acid structure at the 5 ' end, then adds monophosphate structure, such external source line at 5 ' ends
Property RNA head and the tail could be connected circlewise, and specific steps include：
5 ' ends of external source linear rna are removed using calf intestine alkaline phosphatase (being purchased from NEB companies, article No. M0290)
Triphosphoric acid structure, be formulated as follows reaction system：The external source linear rna of 20 μ g purifying and 7U calf intestine alkaline phosphatase, are used
Water is well mixed after supplying 20 μ l, and through of short duration centrifugation, 1 hour is incubated in 37 DEG C, obtains the external source for removing 5 ' end triphosphoric acid structures
Linear rna.5 ' ends are eliminated using Dynabeads mRNA purification kits (being produced by ABI companies, article No. 61006) purifying
The external source linear rna of triphosphoric acid structure, obtain external source linear rna of the 5 ' ends for hydroxyl.
Step 105：Phosphorylation modification is carried out for the external source linear rna of hydroxyl to 5 ' ends, the end of synthesis 5 ' phosphorylation modification
External source linear rna, including：
External source linear rna of the 5 ' end for hydroxyl is modified (by NEB companies, article No. M0201) using T4PNK kinases.With
What enzyme provided together has 10 × T4PNK kinase buffer liquids.In 50 μ l reaction system, following composition is included：1 × T4PNK swashs
Enzyme buffer liquid, 1mM ATP, 10U T4PNK kinases and 5 ' ends of above-mentioned acquisition are the external source linear rna of hydroxyl, are supplied with water
50 μ l are simultaneously well mixed, and through of short duration centrifugation, 30 minutes are incubated in 37 DEG C.Using Dynabeads mRNA purification kits (by ABI
Company produces, article No. 61006) purified, operating method is carried out by the specification of the kit, is obtained 5 ' end phosphorylations and is repaiied
The external source linear rna of decorations.
Step 106：5 ' ends of the external source linear rna of 5 ' end phosphorylation modifications and 3 ' ends are connected, synthesize external source ring-type
RNA, including：
It is linear to the external source of 5 ' the end phosphorylation modification using T4RNA ligases I (NEB companies produce, article No. M0204)
RNA 5 ' ends are attached with 3 ' ends.Also include with the T4RNA ligases I compositions provided：10 × T4RNA ligases
Reaction buffer, 10mM ATP and PEG 8000.Following composition is included in 20 μ l anti-system：1 × T4RNA connection enzyme reactions
Buffer solution, the 10U/ μ l μ l of T4RNA ligases 1,10U/ μ l μ l of RNase inhibitor 0.5, concentration be 10% PEG8000,
20-50 μM of ATP and 5 ' end phosphorylation modifications of above-mentioned acquisition external source linear rna, supply 20 μ l with water and are well mixed, pass through
Of short duration centrifugation, in PCR instrument in 16 DEG C overnight, through 95 DEG C of 2 minutes terminating reactions, obtain external source circular rna.
External source circular rna is purified, specifically, following composition is added into the obtained mixture containing external source circular rna：
5M ammonium acetate (Ambion companies produce, article No. AM9071) 10 μ l, the μ l of ethanol 200 and the μ l of water 80 that concentration is 100%, is mixed
Close uniformly, through of short duration centrifugation, be placed in -80 DEG C of refrigerators (company of Haier produces, model DW-86L626) and place 30 minutes；Take
Through 4 DEG C of 14000rpm centrifugations 25 minutes after going out, supernatant liquor is carefully sucked with the pipette tips of pipettor, 300 μ l are added into precipitation
Concentration is 70% ethanol, is precipitated for cleaning, then sucks supernatant liquor after 4 DEG C of 14000rpm centrifugations 5 minutes, in room temperature
Under, air drying 10 minutes, for removing the ethanol of residual, then dissolved and precipitated without enzyme water with 10 μ l, obtain the external source of purifying
Circular rna.
Step 107：Cut away using RNase R and be not cyclized successful external source linear rna, obtain external source circular rna, specific step
Suddenly include：
In the external source circular rna of the purifying of acquisition, there is part not to be cyclized successful external source linear rna, utilize RNase
R (being produced by Epicentre companies, article No. RNR07250), which is cut away, is not cyclized successful external source linear rna.With RNase R mono-
Also 10 × RNase R the reaction buffers provided are provided, 2 μ 10 × RNases of l are added into the external source circular rna of the purifying of acquisition
R reaction buffers, 1 μ l 20U/ μ l RNase R and the μ l of water 7 and be well mixed, through it is of short duration centrifugation after 40 DEG C be incubated 1 hour,
Obtain the external source circular rna for eliminating external source wire RNA.
Step 108：Purifying eliminates external source wire RNA external source circular rna, including：
Following composition is added into the external source circular rna for eliminating external source wire RNA of acquisition：5M ammonium acetate
(Ambion companies produce, article No. AM9071) 10 μ l, the μ l of ethanol 200 and the μ l of water 80 that concentration is 100% are simultaneously well mixed, and are passed through
Of short duration centrifugation, it is placed in -80 DEG C of refrigerators (company of Haier produces, model DW-86L626) and places 30 minutes, passed through after taking-up
4 DEG C of 14000rpm is centrifuged 25 minutes, carefully sucks supernatant liquor with the pipette tips of pipettor, 300 μ l concentration are added into precipitation is
70% ethanol, precipitated for cleaning, then supernatant liquor, at room temperature, air are sucked after 4 DEG C of 14000rpm centrifugations 5 minutes
Middle drying 10 minutes, for removing the ethanol of residual, dissolved and precipitated without enzyme water with 50 μ l, obtain the external source circular rna of purifying.
The method provided in an embodiment of the present invention for preparing external source circular rna, in order to determine the expression quantity people of endogenous circular rna
Work synthesizes external source circular rna as reference gene, is proportionally added into total serum IgE, with the endogenous ring-type of stable expression in analogue body
RNA.In artificial synthesized external source circular rna provided in an embodiment of the present invention, first, designed external source circular rna and external source
Linear rna in vivo should be without the high sequence of identical sequence and homology, because after adding total serum IgE, being can not be by external source
What RNA and endogenous RNA distinguished, if the two exist it is overlapping, will this real-time quantitative be substantially false with " the constant expression of reference gene "
If contradiction, later stage quantitative accuracy is directly affected.In addition, foreign gene length can not be oversize, cyclisation efficiency is otherwise influenceed.
It is larger in view of the RNA amounts of synthesis, and need to be commonly used, DNA sequence dna corresponding to RNA is transferred to plasmid by the embodiment of the present invention
In, plasmid is transferred in Escherichia coli, breeds plasmid as Escherichia coli breed, meanwhile, corresponding DNA amount is added, from
And flexibly obtain a large amount of DNA sequence dnas.Again by in-vitro transcription, DNA is changed into RNA.Therefore, the body in DNA Front-end Designs
Outer transcriptional promoter sequence.The RNA being transcribed into needs to purify, and in order to realize this purpose, and devises oligomerization in DNA end
T, forms oligomerization A tails after transcription, oligomerization A can be used for purifying RNA.The exogenous RNA of synthesis is wire, it is necessary to could be into after being cyclized
For circular rna.5 ' the ends of RNA during due to synthesis are triphosphoric acid group, and when connecting, it is desirable that monophosphate group, therefore,
After the triphosphoric acid group for eliminating exogenous RNA, along with this step of monophosphate group, so as to synthesize the precursor that can be cyclized cyclization
RNA state, most the head and the tail connection of the RNA, closure cyclization, preparation turn into external source circular RNA molecule at last.By the external source of preparation
Circular rna is added in the total serum IgE of sample to be checked, and is together expanded with the total serum IgE of sample to be checked, therefore, it is possible in being used as
The reference gene of source circular rna, for weighing the expression quantity of endogenous circular rna, so as to quantitatively be detected for endogenous circular rna.
Embodiment two
The embodiments of the invention provide a kind of method of the quantitative detection circular rna for non-disease diagnostic purpose, such as Fig. 2
Shown, this method includes：
Step 100：The method provided using the embodiment of the present invention one prepares external source circular rna.
Step 200：Quality control is carried out to external source circular rna.
Step 300：External source circular rna, total serum IgE and external source ring-type are added into the total serum IgE of sample to be checked and check sample
RNA mass ratio is 2000:1, obtain the mixture of external source circular rna and total serum IgE.
Step 400：Detect the target in the mixture of external source circular rna and total serum IgE respectively using real time quantitative PCR method
The content of circular rna and external source circular rna.
Step 500：Using external source circular rna as reference gene, the relative expression of the target circular rna in sample to be checked is determined
Amount.
Wherein, sample to be checked provided in an embodiment of the present invention is that detection expression change is needed in real-time quantitative PCR detection
Sample, check sample is sample for reference when real-time quantitative PCR detects changes in gene expression in sample to be checked.
In early-stage Study, the ring-type that an expression quantity is higher in Chlamydomonas reinhardtii is found using the mode of high-flux sequence
RNA, be named as ring-type cRNA006, cRNA006 linear order such as sequence table in SEQ ID NO:, will be linear shown in 8
CRNA006 5 ' ends join end to end with 3 ' ends, just form ring-type cRNA006.Shown by technical Analysis such as high-flux sequences：
With normal growing conditions (check sample：Growth temperature is 25 DEG C) compare, Chlamydomonas reinhardtii (treats sample under low temperature growth conditions
This：Growth temperature is 5 DEG C, and the other conditions all same of sample to be checked and check sample), under ring-type cRNA006 expression quantity
More than 2 times have been dropped, it is the circular RNA molecule of a low temperature response to show cRNA006, may have cold-resistant biological function,
It is worth further research, it is then desired to further confirm that it expresses variation tendency.The present embodiment utilizes prepared external source ring-type
RNA is reference gene, demonstrates the downward expression trend of cRNA006 under cryogenic.
Step 200：Quality control is carried out to external source circular rna, including：
1st, reverse transcription is carried out to the external source circular rna of synthesis using random primer, synthesizes external source cDNA, specifically, utilized
RNA quant programs in spectrophotometer (Quawell companies of the U.S. produce, model Q5000), determine and obtain the external source of purifying
The mass concentration of circular rna, the mass concentration for the external source circular rna that the present embodiment provides is 355ng/ μ l.
The μ g of external source circular rna 0.5 for obtaining and purifying are taken, are mixed with following ingredients：5 μ l concentration are that 1 μM of 6 bases are random
Reverse transcriptase primer (being synthesized by Sangon Biotech (Shanghai) Co., Ltd.), 2 μ l concentration are inverse for 10mM dNTP, 20U's
Transcriptase (Invitrigen companies of the U.S. produce, article No. 18064-014), the DL- dithiothreitol (DTT)s that 5 μ l concentration are 100mM
(DL-Dithiothreitol, DTT, being provided with reverse transcriptase by Invitrigen companies of the U.S.) and 10 5 × reverses of μ l
Reaction buffer (being provided with reverse transcriptase by Invitrigen companies of the U.S.) is recorded, after supplying 50 μ l mixings with water, through 42
DEG C insulation 2 hours, 75 DEG C are incubated 15 minutes, inactivate enzyme, synthesis external source cDNA.
2nd, using the first primer and the second primer, cDNA linear to external source and external source ring-type cDNA carries out real-time quantitative respectively
PCR is expanded, and obtains C respectivelyT1 value and CT2 values, the first primer are included such as SEQ ID NO in sequence table:First shown in 2 is positive
SEQ ID NO in primer and such as sequence table:The first reverse primer shown in 3, second primer are included such as SEQ in sequence table
ID NO:SEQ ID NO in the second forward primer and such as sequence table shown in 4:The second reverse primer shown in 5；Wherein, first
Cyclisation tie point of the amplified production of primer not across external source circular rna so that both amplifiable external source is linear for first primer
CDNA, and amplifiable external source ring-type cDNA；Second primer or its amplified production cross over the cyclisation tie point of external source circular rna,
So that second primer can only expand external source ring-type cDNA.Real-time quantitative PCR is expanded including two classes, and one kind is expanded using the first primer
Increase, another kind of to be expanded using the second primer, two classes expand parallel progress, in addition to primer is different, other all other reaction compositions
With condition all same.Specific amplification procedure is as follows：Take the above-mentioned external source cDNA of 2 μ l, the first primer that 3 μ l concentration are 1 μM, 10 μ l determine
50 times of amount PCR mixtures (being produced by Toyobo companies, article No. QPS-201) and 0.4 μ l ROX fluorescence corrections dyestuff (by
Toyobo companies provide with QPS-201) it is well mixed after, through of short duration centrifugation, in ABI StepOne real-time PCRs
In by following amplification program carry out real-time quantitative PCR amplification：95 DEG C 20 seconds；95 DEG C 3 seconds, 60 DEG C 20 seconds, rear two step (95 DEG C 3 seconds,
60 DEG C 20 seconds) totally 40 circulations, collect fluorescence signal in the final step that circulates each time, the power of the fluorescence signal is used to weigh
Measure expression quantity number.
3rd, C is passed throughT1 value and CT2 values and formulaCalculate the cyclisation of the external source circular rna of synthesis
Ratio, when the ratio of cyclisation is more than 90%, external source circular rna can use.Specific steps include：Obtained by the amplification of the first primer
Obtain CT1 value, the CT1 value is 21.55, and the second primer is expanded using same method, and obtains CT2 values, the CT2 values are
21.45 by CT1 value and CT2 values substitute into formula calculate external source circular rna cyclisation ratio be：According to the cyclisation ratio of external source circular rna more than 90%
When, the external source circular rna can use, if the cyclisation ratio of external source circular rna is not less than 90%, prove external source ring-type
RNA cyclisation is unqualified, i.e., can not use, it is necessary to prepare external source circular rna again.Judge the cyclisation ratio of the external source circular rna
The experimental experience of example is made, and the cyclisation ratio can be adjusted according to specific requirement of experiment.
Step 300：External source circular rna, total serum IgE and external source ring-type are added into the total serum IgE of sample to be checked and check sample
RNA mass ratio is 2000:1, the mixture of external source circular rna and total serum IgE is obtained, including：
1st, extract and purify the total serum IgE of sample to be checked, including：Take the logarithm the Chlamydomonas reinhardtii 5ml in growth period, and the Lay taken
The number of mattress chlamydomonas is less than 40,000,000, if more than 40,000,000, accordingly down-samples volume, and 1 is centrifuged through 4000rpm normal temperature
Minute, after removing the nutrient solution on upper strata, (produced using Trizol reagents by Invirtigen companies, article No. 15596-018)
The total serum IgE for extracting Chlamydomonas reinhardtii is simultaneously dissolved in the water of 50 μ l nuclease frees, the method for the extraction and purification of total serum IgE according to
The operation manual of Trizol reagents is carried out.
2nd, the mass concentration of the total serum IgE of measurement after purification, including：Using spectrophotometer, (spectrophotometer is by the U.S.
Quawell companies produce, model Q5000) in RNA quant programs, determine and obtain the total serum IgE of the Chlamydomonas reinhardtii of said extracted
Mass concentration.In the present embodiment, Chlamydomonas reinhardtii (5 DEG C) (sample to be checked) and normal growing conditions under cryogenic are determined
Under the mass concentration of total serum IgE of (25 DEG C) (check sample) be respectively 1.52 μ g/ μ l and 2.00 μ g/ μ l.Thus calculate, extraction
The amount of the total serum IgE of (25 degree) is respectively 76 μ g and 100 μ g to Chlamydomonas reinhardtii under (5 DEG C) and normal growing conditions under cryogenic.
3rd, the quality according to the mass concentration of sample to be checked and the total serum IgE of check sample according to total serum IgE and external source circular rna
Than for 2000:1 mixes with external source circular rna, obtains the mixture of external source circular rna and total serum IgE.External source circular rna is mixed into
After total serum IgE, external source circular rna substantial equivalence is in endogenous circular rna, and therefore, external source circular rna can be as quantitatively detecting
Reference gene.1/2000 mixed proportion both ensure that enough external source circular rnas can be used for detecting, and will not also take too
The ratio of more endogenous circular rnas, detection is caused to waste.Specific steps include：
Total serum IgE is 2000 with the mass ratio for mixing exogenous RNA:1.Specifically, to (5 DEG C) lifes under 76 μ g cryogenic conditions
Under long chlamydomonas total serum IgE and 100 μ g normal conditions 38ng and 50ng external source rings are separately added into the chlamydomonas total serum IgE of (25 DEG C) growths
Shape RNA, it is well mixed after addition, through of short duration centrifugation, the mixture one for respectively obtaining external source circular rna and total serum IgE (is mixed with external source
The sample to be checked of circular rna) and mixture two (check sample for being mixed with external source circular rna).
Step 400：Detect the target in the mixture of external source circular rna and total serum IgE respectively using real time quantitative PCR method
The content of circular rna and external source circular rna, including：
1st, using the mixture one and mixture two described in random primer reverse transcription step 300, reverse transcription is respectively obtained
Product one and reverse transcription product two, reverse transcription product include target ring-type cDNA and external source ring-type cDNA.This two classes reverse transcription
Only it is the object difference of reverse transcription, other reaction condition all sames, the reaction of two classes while and parallel progress.
Reverse transcription reaction process is：The mixture one obtained in 0.5 μ g steps 300 is taken to be used as template, and and following ingredients
Mixing：5 μ l concentration be 1 μM 6 base random primers (being synthesized by Sangon Biotech (Shanghai) Co., Ltd.), 2 μ l it is dense
Spend reverse transcriptase (Invitrigen companies of the U.S. produce, article No. 18064-014), the 5 μ l concentration of dNTP, 20U for 10mM
DTT (being provided with reverse transcriptase) and 10 μ l 5 × reverse transcription reaction buffer solutions for 100mM (carry with reverse transcriptase
For), after supplying 50 μ l mixings with water, 2 hours are incubated in 42 DEG C, 75 DEG C are incubated 15 minutes, inactivate enzyme.The reverse transcription obtained
Product is reverse transcription product one.Removing template is changed to outside the mixture two obtained in step 300, by identical method, is reversed
Record product two.In reverse transcription product one and reverse transcription product two, including target ring-type c DNA and external source ring-type c DNA.
2nd, the target ring-type c DNA in reverse transcription product one and reverse transcription product two are examined respectively using three-primer
Survey；The external source ring-type c DNA in reverse transcription product one and reverse transcription product two are detected respectively using the second primer.To keep away
Exempt from the interference of linear rna molecule, the cyclisation that the second primer spans external source cyclisation RNA with the amplified production of three-primer is connected
Point, circular RNA molecule can only be expanded.Three-primer is included such as SEQ ID NO in sequence table:The 3rd shown in 6 is positive
SEQ ID NO in primer and such as sequence table:The 3rd reverse primer shown in 7, three-primer is by giving birth to work bioengineering (Shanghai) stock
The synthesis of part Co., Ltd.
The real-time quantitative PCR includes four classes：The first kind is three-primer to the target ring-type cDNA in reverse transcription product one
Quantitative PCR detection is carried out, C is obtained after amplificationT3 values；Second class is three-primer to the target ring-type in reverse transcription product two
CDNA carries out quantitative PCR detection, and C is obtained after amplificationT4 values；3rd class is the external source ring in the second primer pair reverse transcription product one
Shape cDNA enters performing PCR amplification, and C is obtained after amplificationT5 values；4th class is the external source ring-type in the second primer pair reverse transcription product two
CDNA enters performing PCR amplification, and C is obtained after amplificationT6 values.During four class real-time quantitative PCRs, except above primer and reverse transcription produce
Thing is different outer, and other compositions are just the same, and the reaction of this four class while and parallel progress.
Further, first kind real-time quantitative PCR reacts：It is 1 μM that 2 μ l reverse transcription products one, which are taken, as template, 3 μ l concentration
Three-primer (mixture of forward primer and reverse primer), 10 μ l quantitative PCR mixtures (are given birth to by Toyobo, Japan
Production, article No. QPS-201) and 50 times of 0.4 μ l ROX fluorescence corrections dyestuff (produced by Toyobo, Japan, and with calmly
Amount PCR mixtures provide together).Thing mixed above is well mixed, of short duration centrifugation, in ABI StepOne real-time PCRs
In by following program carry out real-time quantitative PCR detection：95 DEG C 20 seconds；95 DEG C 3 seconds, 60 DEG C 20 seconds, totally 40 circulation, each time
Fluorescence signal is collected in the final step of circulation, the power of fluorescence signal be used to weighing cDNA amounts number.By procedure above, obtain
Obtain CT3 values are 22.01.Reacted according to first kind real-time quantitative PCR and carry out the reaction of its excess-three class, and obtain CT4 values for 25.45,
CT5 values are 27.33, CT6 values are 29.56.
Step 500：The ratio of target circular rna in sample to be checked is：
Come from this result
See, the Chlamydomonas reinhardtii in the embodiment of the present invention after low-temperature treatment, what ring cRNA006 ratios therein grew at a normal temperature
The expression quantity of annular cRNA006 in Chlamydomonas reinhardtii have dropped 43.22%.This value in early-stage Study using high pass with being measured
Sequence shows that low-temperature treatment have dropped 2 times of result and match, and shows that this programme can be used for correctly evaluating endogenous circular rna expression
The change of amount.
The method provided in an embodiment of the present invention for quantitatively detecting endogenous circular rna, the external source circular rna of preparation is added and treated
In the total serum IgE of sample sheet and check sample, and together expanded and detected with the total serum IgE of sample to be checked and check sample.Cause
And external source circular rna has substantially been equal to the endogenous circular rna of stable expression, can be used as different samples between detect
Reference gene during endogenous circular rna expression quantity, solve the problem of no endogenous circular rna reference gene, it is achieved thereby that
To the purpose of endogenous circular rna expression quantity detection.The present invention realizes endogenous circular rna real-time quantitative PCR detection first, in addition
Source circular rna is used to accurately detect endogenous circular rna expression quantity as reference gene.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.
Claims (7)
1. a kind of method of endogenous circular rna of quantitative detection for non-disease diagnostic purpose, it is characterised in that methods described bag
Include：
External source circular rna is prepared, the method for preparing external source circular rna includes：
Select external source linear rna gene, DNA sequence dna such as sequence table SEQ ID NO corresponding to the external source linear rna gene:1 institute
Show；
By the external source linear rna gene cloning into prokaryotic expression carrier, the external source linear rna gene of clone is obtained；
The external source linear rna gene of the clone is transcribed in vitro, synthesizes external source linear rna；
The triphosphoric acid structure at 5 ' ends of the external source linear rna is removed, and synthesis of hydroxy is held in 5 ' in the external source linear rna；
Phosphorylation modification is carried out for the external source linear rna of hydroxyl to 5 ' ends, the described outer of phosphorylated modification is held in synthesis 5 '
Source linear rna；
5 ' ends of the external source linear rna of described 5 ' the phosphorylated modifications in end and 3 ' ends are connected, synthesize external source circular rna；
Cut away and be not cyclized the successful external source linear rna, obtain external source circular rna；
Quality control is carried out to the external source circular rna, the external source circular rna of synthesis reversed using random primer
Record, synthesis external source cDNA；Utilize the first primer and the second primer cDNA linear to the external source and the external source ring-type respectively
CDNA carries out real-time quantitative PCR amplification, obtains CT1 values and CT2 values respectively, first primer is by SEQ ID in such as sequence table
NO:SEQ ID NO in the first forward primer and such as sequence table shown in 2:The first reverse primer composition shown in 3, described second
Primer is by SEQ ID NO in such as sequence table:SEQ ID NO in the second forward primer and such as sequence table shown in 4:Shown in 5
Two reverse primers form；Pass through CT1 value and CT2 values and formulaCalculate the external source ring-type of synthesis
RNA cyclisation ratio, and select the external source circular rna of the cyclisation ratio more than 90%；
The external source circular rna, the total serum IgE and the external source ring-type are added into the total serum IgE of sample to be checked and check sample
RNA mass ratio is 2000:1, obtain the mixture of the external source circular rna and the total serum IgE；
Detect the external source circular rna and the target ring-type in the mixture of the total serum IgE respectively using real time quantitative PCR method
RNA and the external source circular rna content；
Using the external source circular rna as reference gene, the expression quantity of target circular rna described in the sample to be checked is determined.
2. according to the method for claim 1, it is characterised in that cut away using RNase R and be not cyclized the successful external source line
Property RNA.
3. according to the method for claim 1, it is characterised in that to the described total of the sample to be checked and the check sample
Add the external source circular rna in RNA, the mass ratio of the total serum IgE and the external source circular rna is 2000:1, obtain described
The mixture of external source circular rna and the total serum IgE, including：
Extract and purify the total serum IgE of the sample to be checked and the check sample；
The mass concentration of the total serum IgE of measurement after purification；
According to the mass concentration of the sample to be checked and the total serum IgE of the check sample according to the total serum IgE and the external source ring
Shape RNA mass ratio is 2000:1 ratio mixes with the external source circular rna, obtain the external source circular rna with it is described total
RNA mixture.
4. according to the method for claim 3, it is characterised in that using sample to be checked described in spectrophotometer measurement with it is described
The mass concentration of the total serum IgE of check sample.
5. according to the method for claim 1, it is characterised in that the utilization real time quantitative PCR method detects described respectively
External source circular rna and the target circular rna and the content of the external source circular rna in the mixture of the total serum IgE, including：
Reverse transcription is carried out to the total serum IgE for adding the external source circular rna using the random primer, obtains reverse transcription production
Thing, the reverse transcription product include the target ring-type cDNA and external source ring-type cDNA；
Quantitative PCR is carried out to the target ring-type cDNA of the sample to be checked and the check sample respectively using three-primer
Detection, obtains C respectively after amplificationT3 values and CT4 values, using second primer respectively to the sample to be checked and the control sample
This external source ring-type cDNA carries out quantitative PCR detection, obtains CT after amplification respectively5Value and CT6Value；
The amplified production of the three-primer and second primer spans the cyclisation node of circular rna.
6. according to the method for claim 5, it is characterised in that according to formulaCalculate institute
State the relative expression quantity of the target circular rna in sample to be checked.
7. according to the method for claim 5, it is characterised in that the amplification program of the real-time quantitative PCR is：95℃20
Second；95 DEG C 3 seconds, 60 DEG C 20 seconds, totally 40 circulation.
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| CN105063210A (en) * | 2015-08-12 | 2015-11-18 | 中国农业科学院北京畜牧兽医研究所 | Circular RNA identification method |
| CN105755042B (en) * | 2016-03-25 | 2019-03-05 | 广州吉赛生物科技股份有限公司 | The fluorescence report system and its construction method of screening and identification circular rna translated protein |
| CN107815495B (en) * | 2017-12-13 | 2021-11-12 | 南京医科大学 | Method for detecting plasma circular RNA marker related to non-small cell lung cancer |
| CN109957621A (en) * | 2019-04-01 | 2019-07-02 | 中国人民解放军海军军医大学国家肝癌科学中心 | It is a kind of for measuring the method and its kit of people's circulating excretion body circular rna |
| CN112301109B (en) * | 2019-07-29 | 2024-02-13 | 中国科学院昆明动物研究所 | Method for detecting RNA cyclization efficiency of genotype |
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