CN104328185A - Method for monitoring removal of linear RNA in total RNA - Google Patents

Method for monitoring removal of linear RNA in total RNA Download PDF

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CN104328185A
CN104328185A CN201410606600.2A CN201410606600A CN104328185A CN 104328185 A CN104328185 A CN 104328185A CN 201410606600 A CN201410606600 A CN 201410606600A CN 104328185 A CN104328185 A CN 104328185A
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rna
ercc
external source
linear
primer
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CN104328185B (en
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周俊飞
李利利
陈利红
高利芬
张继
方治伟
章伟雄
卢龙
李甜甜
彭海
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Jianghan University
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Jianghan University
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    • C12Q1/6844—Nucleic acid amplification reactions

Abstract

The invention discloses a method for monitoring removal of linear RNA in total RNA. The method comprises the following steps: artificially synthesizing exogenous linear ERCC-0004-RNA and exogenous circular ERCC-0013-RNA; carrying out mass control on the exogenous circular ERCC-0013-RNA; mixing the exogenous linear ERCC-0004-RNA and the exogenous circular ERCC-0013-RNA according to an equimolar ratio to obtain mixed exogenous RNA; adding the mixed exogenous RNA to the total RNA of a sample to be detected to obtain a first mixture; removing ribosome RNA out of the first mixture to obtain a second mixture; removing the linear RNA including the linear RNA in the total RNA and the exogenous linear ERCC-0004-RNA, thus obtaining a third mixture; separately detecting the contents of the exogenous linear ERCC-0004-RNA and exogenous circular ERCC-0013-RNA in the first mixture and the third mixture; and calculating the removal ratio of the linear ERCC-0004-RNA in the total RNA according the contents. The method can effectively monitor the removal degree of the linear RNA in the total RNA.

Description

A kind of method monitoring wire RNA elimination in total serum IgE
Technical field
The present invention relates to biology field, particularly a kind of method monitoring wire RNA elimination in total serum IgE.
Background technology
In organism, most RNA is linear, but has small part RNA head and the tail to be connected to form ring-type, is called as circular rna.In recent years, circular rna becomes rapidly the focus of RNA world research, this is because high throughput sequencing technologies can be used to detect circular rna, this greatly accelerates the discovery of circular rna and the analysis to circular rna function.
The prerequisite of research circular rna is separated in organism by circular rna, also do not have the direct method obtaining circular rna in organism at present, can only remove linear rna from total serum IgE, indirectly reach the object of enrichment circular rna.Usually, circular rna accounts for the ratio of total serum IgE less than 1%, before high-flux sequence detects circular rna, needs the removal degree monitoring total serum IgE neutral line RNA.At present, a circular rna sample high-flux sequence testing cost is more than 100,000 yuan.If there is more linear rna to exist, then linear rna can take a large amount of order-checking resources in high-flux sequence process, produces invalid data, causes a large amount of wastes of human and material resources and financial resources.But, also do not monitor the method for the elimination of total serum IgE neutral line RNA at present, cause waste to be difficult to avoid.
Summary of the invention
Cannot the problem of removal degree of effective monitoring total serum IgE neutral line RNA in order to solve in prior art, embodiments provide and a kind ofly monitor the method that in total serum IgE, wire RNA eliminates.Described technical scheme is as follows:
Embodiments provide a kind of method monitoring wire RNA elimination in total serum IgE, described method comprises:
The linear ERCC-0004-RNA of synthetic external source and external source ring-type ERCC-0013-RNA;
Quality control is carried out to described external source ring-type ERCC-0013-RNA;
By linear for described external source ERCC-0004-RNA and described external source ring-type ERCC-0013-RNA by equimolar ratio mixing, obtain mixing exogenous RNA;
In the total serum IgE of sample to be checked, add described mixing exogenous RNA, described total serum IgE is 2000:1 with the described mass ratio mixing exogenous RNA, obtains the first mixture;
Remove the ribosome-RNA(rRNA) in described first mixture, obtain the second mixture;
Remove the linear rna in described second mixture, described linear rna comprises endogenous linear rna in described total serum IgE and the linear ERCC-0004-RNA of described external source, obtains the 3rd mixture;
Detect the content of the linear ERCC-0004-RNA of described external source in the content of the linear ERCC-0004-RNA of described external source in described first mixture and described external source ring-type ERCC-0013-RNA and described 3rd mixture and described external source ring-type ERCC-0013-RNA respectively;
The removal ratio of linear rna described in total serum IgE according to described cubage.
Particularly, the linear ERCC-0004-RNA of described synthetic external source and external source ring-type ERCC-0013-RNA, comprising:
Select external source ERCC-0004 gene and external source ERCC-0013 gene, described external source ERCC-0004 gene is as shown in SEQ ID NO:1 in sequence table, and described external source ERCC-0013 gene is as shown in SEQ ID NO:2 in sequence table;
Described external source ERCC-0004 gene and described external source ERCC-0013 gene are cloned in prokaryotic expression carrier respectively, obtain the external source ERCC-0004 gene of clone and the external source ERCC-0013 gene of clone respectively;
The external source ERCC-0004 gene of described clone obtained and the external source ERCC-0013 gene of described clone are transcribed respectively in vitro, synthesizes the linear ERCC-0004-RNA of described external source and the linear ERCC-0013-RNA of external source respectively;
Remove the linear ERCC-0013-RNA of described external source 5 ' the triphosphoric acid structure of holding, and hold synthesis of hydroxy at 5 ' of the linear ERCC-0013-RNA of described external source;
Carry out phosphorylation modification to the linear ERCC-0013-RNA of described external source that 5 ' end is hydroxyl, synthesis 5 ' end is through the linear ERCC-0013-RNA of described external source of phosphorylation modification;
5 ' the end of described 5 ' end through the linear ERCC-0013-RNA of external source of phosphorylation modification is connected with 3 ' end, synthesizes described external source ring-type ERCC-0013-RNA;
Utilize RNA enzyme R to cut away the linear ERCC-0013-RNA of the successful described external source of non-cyclisation, obtain described external source ring-type ERCC-0013-RNA.
Further, there is not gene that is identical with described external source ERCC-0013 gene with described external source ERCC-0004 gene or homology in the gene of described sample to be checked.
Particularly, described quality control is carried out to described external source ring-type ERCC-0013-RNA, comprising:
Utilize the described external source ring-type ERCC-0013-RNA of random primer to synthesis to carry out reverse transcription, synthesis external source ERCC-0013-cDNA, described external source ERCC-0013-cDNA comprise the linear ERCC-0013-cDNA of external source and external source ring-type ERCC-0013-cDNA;
The first primer and the second primer is utilized to carry out real-time quantitative PCR amplification to described external source ERCC-0013-cDNA respectively, described first primer comprises the first forward primer as shown in SEQ ID NO:3 in sequence table and the first reverse primer as shown in SEQ ID NO:4 in sequence table, described second primer comprises the second forward primer as shown in SEQ ID NO:5 in sequence table and the second reverse primer as shown in SEQ ID NO:6 in sequence table, described first primer is for the linear ERCC-0013-cDNA of described external source and described external source ring-type ERCC-0013-cDNA that increases, described second primer is for the described external source ring-type ERCC-0013-cDNA that increases, C is obtained by described first primer amplification t1 value, obtains C by described second primer amplification t2 values,
Pass through C t1 value and C t2 value and formula calculate the cyclisation ratio of the described external source ring-type ERCC-0013-RNA of synthesis, and described external source ring-type ERCC-0013-RNA when selecting cyclisation ratio more than 90%.
Particularly, the linear rna in described second mixture of RNA enzyme R removal is adopted.
Particularly, described detect the linear ERCC-0004-RNA of described external source in described first mixture and described external source ring-type ERCC-0013-RNA respectively content and described 3rd mixture in the linear ERCC-0004-RNA of described external source and the content of described external source ring-type ERCC-0013-RNA, comprising:
Reverse transcriptase primer 0004 and reverse transcriptase primer 0013 is adopted to carry out reverse transcription to described first mixture and described 3rd mixture respectively, obtain described first mixture containing cDNA and described 3rd mixture containing cDNA, described reverse transcriptase primer 0004 is as shown in SEQ ID NO:7 in sequence table, and described reverse transcriptase primer 0013 is as shown in SEQ ID NO:8 in sequence table;
Adopt three-primer 0004 and the 4th primer 0013 to carry out quantitative PCR detection to described the first mixture containing cDNA respectively, obtain C respectively t3 value and C t4 values, adopt described three-primer 0004 and described 4th primer 0013 to carry out quantitative PCR detection to described the 3rd mixture containing cDNA respectively, obtain C respectively t5 value and C t6 values, described three-primer 0004 comprises the 3rd forward primer 0004 as shown in SEQ ID NO:9 in sequence table and the 3rd reverse primer 0004 as shown in SEQ ID NO:10 in sequence table, and described 4th primer 0013 comprises the 4th forward primer 0004 as shown in SEQ ID NO:11 in sequence table and the 4th reverse primer 0004 as shown in SEQ ID NO:12 in sequence table.
Further, the method of described reverse transcription comprises: get described first mixture 0.5 μ g and mix with following ingredients: dNTP, the 5 μ l concentration of 5 μ l concentration to be described reverse transcriptase primer 0004, the 2 μ l concentration of 1 μM be 10mM are the DL-dithiothreitol (DTT) of 100mM, the reversed transcriptive enzyme of 20U and 10 μ l 5 × reverse transcription reaction damping fluids, mix after supplying 50 μ l with water, in 42 DEG C of insulations 2 hours, 75 DEG C were incubated 15 minutes;
Get described 3rd mixture 0.5 μ g to mix with following ingredients: dNTP, the 5 μ l concentration of 5 μ l concentration to be described reverse transcriptase primer 0013, the 2 μ l concentration of 1 μM be 10mM are the DL-dithiothreitol (DTT) of 100mM, the reversed transcriptive enzyme of 20U and 10 μ l 5 × reverse transcription reaction damping fluids, mix after supplying 50 μ l with water, in 42 DEG C of insulations 2 hours, 75 DEG C were incubated 15 minutes.
Further, according to formula calculate the removal ratio of linear rna described in described total serum IgE.
Further, the amplification program of described real-time quantitative PCR amplification is: 95 DEG C 20 seconds; 95 DEG C 3 seconds, 60 DEG C 20 seconds, totally 40 circulations.
The beneficial effect that the technical scheme that the embodiment of the present invention provides is brought is: embodiments provide a kind of method monitoring wire RNA elimination in total serum IgE, the method utilizes the linear ERCC-0004-RNA of synthetic external source and external source ring-type ERCC-0013-RNA to mix with the total serum IgE of sample to be checked, together removes the linear rna in mixture.The linear ERCC-0004-RNA of external source in solution before detection removal linear rna and the content of external source ring-type ERCC-0013-RNA, and the content of the linear ERCC-0004-RNA of external source in solution after removal linear rna and external source ring-type ERCC-0013-RNA, extrapolated the removal ratio of total serum IgE neutral line RNA relative to the content of external source ring-type ERCC-0013-RNA by the linear ERCC-0004-RNA of external source, thus the waste of avoid adopting the bad sample of linear rna removal effect to carry out huge human and material resources that high-flux sequence causes and financial resources.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing used required in describing embodiment is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is the method flow diagram that in the monitoring total serum IgE that provides of the embodiment of the present invention, wire RNA eliminates.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly, below in conjunction with accompanying drawing, embodiment of the present invention is described further in detail.
Reagent in the embodiment of the present invention is commercial reagent.
Embodiment
Embodiments provide a kind of method monitoring wire RNA elimination in total serum IgE, the sample to be checked that the embodiment of the present invention provides is Chlamydomonas reinhardtii, and the product of this Chlamydomonas reinhardtii are that CC503 is (purchased from chlamydomonas resource center, network address is: http://chlamycollection.org/), as shown in Figure 1, concrete grammar comprises:
Step 100: the linear ERCC-0004-RNA of synthetic external source and external source ring-type ERCC-0013-RNA.Existing RNA research is all that the endogenous RNA of an employing stably express is as reference.Eliminating whether clean to monitor wire RNA, can to detect and the endogenous linear rna calculating stably express is removing the ratio before and after linear rna respectively with endogenous circular rna, whether totally monitor wire RNA removal by this ratio.But, also do not find the endogenous circular rna of stably express at present, thus cannot directly utilize endogenous circular rna to monitor.In embodiments of the present invention, in order to monitor the removal degree of endogenous linear rna, synthetic external source circular rna and external source wire RNA, add in proportion after total serum IgE as reference, with the endogenous circular rna of stably express in analogue body and endogenous wire RNA, monitoring linear rna is removed and is provided with realistic feasibility.For the synthetic exogenous RNA that the embodiment of the present invention provides, need to consider some: designed external source circular rna and external source linear rna do not have identical sequence or the higher sequence of homology in vivo, because after adding total serum IgE, exogenous RNA and endogenous RNA cannot be distinguished, if the two exists overlap, result will be caused inaccurate; In addition, foreign gene length is also unsuitable oversize, otherwise affects cyclisation efficiency.Consider that the RNA amount of synthesis is comparatively large, and need often to use, DNA sequence dna corresponding for RNA proceeds in plasmid by the embodiment of the present invention, the amount of corresponding DNA is increased by plasmid propagation, thus obtain a large amount of DNA sequence dna flexibly, then by in-vitro transcription, plasmid DNA is become RNA.For this reason, the vitro transcription promoters sequence in DNA Front-end Design.The RNA be transcribed into needs purifying, in order to realize this purpose, devises oligomerization T again in the end of DNA, and transcribe rear formation oligomerization A tail, oligomerization A can be used for purifying RNA.The exogenous RNA of synthesis is wire, just can become circular rna after needing cyclisation.Because the 5 ' end of RNA during synthesis is triphosphoric acid group, and when connecting, it is desirable that monophosphate group, for this reason, after eliminating the triphosphoric acid group of exogenous RNA, add this step of monophosphate group, thus synthesis can be cyclized into the state of the precursor RNA of ring.Concrete operation step is as follows:
1, select external source ERCC-0004 gene and external source ERCC-0013 gene, this external source ERCC-0004 gene is as shown in SEQ ID NO:1 in sequence table, and this external source ERCC-0013 gene is as shown in SEQ ID NO:2 in sequence table.
Above-mentioned SEQ ID NO:1, SEQ ID NO:2 and complementary strand thereof are synthesized by Sangon Biotech (Shanghai) Co., Ltd..
Retrieved by the blast program of NCBI, do not find the homologous gene with external source ERCC-0004 gene and external source ERCC-0013 gene in biology.The external source ERCC-0004 gene selected and the length of external source ERCC-0013 gene are no more than 1000bp.Hold at the 5 ' end and 3 ' of external source ERCC-0004 gene and external source ERCC-0013 gene respectively and with the addition of ctcgag and aagctt sequence respectively, this ctcgag and aagctt sequence is respectively the restriction enzyme site of restriction enzyme XhoI and HindIII, external source ERCC-0004 gene and external source ERCC-0013 gene clone is convenient to enter prokaryotic expression carrier, 5 ' the taatacgactcactata sequence of holding of external source ERCC-0004 gene and external source ERCC-0013 gene is the promoter sequence of T7 transcriptase, and all the other sequences are transcribed sequence.After taatacgactcactata sequence, be connected with ggg sequence, ggg sequence can increase the transcriptional efficiency of T7 transcriptase.
2, external source ERCC-0004 gene and external source ERCC-0013 gene are cloned in prokaryotic expression carrier respectively, obtain external source ERCC-0004 gene and the ERCC-0013 gene of clone, comprising:
Selected cloning vector is that pBluescript II SK (+) (buy from Changsha Yingrun Biological Technology Co., Ltd. by this cloning vector, article No. is VKS0288), restriction endonuclease XhoI is adopted (to buy from NEB company, article No. is R0146) and restriction endonuclease HindIII (buy from NEB company, article No. is R0104) double digestion is carried out to pBluescript II SK (+) carrier.Particularly, in the reaction system of 20 μ l, 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 company).Above-mentioned reaction system is mixed, through of short duration centrifugal, in 37 DEG C of insulations after 1 hour, through 80 DEG C 20 minutes by enzyme-deactivating, and obtaining linear pBluescript II SK (+) carrier, the concentration of this linear pBluescript II SK (+) carrier is 10/20=0.5 μ g/ μ l.
After external source ERCC-0004 gene and external source ERCC-0013 gene are dissolved with sterilized water, be mixed with the solution that concentration is 0.5 μ g/ μ l respectively, by external source ERCC-0004 gene and complementary strand thereof by volume 1:1 mix, experience successively in PCR instrument 94 DEG C 10 minutes; 65 DEG C 10 minutes and 37 DEG C 10 minutes, form external source ERCC-0004-double-stranded DNA gene; External source ERCC-0013 gene and complementary strand thereof are processed by identical method, obtains external source ERCC-0013-double-stranded DNA gene.
Comprise linear pBluescript II SK (+) carrier 10 μ l in the reaction system of 20 μ l, external source ERCC-0004-double-stranded DNA gene 1 μ l, T4 DNA ligase (buys from NEB company, article No. is M0202) 400U and 1 × T4 DNA ligase damping fluid (buying from NEB company with T4 DNA ligase), 16 DEG C be incubated overnight after, 65 DEG C 10 minutes, T4 DNA ligase, through heat inactivation, obtains pBluescript II SK (+) plasmid vector containing external source ERCC-0004 gene.In the same way, pBluescript II SK (+) plasmid vector containing external source ERCC-0013 gene is obtained.
Utilize heat shock method to be transformed by pBluescript II SK (+) plasmid vector containing external source ERCC-0004 gene and enter intestinal bacteria, concrete grammar is as follows: get 50 μ l competent cells and (produced by Beijing Quanshijin Biotechnology Co., Ltd, catalog number (Cat.No.) is CD501) naturally thaw in ice bath, add pBluescript II SK (+) plasmid vector containing external source ERCC-0004 gene, mix gently, ice bath is hatched 30 minutes, 42 DEG C of heat shocks 30 seconds, ice bath 3 minutes in fast transfer to ice bath, centrifuge tube is not shaken in this process, then 300 μ l are added not containing antibiotic aseptic LB liquid nutrient medium (LB liquid nutrient medium composition: extractum carnis 0.5g, peptone 1.0g, sodium-chlor 0.5g, distilled water 100mL, pH7.2 ~ 7.5), in 37 DEG C 200 revs/min, recover 1 hour, (LB solid medium comprises: Tryptones 10g/L to get the LB solid medium that the bacterium liquid 200 μ l after recovery is spread evenly across containing amicillin resistance, yeast extract 5g/L, sodium-chlor 10g/L and agar powder 15g/L) on, be inverted in 37 DEG C of constant incubators after bacterium liquid absorbs completely, light culture spends the night.Mono-clonal longer on picking LB solid medium containing in the LB liquid nutrient medium of penbritin to 5ml, through 37 DEG C 200 revs/min, shakes bacterium amplification breeding 6-8 hour, is prepared into competent cell.
The little extraction reagent kit of the rapid plasmid utilizing TIANGEN Biotech (Beijing) Co., Ltd. to produce (article No. is DP105) is from above-mentioned competent cell, extract also purifying and contain the DNA of pBluescript II SK (+) plasmid vector of external source ERCC-0004 gene, the method for extraction and purification is carried out according to the specification sheets provided with test kit.Utilize the double-stranded DNA program in spectrophotometer (Quawell company of the U.S. produces, and model is Q5000), detect the mass concentration of pBluescript II SK (+) plasmid after purifying.The PCR primer of external source ERCC-0004 gene is utilized to increase external source ERCC-0004 gene from pBluescript II SK (+) plasmid after purifying.Particularly, the amplification system of 20 μ l comprises: the PCR primer (this PCR primer is the mixture of forward primer and reverse primer) of the external source ERCC-0004 gene of pBluescript II SK (+) the plasmid 50ng after purifying, 10 μ l Q5 high-fidelity amplification mixtures (purchased from NEB company, article No. is M0492) and 10 μMs.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) × 30 circulations.Alcohol settling PCR primer is utilized to carry out purifying, purified product is dissolved in 10 μ l without in enzyme water, obtain the PCR primer of the linear ERCC-0004 gene of external source, (Quawell company of the U.S. produces to utilize Q5000, model is Q5000) in two long, rectangular bag DNA quant programs detection by quantitative is carried out to PCR primer, detected result shows: PCR primer concentration of the linear ERCC-0004 gene of external source that this amplification obtains is 610ng/ μ l.Amplified production delivers to the order-checking of Wuhan Qing Ke Bioisystech Co., Ltd, confirm the exactness of the ERCC-0004 of clone, thus obtain containing Bluescript II SK (+) plasmid of correct cloned foreign ERCC-0004 gene and the PCR primer of the linear ERCC-0004 gene of external source.In the same way, obtain containing Bluescript II SK (+) plasmid of correct cloned foreign ERCC-0013 gene and the PCR primer of the linear ERCC-0013 gene of external source.
Wherein, the forward primer of the PCR primer of external source ERCC-0004 gene is as shown in SEQ ID NO:13 in sequence table, and the reverse primer of the PCR primer of external source ERCC-0004 gene is as shown in SEQ ID NO:14 in sequence table; The forward primer of the PCR primer of external source ERCC-0013 gene is as shown in SEQ ID NO:13 in sequence table, and the reverse primer of the PCR primer of external source ERCC-0013 gene is as shown in SEQ ID NO:15 in sequence table.Wherein, the poly T sequence in SEQ ID NO:13 and SEQ ID NO:15 is to obtain poly A when transcribing, and for simulating the poly A tract bar of biological RNA, this poly A tract bar can be used for reverse transcription and the purifying of RNA.
3, the external source ERCC-0004 gene of clone and ERCC-0013 gene are passed through in-vitro transcription, the synthesis linear ERCC-0004-RNA of external source and the linear ERCC-0013-RNA of external source, comprising:
Adopt T7RNA rapidly and efficiently synthetic agent box (purchased from NEB company, article No. is E2050) transcribe synthesis external source ERCC-0004-RNA.Composition in this test kit comprises: without enzyme water, buffer solution mixture containing NTP and t7 rna polymerase mixture.Reaction system comprises: the PCR primer of the linear ERCC-0004 gene of the above-mentioned external source of 1 μ g, 10 μ l contain buffer solution mixture and the 2 μ l T7 RNA polymerase mixtures of NTP, add water and supply 20 μ l and mix, and it is of short duration centrifugal, through 37 DEG C of insulations after 2 hours, add 30 μ l without enzyme water and 2 μ l DNase I (purchased from NEB company, article No. is M0303), after mixing, through 37 DEG C of insulations 15 minutes, remove DNA profiling.Recycling Dynabeads mRNA purification kit (produced by ABI company, article No. is 61006) carries out purifying, and with 50 μ l without enzyme water dissolution purified product, obtains the linear ERCC-0004-RNA of external source of purifying.Adopt and use the same method, obtain the linear ERCC-0013-RNA of external source of purifying.
4, remove 5 ' the triphosphoric acid structure of holding of the linear ERCC-0013-RNA of external source, and hold synthesis of hydroxy at 5 ' of the linear ERCC-0013-RNA of external source.5 ' the end of the linear ERCC-0013-RNA of the external source because of external synthesis is triphosphoric acid structure, make 5 ' of the linear ERCC-0013-RNA of external source end cannot hold and be connected to form ring-type with 3 ', so this 5 ' triphosphoric acid structure of holding must be removed, monophosphate structure is added again at 5 ' end, so just can make that the linear ERCC-0013-RNA's of external source join end to end circlewise, concrete steps comprise:
The triphosphoric acid structure adopting calf intestine alkaline phosphatase (purchased from NEB company, article No. is M0290) to remove 5 ' of the linear ERCC-0013-RNA of external source to hold.Reaction system comprises: the calf intestine alkaline phosphatase of linear ERCC-0013-RNA and 7U of external source of 20 μ g purifying, mix after supplying 20 μ l with water, through of short duration centrifugal, in 37 DEG C of insulations 1 hour, obtain the linear ERCC-0013-RNA of external source of the triphosphoric acid structure that removal 5 ' is held.Utilize Dynabeads mRNA purification kit (produced by ABI company, article No. is 61006) purifying to remove the linear ERCC-0013-RNA of external source of the triphosphoric acid structure that 5 ' holds, obtain the linear ERCC-0013-RNA of external source that 5 ' end is hydroxyl.
5, carry out phosphorylation modification to the linear ERCC-0013-RNA of external source that 5 ' end is hydroxyl, the linear ERCC-0013-RNA of external source of synthesis 5 ' end phosphorylation modification, comprising:
Utilizing T4 PNK kinases (by NEB company, article No. is M0201) to modify this 5 ' end is the linear ERCC-0013-RNA of external source of hydroxyl.What provide with enzyme has 10 × T4 PNK kinase buffer liquid.In the reaction system of 50 μ l, comprise following composition: the linear ERCC-0013-RNA of external source that 5 ' end of 1 × T4 PNK kinase buffer liquid, 1mM ATP, 10U T4 PNK kinases and above-mentioned acquisition is hydroxyl, supply 50 μ l with water and mix, through of short duration centrifugal, in 37 DEG C of insulations 30 minutes.Utilize Dynabeads mRNA purification kit (produced by ABI company, article No. is 61006) to carry out purifying, working method is undertaken by the specification sheets of this test kit, obtains the linear ERCC-0013-RNA of external source of 5 ' end phosphorylation modification.
6, be connected with 3 ' end by the 5 ' end of the linear ERCC-0013-RNA of external source of 5 ' end phosphorylation modification, synthesis external source ring-type ERCC-0013-RNA, comprising:
Utilize the 5 ' end and 3 ' of T4 RNA ligase I (NEB company produces, and article No. is M0204) to the linear ERCC-0013-RNA of external source of this 5 ' end phosphorylation modification to hold to be connected.The composition provided with this T4 RNA ligase I also comprises: the RNA enzyme inhibitors of 10 × T4 RNA ligase reaction buffer, 10mM ATP, 10U/ μ l and PEG 8000.Following composition is comprised: the T4 RNA ligase 1 μ l of 1 × T4 RNA ligase reaction buffer, 10U/ μ l, the RNA enzyme inhibitors 0.5 μ l of 10U/ μ l, concentration are the linear ERCC-0013-RNA of external source of the ATP of 10% PEG8000,20-50 μM and 5 ' end phosphorylation modification of above-mentioned acquisition in the anti-system of 20 μ l, supply 20 μ l with water and mix, through of short duration centrifugal, spend the night in 16 DEG C in PCR instrument, through 95 DEG C of 2 minutes termination reactions, obtain external source ring-type ERCC-0013-RNA.
Purifying external source ring-type ERCC-0013-RNA, particularly, following composition is added containing in the mixture of external source ring-type ERCC-0013-RNA: (Ambion company produces the ammonium acetate of 5M to what obtain, article No. is AM9071) 10 μ l, concentration be 100% ethanol 200 μ l and water 80 μ l, mix, through of short duration centrifugal, be placed in-80 DEG C of refrigerators (company of Haier produces, and model is DW-86L626) and place 30 minutes; After taking-up through 14000rpm 4 DEG C centrifugal 25 minutes, carefully supernatant liquid is sucked with the rifle head of pipettor, in precipitation, add 300 μ l concentration is the ethanol of 70%, for washing and precipitating, then suck supernatant liquid, through 14000rpm 4 DEG C under room temperature after centrifugal 5 minutes, air drying 10 minutes, for removing residual ethanol, then with 10 μ l without enzyme water dissolution precipitation, obtain the external source ring-type ERCC-0013-RNA of purifying.
7, RNA enzyme R is utilized to cut away the linear ERCC-0013-RNA of the successful external source of non-cyclisation.Concrete steps comprise:
In the external source ring-type ERCC-0013-RNA of the purifying obtained, also has the linear ERCC-0013-RNA of the part successful external source of non-cyclisation, RNA enzyme R (produced by Epicentre company, article No. is RNR07250) is utilized to cut away the linear ERCC-0013-RNA of the successful external source of non-cyclisation.What provide with this RNA enzyme R also has 10 × RNA enzyme R reaction buffer, in the external source ring-type ERCC-0013-RNA of the purifying obtained, add 2 μ l 10 × RNA enzyme R reaction buffers, the RNA enzyme R of 1 μ l 20U/ μ l and 7 μ l water mix, through of short duration centrifugal after in 40 DEG C of insulations 1 hour, obtain and eliminate the external source ring-type ERCC-0013-RNA of external source wire ERCC-0013-RNA.
8, purifying eliminates the external source ring-type ERCC-0013-RNA of external source wire ERCC-0013-RNA, comprising:
Following composition is added: (Ambion company produces the ammonium acetate of 5M to eliminating in the external source ring-type ERCC-0013-RNA of external source wire ERCC-0013-RNA of acquisition, article No. is AM9071) 10 μ l, concentration is ethanol 200 μ l and the water 80 μ l mixing of 100%, through of short duration centrifugal, (company of Haier produces to be placed in-80 DEG C of refrigerators, model is DW-86L626) in place 30 minutes, after taking-up through 14000rpm 4 DEG C centrifugal 25 minutes, carefully supernatant liquid is sucked with the rifle head of pipettor, in precipitation, add 300 μ l concentration is the ethanol of 70%, for washing and precipitating, supernatant liquid is sucked after centrifugal 5 minutes again through 14000rpm 4 DEG C, under room temperature, air drying 10 minutes, for removing residual ethanol, precipitate without enzyme water dissolution with 50 μ l, obtain the external source ring-type ERCC-0013-RNA of purifying.
Step 200: quality control is carried out to external source ring-type ERCC-0013-RNA.Even have employed the step of the linear ERCC-0013-RNA of above removal, in external source ring-type ERCC-0013-RNA, still may be mixed with external source wire ERCC-0013-RNA, therefore need to carry out quality control to external source ring-type ERCC-0013-RNA.Concrete steps comprise:
1, the external source ring-type ERCC-0013-RNA of random primer to synthesis is utilized to carry out reverse transcription, synthesis external source ERCC-0013-cDNA, external source ERCC-0013-cDNA comprises the linear ERCC-0013-cDNA of external source and external source ring-type ERCC-0013-cDNA, and concrete steps are as follows:
(Quawell company of the U.S. produces to utilize spectrophotometer, model is Q5000) middle RNA quant program, measure and obtain the mass concentration of the external source ring-type ERCC-0013-RNA of above-mentioned purifying, the mass concentration of the external source ring-type ERCC-0013-RNARNA that the present embodiment provides is 8.99ng/ μ l.
Get the external source ring-type ERCC-0013-RNA 0.1 μ g obtaining purifying, mix with following ingredients: 5 μ l concentration are the random reverse transcriptase primer of 6 base (being synthesized by Sangon Biotech (Shanghai) Co., Ltd.) of 1 μM, 2 μ l concentration are the dNTP of 10mM, (American I nvitrigen company produces the reversed transcriptive enzyme of 20U, article No. is 18064-014), 5 μ l concentration are the DL-dithiothreitol (DTT) (DL-Dithiothreitol of 100mM, DTT, thered is provided by American I nvitrigen company with reversed transcriptive enzyme) and 10 μ l 5 × reverse transcription reaction damping fluids (being provided by American I nvitrigen company with reversed transcriptive enzyme), after supplying 50 μ l mixings with water, through 42 DEG C of insulations 2 hours, 75 DEG C are incubated 15 minutes, make enzyme deactivation, synthesis external source ERCC-0013-cDNA.
2, the first primer and the second primer is utilized to carry out real-time quantitative PCR amplification to external source ERCC-0013-cDNA respectively, first primer comprises the first forward primer as shown in SEQ ID NO:3 in sequence table and the first reverse primer as shown in SEQ ID NO:4 in sequence table, second primer comprises the second forward primer as shown in SEQ ID NO:5 in sequence table and the second reverse primer as shown in SEQ ID NO:6 in sequence table, first primer is for the linear ERCC-0013-cDNA of the external source that increases and external source ring-type ERCC-0013-cDNA, second primer is for the external source ring-type ERCC-0013-cDNA that increases, C is obtained by the first primer amplification t1 value, obtains C by the second primer amplification t2 values.
Wherein, the amplified production of the first primer does not cross over the cyclisation tie point of external source ring-type ERCC-0013-RNA, and this first primer can be increased the linear ERCC-0013-cDNA of external source, and can increase again external source ring-type ERCC-0013-cDNA; Second primer or its amplified production cross over the cyclisation tie point of external source ring-type ERCC-0013-RNA, and this second primer can only be increased external source ring-type ERCC-0013-cDNA.
Wherein, the first primer and the second primer are parallel in two reaction systems respectively to carry out, and reaction system and reaction conditions are all identical, and only primer is different.Particularly, get 2 μ l external source ERCC-0013-cDNA, 3 μ l concentration are that the first primer, the 10 μ l quantitative PCR mixtures of 1 μM (are produced by Toyobo company, article No. is QPS-201) and after the ROX fluorescence correction dyestuff (being provided with QPS-201 by Toyobo company) of 0.4 μ l 50 times mixes, through of short duration centrifugal, in ABI StepOne real-time PCR, carry out real-time quantitative PCR amplification by following amplification program: 95 DEG C 20 seconds; 95 DEG C 3 seconds, 60 DEG C 20 seconds, totally 40 circulations, collect fluorescent signal in the final step that circulating each time, the power of this fluorescent signal for weigh expression amount number.
3, C is passed through t1 value and C t2 value and formula calculate the cyclisation ratio of the external source ring-type ERCC-0013-RNA of synthesis, when cyclisation ratio is more than 90%, external source ring-type ERCC-0013-RNA can use.
C is obtained by the first primer amplification t1 value, this C t1 value is 21.55; Adopt the second primer to increase in the same way, obtain C t2 values, this C t2 values are 21.45.By C t1 value and C tthe cyclisation ratio that 2 values substitute into formulae discovery external source ring-type ERCC-0013-RNA is: =93.30%.If the cyclisation ratio of external source ring-type ERCC-0013-RNA is more than 90%, this external source ring-type ERCC-0013-RNA can use, otherwise this external source ring-type ERCC-0013-RNA can not use, and need again prepare external source ring-type ERCC-0013-RNA.Wherein, the cyclisation ratio of 90% is an empirical value as the standard that external source ring-type ERCC-0013-RNA is qualified, and this ratio can be made adjustment as the case may be.
Step 300: by linear for external source ERCC-0004-RNA and external source ring-type ERCC-0013-RNA 1:1 mixing in molar ratio, obtain mixing exogenous RNA.Concrete operations are as follows:
Utilize Qubit RNA assay kit (produced by Invitrigen company, article No. is Q32852) to measure the mass concentration of the linear ERCC-0004-RNA of external source and external source ring-type ERCC-0013-RNA, measuring method is undertaken by the specification sheets of this test kit.In the present embodiment, the mass concentration detecting the linear ERCC-0004-RNA of external source and the external source ring-type ERCC-0013-RNA obtained is respectively 13393pg/ μ l and 5298pg/ μ l.The molecular weight utilizing website tools (network address is: http://www.basic.northwestern.edu/biotools/oligocalc.html) to calculate the linear ERCC-0004-RNA of external source and external source ring-type ERCC-0013-RNA is respectively 168246.6 and 262445.9, the volumetric molar concentration being calculated the linear ERCC-0004-RNA of external source and external source ring-type ERCC-0013-RNA by molecular weight is respectively respectively 79603.39pM and 20187.02pM, calculate thus, mix by equimolar ratio to ask the linear ERCC-0004-RNA of external source and external source ring-type ERCC-0013-RNA, so, the volume ratio of the two mixing should be 1:3.94.Particularly, get after external source linear ERCC-0004-RNA 10 μ l mixes with external source ring-type ERCC-0013-RNA 39.4 μ l, through of short duration centrifugal, obtain mixing exogenous RNA.The volumetric molar concentration calculating this mixing exogenous RNA is 32214.62pM, and mass concentration is 6936.66pg/ μ l.
Step 400: add this mixing exogenous RNA in the total serum IgE of Chlamydomonas reinhardtii, total serum IgE is 2000:1 with the mass ratio mixing exogenous RNA, obtains the first mixture.Existing external source ring-type ERCC-0013-RNA in this first mixture, has again the linear ERCC-0004-RNA of external source, simulates endogenous circular rna and endogenous wire RNA.The blending ratio of 1/2000 both ensure that the linear ERCC-0004-RNA of enough external sources and external source ring-type ERCC-0013-RNA can detect, be unlikely to again to add the linear ERCC-0004-RNA of too much external source and external source ring-type ERCC-0013-RNA, to such an extent as to the testing cost of waste later stage high-flux sequence.Concrete steps comprise:
1, the total serum IgE of Chlamydomonas reinhardtii is extracted:
The Chlamydomonas reinhardtii 5ml taken the logarithm vegetative period, and the number of the Chlamydomonas reinhardtii got is less than 4,000 ten thousand, if more than 4,000 ten thousand, then corresponding minimizing sample volume.Centrifugal 1 minute of 4000rpm normal temperature, after removing the nutrient solution on upper strata, Trizol reagent is utilized (to be produced by Invirtigen company, article No. is 15596-018) to extract the total serum IgE of Chlamydomonas reinhardtii and be dissolved in the water of 50 μ l nuclease free, the method for the extraction and purification of total serum IgE is carried out according to the operational manual of Trizol reagent.
Utilize RNA quant program in spectrophotometer (spectrophotometer is produced by Quawell company of the U.S., and model is Q5000), measure and obtain the mass concentration of the total serum IgE of the Chlamydomonas reinhardtii of said extracted.In the present embodiment, the mass concentration of the total serum IgE of the Chlamydomonas reinhardtii of acquisition is 1.52 μ g/ μ l.Calculate thus, the amount of the total serum IgE of the Chlamydomonas reinhardtii of extraction is 76 μ g.
2, in the Chlamydomonas reinhardtii total serum IgE extracted, add mixing exogenous RNA, comprising:
Total serum IgE is 2000:1 with the mass ratio mixing exogenous RNA, particularly, 38ng mixing exogenous RNA is added in the total serum IgE of 76 μ g Chlamydomonas reinhardtiis, mass concentration (6936.66pg/ μ l) according to mixing exogenous RNA calculates, the volume of the mixing exogenous RNA that should add is 5.48 μ l, mix after adding, through of short duration centrifugal, obtain the first mixture.
Step 500: remove the ribosome-RNA(rRNA) in the first mixture, obtains the second mixture.In total serum IgE, the overwhelming majority is all ribosome-RNA(rRNA) (>95%), and this ribosome-RNA(rRNA) is not the RNA of ring-type, therefore, needs to eliminate.Concrete operations are as follows:
Utilize plant ribosome RNA to remove test kit (produced by Epicentre company, article No. is MRZPL116-6) and remove ribosome-RNA(rRNA) in the first mixture.
This test kit comprises: RNA enzyme inhibitors (100U/ μ l), ribosome-RNA(rRNA) remove liquid, reaction buffer, glycogen (10mg/ml), sodium-acetate (3M), the water of nuclease free, magnetic bead and magnetic bead magnetic supernatant liquid.
Magnetic bead pre-treatment: get 225 μ l magnetic beads, be placed on magnet stand, and at room temperature leave standstill 1 minute, clean once with the water of 225 μ l nuclease free, the water reusing 225 μ l nuclease free cleans once again, magnetic bead vibration is suspended in 60 μ l magnetic bead magnetic supernatant liquids, then adds 1 μ l glycogen, obtain through pretreated magnetic bead.
Sample pretreatment: in the reaction system of 40 μ l, add following composition: 5 μ g first mixtures, 8-10 μ l ribosome-RNA(rRNA) remove liquid and 4 μ l reaction buffers, 40 μ l are supplied with water, through of short duration centrifugal after mixing, 68 DEG C are incubated 10 minutes, after taking out, room temperature places 5 minutes, obtains through pretreated sample mixture.
Remove ribosome-RNA(rRNA): to adding through pretreatment sample mixture in pretreated magnetic bead, mix several times with the rifle head piping and druming of pipettor, through of short duration centrifugal, in incubated at room temperature 5 minutes, carry out vortex concussion, 50 DEG C of insulations 5 minutes, be placed on magnet stand, in left at room temperature 1 minute, abandon the magnetic bead of absorption, obtain the mixture removing ribosome-RNA(rRNA).
Purification of samples: add following composition in the mixture of removal ribosome-RNA(rRNA): (Ambion company produces the ammonium acetate of 5M, article No. is AM9071) 10 μ l, the ethanol 200 μ l of 100% and water 60 μ l also mixes, through of short duration centrifugal, (company of Haier produces to be placed in-80 DEG C of refrigerators, model is DW-86L626) in place 30 minutes, centrifugal 25 minutes of 14000rpm 4 DEG C, carefully supernatant liquid is sucked with the rifle head of pipettor, adding 300 μ l concentration is again the ethanol of 70%, for washing and precipitating, supernatant liquid is removed after centrifugal 5 minutes again through 14000rpm 4 DEG C, under room temperature, air drying 10 minutes, for removing residual ethanol, precipitate without enzyme water dissolution with 10 μ l, obtain first mixture eliminating ribosome-RNA(rRNA) of purifying, i.e. the second mixture.
Wherein, the linear rna in total serum IgE comprises: the linear ERCC-0004-RNA of external source and endogenous linear rna, and circular rna comprises: external source ring-type ERCC-0013-RNA and endogenous circular rna.
Step 600: remove the linear rna in the second mixture, linear rna comprises endogenous linear rna in total serum IgE and the linear ERCC-0004-RNA of external source, obtains the 3rd mixture.In the second mixture, the endogenous linear rna in total serum IgE and the linear ERCC-0004-RNA of external source are mixture state, therefore, carry out the process of removing linear rna, carried out identical process simultaneously to the two to the second mixture.Therefore, the removal degree of the linear ERCC-0004-RNA of external source is identical with the removal degree of the endogenous linear rna in total serum IgE, by the removal ratio of the linear ERCC-0004-RNA of monitoring external source, can reach the object of the removal ratio monitoring endogenous linear rna.Now, the linear ERCC-0004-RNA of external source has been equal to endogenous reference gene.Specifically comprise:
Utilize RNA enzyme R (Epicentre company produce, article No. is RNR07250) can the characteristic of digestions linear rna, remove the linear rna in the second mixture.What provide with RNA enzyme R also has 10 × RNA enzyme R reaction buffer.In above-mentioned second mixture, add 2 μ l 10 × RNA enzyme R reaction buffers, the RNA enzyme R of 1 μ l 20U/ μ l and water 7 μ l, mix, through of short duration centrifugal after 40 DEG C of insulations 1 hour, obtain the second mixture removing linear rna.
Purifying removes the second mixture of linear rna: in the second mixture of above-mentioned removal linear rna, add following composition: (Ambion company produces the ammonium acetate of 5M, article No. is AM9071) 10 μ l, concentration is ethanol 200 μ l and the water 80 μ l of 100%, mix, through of short duration centrifugal, (company of Haier produces to be placed in-80 DEG C of refrigerators, model is DW-86L626) in place 30 minutes, centrifugal 25 minutes of 14000rpm 4 DEG C, carefully supernatant liquid is sucked with the rifle head of pipettor, adding 300 μ l concentration is the ethanol of 70%, for washing and precipitating, supernatant liquid is removed after centrifugal 5 minutes again through 14000rpm 4 DEG C, under room temperature, air drying 10 minutes, for removing residual ethanol, precipitate without enzyme water dissolution with 10 μ l, obtain second mixture eliminating linear rna of purifying, i.e. the 3rd mixture.
Step 700: the content detecting the linear ERCC-0004-RNA of external source in the content of the linear ERCC-0004-RNA of external source in the first mixture and external source ring-type ERCC-0013-RNA and the 3rd mixture and external source ring-type ERCC-0013-RNA, calculates the removal ratio of total serum IgE neutral line RNA.First mixture is the solution before removing linear rna, 3rd mixture is the solution after removing linear rna, and in these two kinds of solution, the amount of circular rna is not by the impact of wire RNA Transformatin, content constant can be kept constant, therefore can remove the reference of degree as wire RNA.Detected their amount by real-time quantitative PCR, first calculate the ratio of the content of the linear ERCC-0004-RNA of external source and external source ring-type ERCC-0013-RNA in the 3rd mixing and the first mixture afterwards, the business between these two ratios is wire RNA and removes ratio.In order to avoid the linear ERCC-0013-RNA of the external source of non-cyclisation being detected, span cyclisation tie point for the real-time quantitative PCR primer designed by external source ring-type ERCC-0013-RNA.Concrete operations are as follows:
1, reverse transcription.Particularly, reverse transcriptase primer: reverse transcriptase primer 0004 and reverse transcriptase primer 0013, reverse transcriptase primer 0004 and reverse transcriptase primer 0013 is adopted to carry out reverse transcription to the first mixture and the 3rd mixture respectively, obtain the first mixture containing cDNA and the 3rd mixture containing cDNA, reverse transcriptase primer 0004 is as shown in SEQ ID NO:7 in sequence table, reverse transcriptase primer 0013 is as shown in SEQ ID NO:8 in sequence table, and reverse transcriptase primer 0013 requirement can only reverse transcription external source cyclisation ERCC-0013-RNA, therefore, this reverse transcriptase primer 0013 spans the cyclisation tie point of external source cyclisation ERCC-0013-RNA, reverse transcriptase primer 0004 and reverse transcriptase primer 0013 sequence are synthesized by Sangon Biotech (Shanghai) Co., Ltd..
This process of reverse-transcription comprises two classes, and the first kind is with the first mixture for template, and the first mixture is carried out reverse transcription by reverse transcriptase primer 0004, and the product of acquisition is the first mixture containing cDNA; Equations of The Second Kind is with the 3rd mixture for template, and the 3rd mixture is carried out reverse transcription by reverse transcriptase primer 0013, and the product obtained is the 3rd mixture containing cDNA.In two class process of reverse-transcription, outside different from primer divided by top plate, other composition is all just the same, and two classes reactions simultaneously and parallelly to carry out.
First kind reverse transcription reaction process is as follows: get 0.5 μ g first mixture as template, and mix with following ingredients: 5 μ l concentration are the reverse transcriptase primer 0004 of 1 μM, 2 μ l concentration are the dNTP of 10mM, (American I nvitrigen company produces the reversed transcriptive enzyme of 20U, article No. is 18064-014), 5 μ l concentration are DL-dithiothreitol (DTT) (providing with reversed transcriptive enzyme) and 10 μ l 5 × reverse transcription reactions damping fluid (providing with reversed transcriptive enzyme) of 100mM, after supplying 50 μ l mixings with water, in 42 DEG C of insulations 2 hours, 75 DEG C are incubated 15 minutes, make enzyme deactivation, and the reverse transcription of Equations of The Second Kind is carried out according to identical method, namely get the 3rd mixture 0.5 μ g to mix with following ingredients: 5 μ l concentration are the described reverse transcriptase primer 0013 of 1 μM, 2 μ l concentration are the dNTP of 10mM, 5 μ l concentration are the DL-dithiothreitol (DTT) of 100mM, the reversed transcriptive enzyme of 20U and 10 μ l 5 × reverse transcription reaction damping fluids, mix after supplying 50 μ l with water, in 42 DEG C of insulations 2 hours, 75 DEG C are incubated 15 minutes.
2, real-time quantitative PCR, particularly, quantification PCR primer: three-primer 0004 and the 4th primer 0013, 4th primer 0013 requirement can only be increased external source cyclisation ERCC-0013-RNA, therefore, 4th primer 0013 spans the cyclisation tie point of external source cyclisation ERCC-0013-RNA, three-primer 0004 comprises the 3rd forward primer 0004 as shown in SEQ ID NO:9 in sequence table and the 3rd reverse primer 0004 as shown in SEQ ID NO:10 in sequence table, 4th primer 0013 comprises the 4th forward primer 0004 as shown in SEQ ID NO:11 in sequence table and the 4th reverse primer 0004 as shown in SEQ ID NO:12 in sequence table, three-primer 0004 and the 4th primer 0013 synthesize by Sangon Biotech (Shanghai) Co., Ltd..
Particularly, this real-time quantitative PCR comprises four classes: the first kind is three-primer 0004 and the combination of the first mixture containing cDNA, obtains C after amplification t3 values; Equations of The Second Kind is the combination between the 4th primer 0013 and the first mixture containing cDNA, obtains C after amplification t4 values; 3rd class is three-primer 0004 and the combination of the 3rd mixture containing cDNA, obtains C after amplification t5 values; 4th class is the 4th primer 0013 and the combination of the 3rd mixture containing cDNA, obtains C after amplification t6 values.The first mixture wherein containing cDNA and be template containing the 3rd mixture of cDNA, in four class real-time quantitative PCR processes, outside different from primer divided by top plate, other composition is all just the same, and parallelly to carry out the while of the reaction of this four class.
Further, first kind real-time quantitative PCR reacts: get the first mixture that 2 μ l contain cDNA and (produced by Toyobo, Japan as three-primer 0004 (mixture of forward primer and reverse primer), the 10 μ l quantitative PCR mixtures that template, 3 μ l concentration are 1 μM, article No. is QPS-201) and the ROX fluorescence correction dyestuff (produced by Toyobo, Japan, and provide with quantitative PCR mixture) of 0.4 μ l 50 times.Above mixture is mixed, of short duration centrifugal, in ABI StepOne real-time PCR, carry out real-time quantitative PCR detection by following program: 95 DEG C 20 seconds; 95 DEG C 3 seconds, 60 DEG C 20 seconds, totally 40 circulations, collect fluorescent signal in the final step that circulating each time, the power of fluorescent signal for weigh cDNA amount number.By above program, obtain C t3 is 24.35.
Carry out the reaction of other three classes real-time quantitative PCR by identical method, obtain C t5 be 21.20, C t4 be 24.26, C t6 is 24.33.
Step 800: the removal ratio according to cubage total serum IgE neutral line RNA: the formula calculating the removal ratio of total serum IgE neutral line RNA is:
1 - 2 [ C T 5 + C T 4 - C T 6 - C T 3 ] × 100 % = 1 - 2 [ 21.20 + 24.26 - 24.33 - 24.35 ] × 100 % = 89.27 % . As can be seen here, most of external source linear rna is removed, and infers thus, and endogenous linear rna is also removed comparatively clean, can be used for subsequent high pass amount sequencing analysis.
Further, whether accurate in order to verify the method that in monitoring total serum IgE provided by the invention, wire RNA eliminates, second mixture and the 3rd mixture are built storehouse according to the technical scheme of Proton high-flux sequence and are checked order by the embodiment of the present invention, containing a large amount of ribosome-RNA(rRNA)s in first mixture, this can waste sequencing data amount, and between the second mixture and the first mixture, only have a step to remove the step of ribosome-RNA(rRNA), this can not cause the change of external source linear rna ratio, total data volume of order-checking is 10M, the Reads number of the linear ERCC-0004-RNA of external source in the second mixture and the 3rd mixture is respectively 506 and 5578, and the Reads number of external source ring-type ERCC-0013-RNA in the second mixture and the 3rd mixture is respectively 5946 and 5672, the removal ratio calculating external source linear rna is thus 1-(506/5946)/(5678/5672)=91.37%.It is roughly equal that what the value recorded by the method and the embodiment of the present invention were provided remove ratio by the linear rna monitoring method that in total serum IgE, wire RNA eliminates records 89.27%.As can be seen here, the removal ratio of the linear rna that the present invention obtains is correct, can predict before high-flux sequence whether linear rna is removed totally, avoid to utilize and remove sordid sample and carry out high-flux sequence and the waste of a large amount of human and material resources of causing and financial resources.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (9)

1. monitor the method that in total serum IgE, wire RNA eliminates, it is characterized in that, described method comprises:
The linear ERCC-0004-RNA of synthetic external source and external source ring-type ERCC-0013-RNA;
Quality control is carried out to described external source ring-type ERCC-0013-RNA;
By linear for described external source ERCC-0004-RNA and described external source ring-type ERCC-0013-RNA by equimolar ratio mixing, obtain mixing exogenous RNA;
In the total serum IgE of sample to be checked, add described mixing exogenous RNA, described total serum IgE is 2000:1 with the described mass ratio mixing exogenous RNA, obtains the first mixture;
Remove the ribosome-RNA(rRNA) in described first mixture, obtain the second mixture;
Remove the linear rna in described second mixture, described linear rna comprises endogenous linear rna in described total serum IgE and the linear ERCC-0004-RNA of described external source, obtains the 3rd mixture;
Detect the content of the linear ERCC-0004-RNA of described external source in the content of the linear ERCC-0004-RNA of described external source in described first mixture and described external source ring-type ERCC-0013-RNA and described 3rd mixture and described external source ring-type ERCC-0013-RNA respectively;
The removal ratio of linear rna described in total serum IgE according to described cubage.
2. method according to claim 1, is characterized in that, the linear ERCC-0004-RNA of described synthetic external source and external source ring-type ERCC-0013-RNA, comprising:
Select external source ERCC-0004 gene and external source ERCC-0013 gene, described external source ERCC-0004 gene is as shown in SEQ ID NO:1 in sequence table, and described external source ERCC-0013 gene is as shown in SEQ ID NO:2 in sequence table;
Described external source ERCC-0004 gene and described external source ERCC-0013 gene are cloned in prokaryotic expression carrier respectively, obtain the external source ERCC-0004 gene of clone and the external source ERCC-0013 gene of clone respectively;
The external source ERCC-0004 gene of described clone obtained and the external source ERCC-0013 gene of described clone are transcribed respectively in vitro, synthesizes the linear ERCC-0004-RNA of described external source and the linear ERCC-0013-RNA of external source respectively;
Remove the linear ERCC-0013-RNA of described external source 5 ' the triphosphoric acid structure of holding, and hold synthesis of hydroxy at 5 ' of the linear ERCC-0013-RNA of described external source;
Carry out phosphorylation modification to the linear ERCC-0013-RNA of described external source that 5 ' end is hydroxyl, synthesis 5 ' end is through the linear ERCC-0013-RNA of described external source of phosphorylation modification;
5 ' the end of described 5 ' end through the linear ERCC-0013-RNA of external source of phosphorylation modification is connected with 3 ' end;
Utilize RNA enzyme R to cut away the linear ERCC-0013-RNA of the successful described external source of non-cyclisation, obtain described external source ring-type ERCC-0013-RNA.
3. method according to claim 2, is characterized in that, there is not gene that is identical with described external source ERCC-0013 gene with described external source ERCC-0004 gene or homology in the gene of described sample to be checked.
4. method according to claim 1, is characterized in that, describedly carries out quality control to described external source ring-type ERCC-0013-RNA, comprising:
Utilize the described external source ring-type ERCC-0013-RNA of random primer to synthesis to carry out reverse transcription, synthesis external source ERCC-0013-cDNA, described external source ERCC-0013-cDNA comprise the linear ERCC-0013-cDNA of external source and external source ring-type ERCC-0013-cDNA;
The first primer and the second primer is utilized to carry out real-time quantitative PCR amplification to described external source ERCC-0013-cDNA respectively, described first primer comprises the first forward primer as shown in SEQ ID NO:3 in sequence table and the first reverse primer as shown in SEQ ID NO:4 in sequence table, described second primer comprises the second forward primer as shown in SEQ ID NO:5 in sequence table and the second reverse primer as shown in SEQ ID NO:6 in sequence table, described first primer is for the linear ERCC-0013-cDNA of described external source and described external source ring-type ERCC-0013-cDNA that increases, described second primer is for the described external source ring-type ERCC-0013-cDNA that increases, CT1 value is obtained by described first primer amplification, CT2 value is obtained by described second primer amplification,
Pass through C t1 value and C t2 value and formula calculate the cyclisation ratio of the described external source ring-type ERCC-0013-RNA of synthesis, and select the described external source ring-type ERCC-0013-RNA of cyclisation ratio more than 90%.
5. method according to claim 1, is characterized in that, adopts the linear rna in described second mixture of RNA enzyme R removal.
6. method according to claim 1, it is characterized in that, described detect the linear ERCC-0004-RNA of described external source in described first mixture and described external source ring-type ERCC-0013-RNA respectively content and described 3rd mixture in the linear ERCC-0004-RNA of described external source and the content of described external source ring-type ERCC-0013-RNA, comprising:
Reverse transcriptase primer 0004 and reverse transcriptase primer 0013 is adopted to carry out reverse transcription to described first mixture and described 3rd mixture respectively, obtain described first mixture containing cDNA and described 3rd mixture containing cDNA, described reverse transcriptase primer 0004 is as shown in SEQ ID NO:7 in sequence table, and described reverse transcriptase primer 0013 is as shown in SEQ ID NO:8 in sequence table;
Adopt three-primer 0004 and the 4th primer 0013 to carry out quantitative PCR detection to described the first mixture containing cDNA respectively, obtain C respectively t3 value and C t4 values, adopt described three-primer 0004 and described 4th primer 0013 to carry out quantitative PCR detection to described the 3rd mixture containing cDNA respectively, obtain C respectively t5 value and C t6 values, described three-primer 0004 comprises the 3rd forward primer 0004 as shown in SEQ ID NO:9 in sequence table and the 3rd reverse primer 0004 as shown in SEQ ID NO:10 in sequence table, and described 4th primer 0013 comprises the 4th forward primer 0004 as shown in SEQ ID NO:11 in sequence table and the 4th reverse primer 0004 as shown in SEQ ID NO:12 in sequence table.
7. method according to claim 6, it is characterized in that, the method of described reverse transcription comprises: get described first mixture 0.5 μ g and mix with following ingredients: dNTP, the 5 μ l concentration of 5 μ l concentration to be described reverse transcriptase primer 0004, the 2 μ l concentration of 1 μM be 10mM are the DL-dithiothreitol (DTT) of 100mM, the reversed transcriptive enzyme of 20U and 10 μ l5 × reverse transcription reaction damping fluid, mix after supplying 50 μ l with water, in 42 DEG C of insulations 2 hours, 75 DEG C were incubated 15 minutes;
Get described 3rd mixture 0.5 μ g to mix with following ingredients: dNTP, the 5 μ l concentration of 5 μ l concentration to be described reverse transcriptase primer 0013, the 2 μ l concentration of 1 μM be 10mM are the DL-dithiothreitol (DTT) of 100mM, the reversed transcriptive enzyme of 20U and 10 μ l5 × reverse transcription reaction damping fluid, mix after supplying 50 μ l with water, in 42 DEG C of insulations 2 hours, 75 DEG C were incubated 15 minutes.
8. method according to claim 6, is characterized in that, according to formula calculate the removal ratio of linear rna described in described total serum IgE.
9. method according to claim 4, is characterized in that, the amplification program of described real-time quantitative PCR amplification is: 95 DEG C 20 seconds; 95 DEG C 3 seconds, 60 DEG C 20 seconds, totally 40 circulations.
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