CN107974486A - A kind of breast cancer recurrence risk checking method - Google Patents

A kind of breast cancer recurrence risk checking method Download PDF

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CN107974486A
CN107974486A CN201710589382.XA CN201710589382A CN107974486A CN 107974486 A CN107974486 A CN 107974486A CN 201710589382 A CN201710589382 A CN 201710589382A CN 107974486 A CN107974486 A CN 107974486A
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方仪
王靖
王英男
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Cancer Hospital and Institute of CAMS and PUMC
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Abstract

The present invention provides a kind of breast cancer recurrence risk checking method, (1) prepares the total nucleic acid that can embody biological specimen RNA information；(2) the multiple probe group to match with target RNA transcript is added in total nucleic acid, is hybridized and using nucleic acid ligase excitation coupled reaction；(3) using the connection product of gained in (2) as template, PCR reactions are carried out using universal primer；(4) PCR product in (3) is purified, the sequence abundances information of all probe connection products is obtained using the sequenator compatible with universal linker sequence or chip hybridization scanner；(5) bioinformatic analysis method determines the relative expression quantity and sequence information of different rna transcription sheets.

Description

A kind of breast cancer recurrence risk checking method
Technical field
The present invention relates to biotechnology and biology field, and being related to RT-MLPA, (reverse transcription-multiple rely on expands skill Art) and high throughput sequencing technologies and molecular method quantification it is qualitative, the detection method especially to breast cancer recurrence risk.
Background technology
At present, breast cancer incidence in female malignant is higher and in rising trend.Breast cancer is due to Caused by many factors synthesis, such as the effect of environmental factor, habits and customs, hormonal readiness, inherent cause etc., wherein inherent cause It is the most notable.The treatment of breast cancer is mainly used centered on performing the operation in the past 20 years, and chemotherapy, radiotherapy, endocrine therapy are auxiliary Colligation Therapy Mode, reduce recurrence and the death rate of breast cancer.But there have quite a few chemotherapy to benefit to be very few low multiple Hair risk patient also has to receive chemotherapy, and which results in waste and the over-treatment to the low danger person in part of resource.Patient selects The query of adjuvant chemotherapy whether is needed urgently to need to be resolved when selecting which type for the treatment of, particularly early-stage breast cancer.Inspection It is mainly the relative expression quantity by detecting the transcription product mRNA of 21 genes in tumor tissues to survey breast carcinoma recurring risk, is led to Overexpression amount calculates recurrence scoring RS values.
As for assessing normal living process, pathological development and the index of drug response to treatment, biomarker Can be as differentiation morbid state, progression of disease, the prognosis assessed clinical intervention and effective surrogate end point of prediction.It is raw Thing marker is in extensive range, not only includes the index of cellular level, protein level and metaboilic level, further comprises specific gene Transcriptional level difference and somatic mutation.
Present analysis genetic transcription rna expression amount mainly has rna transcription group horizontal and two detection sides of single-gene rna expression To.The detection of rna transcription group generally uses high throughput RNAseq or expressing gene chip or numeral expression spectral technology；Single-gene turns The detection of record group level relies primarily on reverse transcription-fluorescent quantitative PCR technique.Current study show that rna expression is horizontal as effective Biomarker often need a gene incessantly, but need the expressions of relevant multiple genes as meeting finger Mark, the gene expression detection of transcript profile level are generally used for the biomarker screening of no assumed condition, but since it is higher RNA mass and quantity are not appropriate for as clinical detection.RT-qPCR is often used for as the goldstandard of detection rna expression level The rna expression detection of small throughput, is not suitable for the horizontal joint-detection of multiple gene transcription.
Amplification technique (the muitiplex ligation-dependent probe of multiple join dependency Amplication, MLPA) moderate fluxes gene tester, can be in same reaction tube to much 45 kinds of different nucleosides Acid sequence is detected and quantitative analysis.MLPA technologies extensively should because its accuracy is high, reproducible and easy to operate etc. advantage For fields such as tumor-related gene detections, as single nucleotide polymorphism and detection in Gene Mutation, gene large fragment deletion and Repetition, chromosome number variation, DNA methylation assay analysis etc., become the research hotspot of tumor cells diagnostic field.MLPA technologies It is that the one kind improved and designed on the basis of multiplex amplification probe hybridization technique has high throughput, high efficiency and low cost, can Qualitative to nucleotide sequence to be detected and relative quantitative assay gene analysis technique.Its basic principle is the probe of special synthesis With target sequence DNA hybridization, by the connection of hybridization probe, PCR amplification, obtained PCR product through capillary electrophoresis separation, is received again Collection data analysis is finally drawn a conclusion, and is widely used to the detection of tumor-related gene at present.But due to its detection technique base In the fragmentation assay of Capillary Electrophoresis, so its Multiple detection power limitations is in 45 weights, and can only semi-quantitative analysis, it is fixed The sensitivity of amount is not high.
At present, two generation high throughput sequencing technologies are since it is more accurate, sensitive, has the flux of higher, and with price Continuous reduction, its application range constantly expands, and has been directed to life science and each different aspect of medical research.Profit With high throughput sequencing technologies come to carry out high-throughout genetic test be one of current research hotspot.But two generation sequencing technologies It is required for carrying out the structure in library, then carries out upper machine sequencing using the library built.General step mainly includes DNA/RNA Extraction, fragmentation, end reparation, adjunction head, Piece Selection, amplification and upper machine sequencing, be finally data analysis.And at this In a little links, library construction is more time-consuming.In this way be adapted to genome is sequenced, if to it is therein certain A little genes are interested, or only specific part is sequenced, and this banking process will result in waste, and increase the difficulty of data analysis Degree.Meanwhile the nucleic acid initial amount that this method needs is high, it is difficult to be sequenced while extensive sample, is not also suitable for facing Bed sample.
Specific some gene of interest sequencings are then needed target sequence enrichment etc. it is extra build storehouse step.Pass through hybridization It is the method being most widely used at present to carry out target sequence capture enrichment.Widely used target sequence capture technique master To be based on solid-phase hybridization capture (Choietal.2009) or solution hybridization capture technique (Bainbridge et al.2010).It is existing There is commercialized customization capture agent box to use.These commercialized custom sequence capture agent box price general chargeds are held high It is expensive, fixed once target sequence is detected after chip customization, the selection in site is dumb.
Lllumina companies also provide a kind of different PCR amplification database technology (Truseq coustom amplicon). Its cardinal principle is：A pair of probe in the same direction for carrying general probe of design in the both sides of target sequence.It is special by probe and target 2 probes, are anchored on 5 ' and 3 ' ends of target sequence by different sequence hybridization.Extension, which is carried out, using archaeal dna polymerase fills up two spies Gap between pin, is then sequenced.This method needs to design different probes according to institute's cls gene sequence, its probe Design complex, building storehouse quality can be had a great influence by hybridization efficiency.This every part of sample of banking process needs up to hundreds of The nucleic acid initial amount of ng, is unfavorable for that some concentration are relatively low, rare or difficult acquisition nucleic acid samples are sequenced.
The content of the invention
To solve the above-mentioned problems, the invention discloses a kind of method for detecting breast carcinoma recurring risk.In MLPA probes Capillary Electrophoresis fragmentation assay is replaced using two generation high throughput sequencing technologies in the technical foundation of principle, eliminates PCR product The limitation of clip size, and accurately the relative expression quantity of the transcription product mRNA of 21 genes of breast cancer can be quantified Analysis.
One aspect of the invention is a kind of breast cancer recurrence risk checking method, include the following steps：
(1) total nucleic acid of biological specimen RNA information can be embodied by preparing；
(2) the multiple probe group to match with target RNA transcript is added in total nucleic acid, hybridizes and is connected using nucleic acid Enzyme excites coupled reaction；
(3) using the connection product of gained in (2) as template, PCR reactions are carried out using universal primer；
(4) PCR product in (3) is purified, using the sequenator or biological chip instrument compatible with universal linker sequence, obtained The sequence abundances information of all probe connection products；
(5) by the method for bioinformatic analysis, the relative expression quantity and sequence information of different rna transcription sheets are determined；
The step (5) refers to that the sequence information of all probe connection products will be obtained, and passes through bioinformatic analysis Method, the 21 of clean reads and target gene mRNA will be obtained by sequencing data after low-quality reads is gone out in Quality Control A target sequence is compared and counts reads quantity on each target respectively, determine different rna transcription sheets relative expression quantity and Sequence information.
Preferably, total nucleic acid is the RNA directly extracted in the step (1)；The spy of the multiple probe group of the step (2) The base sequence reverse complemental of isobase sequence and corresponding rna transcription sheet；The nucleic acid ligase has catalysis RNA-DNA Hybridize single stranded DNA nicking coupled reaction ability in chain；The purification process of the step (4) is by being marked with Oligo (dT)N Magnetic bead or the affinity column purifying RNA of (15≤N≤35)-probe double-strand connection product compound.
Preferably, the nucleic acid ligase for T4 DNA Ligase orLigase。
Preferably, the single-stranded cDNA that total nucleic acid obtains for RNA reverse transcriptions in the step (1)；The step (2) it is multiple Specific base sequence should be consistent with the base sequence of corresponding rna transcription sheet in probe groups；The nucleic acid ligase, which has, urges Change single stranded DNA nicking coupled reaction ability in DNA-DNA hybridization chains；Purification process in the step (4) is to use Avidin The magnetic bead of mark or affine column purification cDNA- probes double-stranded products compound.
Preferably, the nucleic acid ligase be selected from T4 DNA Ligase, T3 DNA Ligase, Taq DNA Ligase, T7 DNA Ligase、9°NTMThe one or more of DNA Ligase and Ligase-65.
Preferably, the hybridization temperature in the step (2) is 50-60 DEG C；The preparation method of the step (1) is to pass through examination Agent box extracts or the method for trizol extractions；Universal primer in the step (3) is 5UAP/3UA, the bar of the PCR reactions Part is 98 DEG C of denaturation 30s, and 55-60 DEG C of annealing 30s, 72 DEG C of extension 15s, common 30-35 circulates；The sequenator of the step (4) Or biological chip instrument include Illumina Hiseq, Miseq, Nextseq microarray dataset, Life Ion microarray datasets, Illumina, Affymetrix chip platform.
Preferably, the multiple probe group in the step (2) includes at least two fragments of left probe and right probe；Left probe Including 5 ' universal sequences and 3 ' specific hybridization sequences, 5 ' specific hybridization sequences and 3 ' of the right probe including phosphorylated modification are general Sequence；Close adjacent no base notch, can hybridize in same single nucleic acid strands template at the same time between left probe and right probe, Adjacent probe can be catalyzed by nucleic acid ligase and be connected as same nucleic acid molecules.
Preferably, the phosphorylated modification of 5 ' universal sequences of the left probe and 3 ' specific hybridization sequences and right probe 5 ' specific hybridization sequences and 3 ' universal sequences between can add 8-15 randomized bases (dN)8-15。
Another aspect of the invention is a kind of multiple probe group, including at least two fragments of left probe and right probe；Left spy Pin includes 5 ' universal sequences and 3 ' specific hybridization sequences, and right probe includes 5 ' specific hybridization sequences of phosphorylated modification and 3 ' and leads to Use sequence；Close adjacent no base notch, can hybridize in same single nucleic acid strands template at the same time between left probe and right probe On, adjacent probe can be catalyzed by nucleic acid ligase and be connected as same nucleic acid molecules.
Another aspect of the invention is a kind of purposes of multiple probe group, the probe groups can be used in the wind of following disease Danger detection, the disease include tumour, microorganism infection, self-closing disease and genetic disease.
Beneficial effects of the present invention：This method can be more accurately to the phase of the transcription product mRNA of 21 genes of breast cancer Quantitative analysis is carried out to expression quantity；This method is reproducible, operation is easier, has been widely used for tumor-related gene detection Deng field；This method or a kind of method with high throughput, high efficiency and low cost；Therefore this method can accomplish it is quick, Accurately, the postoperative recurrence risk of breast cancer is predicted easily to operate.Purifying is to remove the enzyme of previous step and buffer bufferings Liquid, prevents from influencing follow-up reaction.
Brief description of the drawings
Fig. 1 is left and right probe combinations structure diagram.
Fig. 2 is the probe combinations detection principle diagram for specific nucleic acid template；
Fig. 3 is that the 11 patient with breast cancer's risks of recurrence calculated respectively with RT-qPCR methods and RT-MLPSeq methods are commented Divide and compare figure；
Fig. 4 is respectively by RT-qPCR methods and RT-MLPSeq methods, to 16 breast cancer phases of 2 patient with breast cancers The expression comparative analysis figure of correlation gene.
Embodiment
Unless stated otherwise, the implication of term used herein understands according to broader sense known to association area. Experimental method used in following embodiments is conventional method unless otherwise specified.Material used in following embodiments, Reagent etc., is commercially available unless otherwise specified.
In order to which the present invention is better described, with reference to the attached drawing in the embodiment of the present invention, in the embodiment of the present invention Technical solution is clearly and completely described.
Embodiment 1：Breast carcinoma recurring risk is assessed using the rna expression amount of probe combinations targeting detection FFPE samples
In order to verify whether the probe combinations of design being capable of the accurately multiple expression for targeting the RNA that detection is seriously degraded Amount, this experiment selects 11 different breast cancer tissue FFPE sample extraction RNA respectively, respectively with RT-qPCR methods and RT- MLPSeq methods are detected, and according to the reliability of following experiment flow assessment final data：
(1) RNA extraction selection 11 different breast cancer tissue's formalin fix, the sample of paraffin embedding (FFPE samples This) 8 μ m-thicks section 5, total serum IgE is extracted using Tiangeng paraffin embedding total RNA extraction reagent box, is measured using Qubit 3.0 dense After degree, -80 DEG C freeze it is spare.
(2) reverse transcription MMLV reverse transcriptases RNase Inhibitor (RNase inhibitor) there are under system by step (1) the breast cancer tissue RNA difference reverse transcriptions that 11 different in are single-stranded cDNA, and reverse transcriptase primer is biotin modification oligo(dN)6。
(3) respectively take 11 different breast cancer sample 7ul and add designed 21 pairs of probe groups hybridization buffer, DNA ligase (including but not limited to T4 DNA Ligase, T3 DNA Ligase, Taq DNA Ligase, T7 DNA Ligase and Ligase-65 etc.) and DNA ligase buffer, 98 DEG C of denaturation 5min after 60 DEG C hybridize and connect 1-3 it is small when. All probe particular sequences are as shown in table 1；Take the cDNA progress RT-qPCR of other 11 different breast cancer tissues of 7ul anti-again Should.
1 multiple probe group sequence table of table
(4) using magnetic beads for purifying connection product to replace buffer solution.
(5) it is template to take connection product 10ul in step 4, is carried out using universal PC R primer pair 50ul PCR reaction systems Storehouse processing is built in last amplification.General PCR primer 5UAP/3UAP sequences are as shown in table 2.
Table 2：Universal primer 5UAP/3UAP sequence tables
(6) library Quality Control and it is sequenced using 500 PE75 of Nextseq, data volume is 1M reads.
(7) data analysis：Sequencing data will obtain clean reads and target after low-quality reads is gone out in Quality Control 21 target sequences of mark gene mRNA are compared and count reads quantity on each target respectively.
(8) result that RT-qPCR methods and RT-MLPASeq methods are drawn is compared and analyzed, as shown in Figure 3.
According to result it is visible using the obtained result of RT-MLPASeq methods with the result that RT-qPCR methods obtain into Found after row contrast, both have very high uniformity, and RT-MLPASeq methods are to measure RT-MLPA technologies and high pass Sequence is combined, and improves flux and accuracy to 21 genetic test of breast cancer.These experimental datas show that the present invention can use Effectively detect breast cancer recurrence risk.
Embodiment 2：The expression of detection FFPE sample multiple rna transcripts is targeted using probe combinations
In order to verify whether the probe combinations of design being capable of the accurately multiple expression for targeting the RNA that detection is seriously degraded Amount, this experiment select the breast cancer tissue FFPE sample extraction RNA of 2 patients (Patient#04 and Patient#05) respectively, Be detected respectively with RT-qPCR methods and RT-MLPSeq methods, and according to following experiment flow assessment final data can By property：
(1) 8 μm of breast cancer tissue's FFPE samples of 2 patients (Patient#04 and Patient#05) of RNA extractions selection Thickness section 5, total serum IgE is extracted using Tiangeng paraffin embedding total RNA extraction reagent box, after measuring concentration using Qubit 3.0 ,- 80 DEG C freeze it is spare.
(2) reverse transcription MMLV reverse transcriptases RNase inhibitor there are under system by 2 patients in step (1) The breast cancer tissue RNA difference reverse transcriptions of (Patient#04 and Patient#05) are single-stranded cDNA, and reverse transcriptase primer is biology The oligo (dN) of element modification6。
(3) 2 different breast cancer sample 7ul are respectively taken and add hybridization buffer, DNA of designed 16 pairs of probe groups Ligase (including but not limited to T4 DNA Ligase, T3 DNA Ligase, Taq DNA Ligase, T7 DNA Ligase And Ligase-65 etc.) and DNA ligase buffer, 98 DEG C of denaturation 5min after 60 DEG C hybridize and connect 1-3 it is small when.It is all more Weight probe groups particular sequence is as shown in table 3；Take the cDNA progress RT-qPCR of other 2 different breast cancer tissues of 7ul anti-again Should.
3 multiple probe group particular sequence table of table
(4) using magnetic beads for purifying connection product to replace buffer solution, buffer solution includes but is not limited to ddH20, TE etc., this DdH20 is selected in embodiment.
(5) it is template to take connection product 10ul in step 4, is carried out using universal PC R primer pair 50ul PCR reaction systems Storehouse processing is built in last amplification.General PCR primer 5UAP/3UAP sequences are as shown in table 2.
(6) library Quality Control and it is sequenced using Nextseq 500PE75, data volume is 0.5M reads.
(7) data analysis：Sequencing data will obtain clean reads and target after low-quality reads is gone out in Quality Control 16 target sequences of mark gene mRNA are compared and count reads quantity on each target respectively.
(8) result that RT-qPCR methods and RT-MLPSeq methods are drawn is compared and analyzed, as shown in Figure 4.
According to result as it can be seen that detecting two patient with breast cancers' respectively with two methods of RT-qPCR methods and RT-MLPSeq 16 cancer related genes, it is found that same gene has similar expression in same sample；And pass through RT-MLPSeq The rna expression of detection is horizontal consistent with IHC results before, therefore, it can be proved that RT-MLPSeq methods can be used for examining The rna expression surveyed in FFPE samples is horizontal.
The primer selected in the application is the primer with P5/P7 sequences, can be with sequenator or biochip analysis instrument On connector it is compatible；And the primer carries both-end index sequences, can better discriminate between different samples.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto, Any one skilled in the art is in the technical scope of present disclosure, the change or replacement that can readily occur in, It should all be included within the scope of the present invention.Therefore, protection scope of the present invention should be with the protection of claims Subject to scope.

Claims (10)

1. a kind of breast cancer recurrence risk checking method, it is characterised in that include the following steps：
(1) total nucleic acid of biological specimen RNA information can be embodied by preparing；
(2) the multiple probe group to match with target RNA transcript is added in total nucleic acid, hybridizes and is swashed using nucleic acid ligase Send out coupled reaction；
(3) using the connection product of gained in (2) as template, PCR reactions are carried out using universal primer；
(4) PCR product in (3) is purified, using the sequenator or biological chip instrument compatible with universal linker sequence, is owned The sequence abundances information of probe connection product；
(5) by the method for bioinformatic analysis, the relative expression quantity and sequence information of different rna transcription sheets are determined.
2. breast cancer recurrence risk checking method according to claim 1, it is characterised in that in the step (1) Total nucleic acid is the RNA directly extracted；The specific base sequence of the multiple probe group of the step (2) and corresponding rna transcription sheet Base sequence reverse complemental；The nucleic acid ligase has single stranded DNA nicking coupled reaction in catalysis RNA-DNA hybridization chains Ability；The purification process of the step (4) is by being marked with Oligo (dT)NThe magnetic bead of (15≤N≤35) or affine column purification RNA- probe double-strand connection product compounds.
3. breast cancer recurrence risk checking method according to claim 2, it is characterised in that the nucleic acid ligase For T4 DNA Ligase orLigase。
4. breast cancer recurrence risk checking method according to claim 1, it is characterised in that in the step (1) Total nucleic acid is the single-stranded cDNA that RNA reverse transcriptions obtain；In the multiple probe group of the step (2) specific base sequence should with it is corresponding Rna transcription sheet base sequence it is consistent；The nucleic acid ligase has single stranded DNA nicking in catalytic dna-DNA hybridization chain Coupled reaction ability；Purification process in the step (4) is the magnetic bead or affine column purification cDNA- spies marked using Avidin Pin double-stranded products compound.
5. breast cancer recurrence risk checking method according to claim 4, it is characterised in that the nucleic acid ligase Selected from T4 DNA Ligase, T3 DNA Ligase, Taq DNA Ligase, T7 DNA Ligase, 9 ° of NTMDNA Ligase and The one or more of Ligase-65.
6. breast cancer recurrence risk checking method according to claim 1, it is characterised in that in the step (2) Hybridization temperature be 50-60 DEG C；The preparation method of the step (1) is the method by kit extraction or trizol extractions； Universal primer in the step (3) is 5UAP/3UA, and the condition of the PCR reactions is 98 DEG C of denaturation 30s, and 55-60 DEG C is annealed 30s, 72 DEG C of extension 15s, common 30-35 circulation；The sequenator or biological chip instrument of the step (4) include Illumina Hiseq, Miseq, Nextseq microarray dataset, Life Ion microarray datasets, Illumina, Affymetrix chip platform.
7. breast cancer recurrence risk checking method according to claim 1, it is characterised in that in the step (2) Multiple probe group include at least two fragments of left probe and right probe；Left probe includes 5 ' universal sequences and 3 ' specific hybridization sequences Row, right probe include 5 ' the specific hybridization sequences and 3 ' universal sequences of phosphorylated modification；It is close between left probe and right probe Adjacent no base notch, can hybridize in same single nucleic acid strands template, adjacent probe can be urged by nucleic acid ligase at the same time Change is connected as same nucleic acid molecules.
8. breast cancer recurrence risk checking method according to claim 7, it is characterised in that the 5 ' of the left probe 5 ' specific hybridization sequences of the phosphorylated modification of universal sequence and 3 ' specific hybridization sequences and right probe and 3 ' universal sequences Between can add 8-15 randomized bases (dN)8-15。
9. a kind of multiple probe group, it is characterised in that including at least two fragments of left probe and right probe；It is logical that left probe includes 5 ' With sequence and 3 ' specific hybridization sequences, right probe includes 5 ' the specific hybridization sequences and 3 ' universal sequences of phosphorylated modification；It is left Close adjacent no base notch, can hybridize in same single nucleic acid strands template, adjacent spy at the same time between probe and right probe Pin can be catalyzed by nucleic acid ligase and be connected as same nucleic acid molecules.
10. the purposes of multiple probe group according to claim 9, it is characterised in that the probe groups can be used in following The risk supervision of disease, the disease include tumour, microorganism infection, self-closing disease and genetic disease.
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