CN109554450A - The method that circulating tumor cell captures and carries out full genome transcript profile detection - Google Patents
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Abstract
The method for being captured the present invention relates to circulating tumor cell (CTC) and carrying out full genome transcript profile detection.Carry out present invention discloses CTC in a kind of capture sample to be tested and to it method that unicellular full genome transcript profile is sequenced and analyzes.Method of the invention is in a simple manner after preliminary concentration CTC in sample to be tested, by carrying out unicellular capture with micro-fluidic chip, prepare Water-In-Oil emulsion droplet, it carries out high-throughput unicellular full genome transcription and sets up library, finally transcript profile data split and to individual cells genetic transcription group analysis.The analysis of the information such as number, source, mutation, the heterogeneity of CTC may be implemented in method of the invention.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of circulating tumor cell (CTC) captures and carries out full genome
The method of transcript profile detection.
Background technique
Entity tumor is during the growth process with the variation of surrounding microenvironment, and a part of cell falls off from thereon, with blood
Liquid circulation or lymphatic system are transferred to body everywhere, wherein it is thin to be referred to as circulating tumor via sanguimotor tumour cell
Born of the same parents (CTC, Circulating Tumor Cells), these cells further breed and form secondary tumors under optimum conditions.
In recent years, as tumour early sieves the proposition of concept, the early detection and detection of CTC has played important work in tumour early prevention
With.Meanwhile the Monitoring of Quantity of CTC cell provides important evidence for the treatment of cancer patient and prognosis evaluation.Therefore, CTC
Effectively capture and Clinical significance of detecting are great.However, circulating tumor cell content in patient's peripheral blood is very low, in every milliliter of whole blood only
There is 1~10 CTC, and individual is heterogeneous higher, how effectively to carry out capture and accurate detection to CTC is the weight currently faced
Big technological challenge.
In recent years, about the capture of circulating tumor cell and identification by domestic and international extensive concern.It is a series of rich for CTC
Collection and the method for detection are reported in succession.Traditional CTC detection method is substantially based on the detection to CTC cell surface antigen and to determine
Amount.In these methods, antibody is attached to first on a solid carrier, due to antibody-antigene interaction antibody and
Cell to be detected forms compound.Same principle, antibody may first pass through antigen-antibody reaction and cell to be checked formed it is compound
Then object is reattached on a solid carrier.Thereafter, the cell caught by antibody is collected and marks to be quantified with this.Than
As immunofluorescence technique detects circulating tumor cell.First be attached to the antibody of magnetic bead or micro-fluid chip by tumour cell from
Enrichment comes out in blood.Then with the antibody marked tumor cell with marker, tumor cell specific and quantitative inspection is carried out
It surveys.In general, marker includes radioactive isotope, dyestuff, fluorescein and enzyme (reacting for enzyme mark) etc..Although being based on
The detection method of antigen capture is clinically widely used, but there are many shortcomings for these methods: (1) detection sensitivity
Low, the detection of antigen enzyme mark is mainly by directly detecting cellular antigens, and the detection limit of these methods is limited, therefore thin
When born of the same parents' quantity is rare, sensitivity is inadequate.(2) flux is low, and the flux of these methods is limited to manually, therefore is not suitable for largely sieving
It looks into.(3) troublesome in poeration and human error is big, and the operating procedure of conventional method is complicated, and manual request is high, therefore unavoidably has very
The experimental error more being artificially introduced, so as to cause the credibility decline of testing result.(4) Multiple detection, to the multiple anti-of sample
Original carries out while detection is the important guiding index of clinical diagnosis and treatment parting, due to the limitation of marker, it is impossible to sample
This antigen carries out large-scale scanning.
It is existing a series of in CTC enrichment and the method detected, the enrichment of CTC mainly to utilize cell surface antigen
Specific binding carry out, including positive prize law and negative prize law.Positive prize law depends on tumor cell surface egg
The expression of white EpCAM, it is tissue-derived by CTC and EpCAM expression is influenced, it is weak for EpCAM expression or do not express
Tumour cell, this method can not be captured effectively.Feminine gender capture rule adsorbs leucocyte, removal using CD54 magnetic bead antibody
It is enriched with after leucocyte, this method can recycle all kinds CTC, but purity is low, can not carry out accurate counting and analysis to CTC.
Therefore, this field urgently conducts further research and improves, to find more preferably enrichment, identification, divide
The method for analysing circulating tumor cell.
Summary of the invention
The method for being captured the purpose of the present invention is to provide circulating tumor cell and carrying out full genome transcript profile detection.
In the first aspect of the present invention, the method that tumour cell carries out transcriptome analysis is provided, which comprises
(1) enrichment cycles tumour cell, unicellular capture are simultaneously packaged into emulsion droplet, and generating includes single celled water in oil emulsion
Drop；
(2) library RNA-seq, sequencing are established to unicellular in emulsion droplet；
(3) transcriptome analysis is carried out.
In a preference of the invention, the capture and detect circulating tumor cell method be it is nondiagnostic,
The method of non-therapeutic.
In another preference of the invention, in step (1), enrichment cycles tumour cell includes: thin according to circulating tumor
The physical characteristic (such as: cell size, electrical property) of born of the same parents carries out preliminary concentration, or, according to the biological nature of circulating tumor cell
(such as: CD45 feminine gender, the EpCAM positive) carries out preliminary concentration, obtains the sample of enriched circulating tumor cell.
In another preference of the invention, in step (1), enrichment cycles tumour cell includes: red to sample removal thin
Born of the same parents, and be enriched with by CD45+ magnetic bead feminine gender, the leucocyte that cell surface has CD45 label is excluded, it is swollen to obtain enriched circulation
The sample of oncocyte.
In another preference of the invention, red blood cell is removed by the method for erythrocyte splitting.
In another preference of the invention, after the sample for obtaining enriched circulating tumor cell, using 1 × PBS and
0.1%BSA mixed liquor is diluted, make its reach 2500 ± 300 cells of final concentration/microlitre.
In another preferred example, after dilution, further includes: the sample for obtaining claim 4 utilizes unicellular micro-fluidic core
Piece carries out unicellular capture, mixes, is distributed into cell cracking reaction solution, RNA reverse transcription primer, amplification enzyme and cell label
Emulsion droplet；Each different cell labels is had comprising single celled emulsion droplet.
In another preference of the invention, the cell label is individual cells specificity, for distinguishing difference
Individual cells；Preferably, the cell label is Cell Barcode.
In another preference of the invention, in step (2), establishing the library RNA-seq includes:
(i) cracking reaction will be carried out comprising the cell in single celled oily packet emulsion droplet, reverse transcription obtains the first chain cDNA；
(ii) the second chain cDNA is synthesized, fragmentation, amplification, purifying are carried out, generates sequencing library.
It in another preferred example, further include carrying out the broken step of emulsion droplet after obtaining the first chain cDNA in step (ii)
Suddenly, the second chain is synthesized again later.
In another preference of the invention, after generating sequencing library, sequencing analysis is carried out, according to different labels,
Individual transcriptome analysis is carried out to single loop tumour cell.
In another preference of the invention, the transcriptome analysis includes: to be swollen according to the single loop that label is distinguished
Oncocyte transcribes group information, carries out gene expression amount, gene mutation analysis, obtains single loop tumour cell transcript profile feature.
In another preference of the invention, gene expression amount, gene mutation analysis are being carried out, it is thin to obtain single loop tumour
After born of the same parents' transcript profile feature, further includes: according to the transcript profile feature of known different cells, distinguish the single loop tumour cell
Source and/or pathological characteristics.
Other aspects of the invention are apparent to those skilled in the art due to this disclosure
's.
Detailed description of the invention
Fig. 1, after being washed to the CTC cell of enrichment, microscopy is carried out after dyeing by platform phenol indigo plant, shows that cell activity exists in figure
90% or more.
Fig. 2, generation the result that is tested and analyzed with Agilent Bioanalyzer 2100 of cDNA library.
Fig. 3, the building library RNA-seq, the sequencing library of generation are detected with Agilent Bioanalyzer 2100
The result of analysis.
Specific embodiment
The present inventor passes through in-depth study, provides whole circulating tumor cells (CTC) in a kind of capture sample to be tested
And the method that unicellular full genome transcript profile is sequenced and analyzes is carried out to it.Method of the invention, in a simple manner to be measured
In sample after preliminary concentration CTC, by carrying out unicellular capture with micro-fluidic chip, Water-In-Oil emulsion droplet is prepared, high pass is carried out
Library is set up in the unicellular full genome transcription of amount, finally split and to individual cells genetic transcription component to transcript profile data
Analysis.The analysis of the information such as number, source, mutation, the heterogeneity of CTC may be implemented in method of the invention.
As used herein, " sample to be tested " or " sample to be tested " refers to the substance for needing to carry out CTC cell analysis, can be with
From individual (such as human or animal, the more specifically blood, pleural effusion of such as patient), it is also possible to other sources, example
As some through processing or untreated lab material or artificial synthesized test article.It should be understood that " sample " or " sample "
Detection be not limited to diagnostic purpose, may also refer to other non-diagnostic purposes.
Capture circulating tumor cell of the invention and the method for carrying out full genome transcriptome analysis, main includes following step
Rapid: (1) enrichment cycles tumour cell, unicellular capture are simultaneously packaged into emulsion droplet, and generating includes single celled Water-In-Oil emulsion droplet；(2)
The library RNA-seq and sequencing are established to unicellular in emulsion droplet；(3) transcriptome analysis is carried out.Capture and detection of the present invention
The method of circulating tumor cell can be nondiagnostic, non-therapeutic method.
Using method of the invention, the requirement to the CTC concentration stage of early period is lower, for example, according to circulating tumor cell
Physical characteristic (such as: cell size, electrical property) carry out preliminary concentration, or according to the biological nature of circulating tumor cell (such as:
CD45 feminine gender, EpCAM positive etc.) preliminary concentration is carried out, the sample obtained containing circulating tumor cell can be used to subsequent
Step.In method of the invention, this preliminary enrichment allow in sample still comprising other than a certain amount of CTC cell other are thin
Born of the same parents.
As preferred embodiment of the invention, enrichment cycles tumour cell includes: to remove red blood cell to sample, and pass through CD45
The enrichment of+magnetic bead feminine gender excludes the leucocyte that cell surface has CD45 label, obtains the sample of enriched circulating tumor cell.
Removing red blood cell can be by conventional method, such as the method for passing through erythrocyte splitting.
As preferred embodiment of the invention, after the sample for obtaining enriched CTC cell, using 1 × PBS and 0.1%BSA
Mixed liquor is diluted, make its reach 2500 ± 300 cells of final concentration/microlitre.The dilution of appropriate solution, debita spissitudo, has
Conducive to so that subsequent step carries out well.
As preferred embodiment of the invention, as after above-mentioned dilution, further includes: react the sample of acquisition with cell cracking
Liquid, RNA reverse transcription primer, amplification enzyme and the mixing of cell label, in case in preparing emulsion droplet.Wherein, the label is single
Cell-specific, for distinguishing different individual cells.For sample and cell cracking reaction solution, RNA reverse transcription primer, expand
The solution for increasing enzyme and the mixing of cell label carries out unicellular capture using unicellular micro-fluidic chip, is distributed into emulsion droplet, obtains
Include single celled Water-In-Oil emulsion droplet.It so operates, the water phase in each emulsion droplet includes: individual cells, cell pyrolysis liquid,
RNA reverse transcription primer distinguishes the cell label (such as Cell Barcode) of cell origin and the enzyme that amplification is required.
After obtaining comprising single celled Water-In-Oil emulsion droplet, the library RNA-seq is established to unicellular in emulsion droplet, comprising:
Cracking including cell, oligodT reverse transcription obtain a chain cDNA.The cDNA that unicellular RNA reverse transcription obtains in each emulsion droplet
All have the cell label (such as Cell Barcode) of unique specificity.According to the broken template of emulsion droplet, two chain cDNA are synthesized,
And final library is obtained by PCR amplification.
After generating sequencing library, sequencing analysis is carried out, according to different labels, single loop tumour cell is carried out single
Only transcriptome analysis.The transcriptome analysis includes: to transcribe group information according to the single loop tumour cell that label is distinguished,
Gene expression amount, gene mutation analysis are carried out, single loop tumour cell transcript profile feature is obtained；Preferably, further include: foundation
It is known difference cell transcript profile feature, distinguish the single loop tumour cell source and/or pathological characteristics.
Method of the invention carries out unicellular capture with micro-fluidic chip and combines the side of the unicellular sequencing of droplet type
Method carries out high-throughput unicellular full genome transcription and sets up library after capturing whole CTC.All cells have single
Barcode information may be implemented by carrying out fractionation and the analysis of individual cells genetic transcription group to transcript profile data to CTC
It carries out accurate counting and the transcription group information of whole CTC individual cells can be obtained.By being somebody's turn to do compared with different cell transcription groups
Method can know the source CTC, and can further appreciate that the gene mutation of CTC, the heterogeneity of CTC, the pathological characteristics etc. of CTC are more
Kind have the information of directive significance to clinic, can be provided for the early screening of tumour, postoperative evaluation and Personalized medicine it is important according to
According to.
It in the prior art, is to be measured using traditional CTC cell surface antigen detection and quantitative approach, analyze CTC
Cell, precision, accuracy are inadequate.And present invention firstly provides carry out unicellular capture with micro-fluidic chip and tie
The method for closing the unicellular sequencing of droplet type, this method is reliable and stable, and accuracy is high.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or
According to the normal condition proposed by manufacturer.
In following embodiment, for the CTC in Pleural Effusion of lung cancer is captured and is carried out full genome transcript profile detection.
Matching used instrument is as follows in embodiment:
DNA quantitative analysis instrument: Agilent Bioanalyzer 2100, for carrying out quality testing and dense to various libraries
Degree analysis.
Unicellular capture instrument: BIO-RAD ddSEQ Single-Cell Isolator, the preparation for Water-In-Oil emulsion droplet
And single celled capture.
PCR instrument: Eppendorf PCR amplification instrument, for the generation of cDNA chain and the amplification of sequencing library.
Embodiment 1, unicellular capture and packing
Unicellular catch is carried out with micro-fluidic chip (being purchased from Illumina, SureCell Cartridge) to CTC cell
It obtains and the process for being distributed into emulsion droplet is as follows:
(1) cell in Lung Cancer Recurrence patient pleural effusion is separated, wherein containing circulating tumor cell；
(2) erythrocyte splitting is carried out to sample, removes red blood cell, and by the negative enrichment of CD45 magnetic bead, eliminated thin
Cellular surface has the leucocyte of CD45 label；
(3) active microscopy, counting, cell dilution pretreatment are carried out to the CTC cell of enrichment, comprising:
(a) cell is washed twice with the cleaning solution of 1 × PBS+0.1%BSA, carries out microscopy after dyeing by platform phenol indigo plant,
Cell activity is 90% or more, such as Fig. 1；
(b) after cell count, the CTC cell of enrichment is diluted with 1 × PBS+0.1%BSA, it is made to reach final concentration
2500 cells/microlitre；
(4) prepare cell cracking reaction solution, system such as table 1.
Table 1
(5) by 4.5 microlitres of final concentrations, 2500 cells/microlitre cell and 21.5 microlitres cell cracking reaction solution mix
And it mixes；
(6) prepare Barcode dilution, system such as table 2.
Table 2
(7) it pre-processes unicellular micro-fluidic chip: ddSEQ chip being packed into chip cartridges, and in 8 holes of an intermediate row
Each ddSEQ Priming Solution for being added 20 microlitres, it is after waiting 1 minute, ddSEQ Priming Solution is whole
It is sucked out；
(8) Barcode dilution is added in the reagent wells of ddSEQ chip, 20 microlitres is added in each hole；
(9) cell being suspended in cell cracking reaction solution is added in the sample well of ddSEQ chip, is added in each hole
20 microlitres；
(10) ddSEQ chip is added in oil liquid (BIO-RAD Droplet Generation Oil for EvaGreen)
Oil liquid hole in, in each hole be added 80 microlitres；
(11) BIO-RAD ddSEQ Single-Cell Isolator is run, unicellular capture is carried out and dispenses cell
Enter emulsion droplet.
Embodiment 2, the building library RNA-seq
In the present embodiment, single celled RNA-seq library construction process is as follows in emulsion droplet:
(1) it is transferred to what is generated in embodiment in 96 hole PCR plates comprising single celled water in oil emulsion dropping liquid；
(2) sample is placed on to the cracking that cell is carried out in PCR instrument, and reverse transcription obtains the first chain cDNA, specifically reacts item
Part is as follows:
37 DEG C 30 minutes；
50 DEG C 60 minutes；
85 DEG C 5 minutes；
Cool to 4 DEG C；
Wherein, the sequence of reverse transcription primer are as follows: 5'-AAGCAGTGGTATCAACGCAG AGTGGG-3'(SEQ ID NO:
1)；Wherein, last three bases are the base of riboguanosine modification, i.e., last three bases G GG are " rGrGrG ".
(3) 20 microlitres of Droplet Disruptor are added in each reaction system, are added 100 microlitres after waiting 30 seconds
Water destroys the emulsion droplet of Water-In-Oil；
(4) 90 microlitres of Purification Beads are added in each reaction system, and mix it in the liquid phase；Room
Temperature is incubated for after ten minutes, is placed on magnetic frame and is stood 10 minutes.Supernatant is removed with pipettor, and with 200 microlitre 80% of ethyl alcohol
It washes twice, purifies a chain cDNA of generation；
(5) 35 microlitres of Resuspension Buffer, the first chain of enriching and purifying cDNA are added；
(6) 36 microlitres of Second Strand Buffer and 18 microlitres of Second Strand is added in each sample
Enzyme synthesizes two chain cDNA in PCR instrument after mixing, specific reaction condition is as follows:
16 DEG C 2 hours；
Cool to 4 DEG C；
(7) purify cDNA library with Purification Beads, and with 10 microlitres of Resuspension Buffer by its
Elution；
(8) cDNA library generated is tested and analyzed with Agilent Bioanalyzer 2100, as a result such as Fig. 2；
(9) prepare fragmentation reagents, system such as table 3.
Table 3
(10) fragmentation cDNA: being added 30 microlitres of fragmentation reagents in the cDNA library reacting hole of each purifying, mixes
The fragmentation cDNA in PCR instrument afterwards, specific reaction condition such as Fig. 2；
55 DEG C 5 minutes；
Cool to 4 DEG C；
(11) 10 microlitres of Tagment Stop Buffer are added in each reaction, are incubated at room temperature 5 minutes；
(12) it expands sequencing library: 30 microlitres of Tagment PCR Mix, 10 microlitres of Tagment being added in each reaction
PCR Adapter, 10 microlitres of DNA adapter expand sequencing library in PCR instrument after mixing, specific reaction condition is as follows:
(13) purify sequencing library with Purification Beads, and will with 51 microlitres of Resuspension Buffer
It is eluted；
(14) sequencing library generated is tested and analyzed with Agilent Bioanalyzer 2100, as a result such as Fig. 3.
Embodiment 3, sequencing data is split and cell transcription group analysis
This example sequencing data is split and cell transcription group analysis process is as follows:
(1) sequencing library established is sequenced with 4000 sequenator of Illumina HiSeq；
(2) sequencing data is split according to different Barcode, and all reads with same Barcode both are from
In the same cell, independent analysis is carried out；
(3) everyone sample sequencing data has about 400 kinds of different Barcode, and representative has about 400 kinds of differences
It is unicellular；
(4) for the cell of all expression EpCAM, determination is circulating tumor cell.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
<110>Shanghai Ai Ke gene technology Co., Ltd
<120>method that circulating tumor cell captures and carries out full genome transcript profile detection
<130> 176246
<160> 1
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Claims (10)
1. a kind of capture and the method for carrying out transcriptome analysis to circulating tumor cell, which is characterized in that the described method includes:
(1) enrichment cycles tumour cell, unicellular capture are simultaneously packaged into emulsion droplet, and generating includes single celled Water-In-Oil emulsion droplet；
(2) library RNA-seq, sequencing are established to unicellular in emulsion droplet；
(3) transcriptome analysis is carried out.
2. the method as described in claim 1, which is characterized in that in step (1), enrichment cycles tumour cell includes: that basis is followed
The physical characteristic of ring tumour cell carries out preliminary concentration, or, carrying out preliminary concentration according to the biological nature of circulating tumor cell, obtains
Obtain the sample of enriched circulating tumor cell.
3. method according to claim 2, which is characterized in that in step (1), enrichment cycles tumour cell includes: to sample
Red blood cell is removed, and is enriched with by CD45+ magnetic bead feminine gender, the leucocyte that cell surface has CD45 label is excluded, is obtained through richness
Collect the sample of circulating tumor cell.
4. method according to claim 2, which is characterized in that after the sample for obtaining enriched circulating tumor cell, using 1 ×
PBS and 0.1%BSA mixed liquor is diluted, make its reach 2500 ± 300 cells of final concentration/microlitre.
5. method as claimed in claim 4, which is characterized in that after dilution, further includes: the sample for obtaining claim 4, benefit
Unicellular capture is carried out with unicellular micro-fluidic chip, with cell cracking reaction solution, RNA reverse transcription primer, amplification enzyme and thin
The mixing of born of the same parents' label, is distributed into emulsion droplet；Each different cell labels is had comprising single celled emulsion droplet.
6. method as claimed in claim 5, which is characterized in that wherein, the cell label is individual cells specificity,
For distinguishing different individual cells；Preferably, the cell label is Cell Barcode.
7. the method as described in claim 1, which is characterized in that in step (2), establishing the library RNA-seq includes:
(i) cracking reaction will be carried out comprising the cell in single celled oily packet emulsion droplet, reverse transcription obtains the first chain cDNA；
(ii) the second chain cDNA is synthesized, fragmentation, amplification, purifying are carried out, generates sequencing library.
8. the method for claim 7, which is characterized in that in step (ii), obtain the first chain cDNA after, further include into
The broken step of row emulsion droplet, synthesizes the second chain again later.
9. the method for claim 7, which is characterized in that after generating sequencing library, sequencing analysis is carried out, according to difference
Label, individual transcriptome analysis is carried out to single loop tumour cell.
10. method as claimed in claim 9, which is characterized in that the transcriptome analysis includes: the list distinguished according to label
A circulating tumor cell transcribes group information, carries out gene expression amount, gene mutation analysis, obtains the transcription of single loop tumour cell
Group feature；Preferably, further include: according to the transcript profile feature of known different cells, distinguish the single loop tumour cell
Source and/or pathological characteristics.
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| WO2021128035A1 (en) * | 2019-12-25 | 2021-07-01 | 苏州绘真生物科技有限公司 | High throughput single cell transcriptome sequencing method and test kit |
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| US20120071335A1 (en) * | 2008-11-04 | 2012-03-22 | Silicon Biosystems S.P.A. | Method for Identification, Selection and Analysis of Tumour Cells |
| CN106456694A (en) * | 2013-12-20 | 2017-02-22 | 通用医疗公司 | Methods and assays relating to circulating tumor cells |
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