CN108611346A - A kind of construction method in unicellular gene expression amount detection library - Google Patents
A kind of construction method in unicellular gene expression amount detection library Download PDFInfo
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Abstract
The invention belongs to biotechnologies, and in particular to a kind of construction method in unicellular gene expression amount detection library.The present invention as little as RNA of pg ranks using in individual cells builds library in such a way that chain specificity and swivel base combine, is detected to the expression quantity of the specific gene of specific cells, to which gene sequencing has been applied to individual cells level as initial amount.Present invention can apply to different types of microarray dataset, library construction initial amount is extremely low, is the nucleic acid amount of individual cells, can be directed to individual cells and carry out gene expression quantifier elimination.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of structure side in unicellular gene expression amount detection library
Method.
Background technology
Traditional sequencing approach, either genetic chip or two generations are sequenced, and are required for carrying from more than 100,000 cells
A large amount of DNA or RNA is taken, and the information provided is the average value of a large amount of cells.RNA sequencings (RNA-seq) have become biology
It learns and the indispensable tool of medicine, RNA sequence information, in the characterization of transcriptional level, is analyzed as body gene for medical diagnosis on disease
Important support is provided with the methods of gene therapy application.Unicellular gene sequencing refers in individual cells level to genome
It is sequenced.2013, Annual Technical was granted by unicellular gene sequencing by Nature Journal, it is believed that the technology will change living nature
With many fields of medical field.
And gene expression amount of certain a kind of even some cell studied, more contribute to we have appreciated that cell
Function, variation, origin etc., it is therefore desirable to establish unicellular gene expression amount detection library.
Invention content
For blank of the existing technology, the present invention provides a kind of structure side in unicellular gene expression amount detection library
Method, it is therefore an objective to using the as little as RNA initial amounts of pg ranks in individual cells come the structure in such a way that chain specificity and swivel base combine
Library is built, the expression quantity of the specific gene of specific cells is detected, to which gene sequencing has been applied to individual cells layer
Face can study the gene expression amount of certain a kind of even some cell, more contribute to the work(we have appreciated that cell
Can, variation, origin etc..
Realize that the construction method in the unicellular gene expression amount detection library of the object of the invention follows the steps below：
(1) individual cells are sorted out by flow cytometer, be placed in Lysis Buffer buffer solutions, individual cells cracking
Discharge RNA；
(2) reverse transcription group needed for the synthesis of the first chain is directly added into containing single celled Lysis Buffer buffer solutions
Point, reverse transcription synthesizes First-Strand cDNA；
(3) amplification system directly is added in upper step system afterwards in step (2)；
(4) magnetic bead is added afterwards in step (3) and carries out PCR product purifying；
(5) swivel base system is added afterwards in step (4), carries out swivel base and adjunction head；
(6) swivel base product is purified after step (5)；
(7) PCR amplification system is added afterwards in step (6), carries out the amplification in library；
(8) magnetic bead is added afterwards in step (7) to purify the library after amplification, product after purification is upper machine text
Library.
Wherein, the cell includes cultured cell in vitro, the free cell in various histocytes and body fluid, cell
Source of species include the mankind, rat, mouse.
The RNA amounts are down to unicellular grade or pg ranks.
The main component of the Lysis Buffer buffer solutions includes：SDS, NP-40, PMSF, NaCl, EDTA, Tris-
HCl。
The reverse transcription component includes：Buffer solution, dNTPs, reverse transcriptase, RNase inhibitor and primer, wherein primer
Primer is redirected including reverse transcriptase primer and template, if reverse transcriptase primer SC-RT-T30 and template redirect primer SC-TSO-GC3G,
3min is denaturalized at 72 DEG C before addition, the end of reverse transcriptase primer 5 ' is sequencing primer sequence, and 3 ' ends are oligodT primer sequences,
Oligo dT primer sequence length is 20-60nt；It is sequencing primer sequence that template, which redirects the end of primer 5 ', and 3 ' ends are to redirect primer sequence
Row redirect primer sequence 3 ' and hold the sequence containing 3-10nt length, and it is the embedding of DNA, RNA or DNA and RNA to redirect series
Close, redirecting series needs to modify, the modification include methylate modification, amination modification, phosphorylation modification, thio repair
Decorations, C3Spacer modifications.
The amplification system includes buffer solution, archaeal dna polymerase, dNTPs and a pair of of upstream and downstream primer.
The swivel base system includes LM Buffer, TN5 swivel bases complex and cDNA, and condition is 55 DEG C needed for swivel base,
15min, wherein TN5 swivel bases complex include TN5 transposases, primer SC-TN5-ADT2 and SC-TN5-CORE.
The PCR amplification system includes buffer solution, archaeal dna polymerase, dNTPs and a pair of of upstream and downstream primer, wherein on
Primer TN5-INDEX is swum, 5 ' ends are P7 joint sequences, and 3 ' ends are specific primer sequence, downstream primer TS-Primer1,5 ' ends
For P5 joint sequences, 3 ' ends are specific primer sequence.
The upper machine library size is in 400-700bp.
The upper machine library, for Roche, Illumina, ThermoFisher, Pacific Biosciences, China
Big gene, Oxford Nanopore Technologies, Hua Yinkang, big desert gene high-flux sequence platform.
Compared with prior art, the features of the present invention and advantageous effect are：
(1) the present invention provides a kind of unicellular transcript profile library constructing methods, and it is flat to can be applied to different types of sequencing
Platform, library construction initial amount is extremely low, is the nucleic acid amount of individual cells, can be directed to individual cells and carry out grinding for gene expression amount
Study carefully.
(2) present invention terminates to complete in a pipe from cell cracking to pre- library PCR, and error rate is significantly reduced,
It is possible to prevente effectively from operational pollution, shortens operating procedure, library construction efficiency is improved.
(3) the entire library construction flow of the present invention is short, easy to operate, time-consuming short, at low cost.
(4) present invention is advantageously implemented automation mechanized operation and structure is concentrated in extensive library.
(5) experimental result of the method for the present invention is reproducible, and the unicellular transcript profile Library Quality of structure is high.
(6) banking process provided by the invention can be used for different microarray datasets, and applicability is wide, easy to spread.
Description of the drawings
Fig. 1 is the structure schematic diagram in the unicellular gene expression amount detection library of the present invention；
Fig. 2 is the structure flow chart in the unicellular gene expression amount detection library of the present invention；
Fig. 3 is that 2% agarose gel electrophoresis detects library result figure in the embodiment of the present invention 1；
Wherein：M:100bp DNA ladder (precious bioengineering (Dalian) Co., Ltd)；1：Experimental group is built slender
Born of the same parents' transcript profile library；2：The unicellular transcript profile library of control group structure.
Specific implementation mode
The embodiment of the present invention is by taking Illumina platforms as an example, and the present invention is further described in conjunction with the embodiments.
Embodiment 1：
The present embodiment is carried out using patient with breast cancer's normal tissue cell (experimental group) and cancerous issue cell (control group)
Unicellular transcript profile library construction and sequencing analysis specifically follow the steps below as depicted in figs. 1 and 2：
(1) the unicellular separation of patient with breast cancer's canceration histocyte and normal tissue cell, by flow cytometer into
The single celled separation of row is placed in the PCR pipe equipped with 5 μ L Lysis Buffer, and mixing places 10-20min on ice；
(2) reverse transcription component needed for the synthesis of the first chain, reverse transcription is directly added to synthesize First- in the PCR pipe of step (1)
Strand cDNA；
Specifically, reverse transcription component needs first to carry out primer denaturation before being added, and table 1 is added in the PCR pipe of step (1) and reacts
System, 72 DEG C of processing 3min of thermal cycler, sets on ice immediately；
1 reaction of degeneration (RD) system of table
| Component | Volume (μ L) |
| SC-RT-T30 | 1 |
| SC-TSO-GC3G | 1 |
| Nuclease free water | 2 |
After reaction of degeneration (RD), it is directly added into 2 reverse transcription reaction system of table, mixing is placed at room temperature for 10min, thermal cycler
In 42 DEG C reaction 90min；
2 reverse transcription reaction system of table
| Component | Volume (μ L) |
| First-strand Buffer | 4 |
| DTT | 2 |
| dNTP | 3.5 |
| RNase Inhibitor | 0.5 |
| Reverse Transcriptase | 1 |
| Total volume | 20 |
(3) amplification system that table 3 is added directly in upper step system afterwards in step (2) carries out pre- amplified library；
3 pre- amplified library reaction system of table
| Component | Volume (μ L) |
| KAPA HiFi HotStart ReadyMix | 25 |
| SC-ISPCR-P1 | 1 |
| SC-ISPCR-P2 | 1 |
| Nuclease free water | 3 |
| Total volume | 50 |
PCR amplification program：
(4) magnetic bead is added afterwards in step (3) and carries out PCR product XP Beads purifying, specifically use 10mM Tris-HCl will
PCR product complements to 50 μ L, and 40 μ L AMPure XP beads are added, and mixing is simultaneously stored at room temperature 5min.Reaction tube is placed in magnetic force
On frame, 3-5min is stood.Supernatant is abandoned, 200 μ L, 80% ethyl alcohol is added to be eluted, is repeated 2~3 times, with 25 μ L10mM Tris-
HCl is eluted.It takes 2 μ L to carry out Qubit to quantify, 5 μ L is taken to carry out 2% agarose gel electrophoresis, 140V, 25min；
(5) swivel base system is added afterwards in step (4), carries out swivel base and adjunction head, specifically：
With 4 reaction systems of tabulating, mixing, 55 DEG C of reaction 15min in thermal cycler.After 20 μ L ddH are added at once2O
It blows and beats mixing and terminates reaction；
4 swivel base reaction system of table
| Component | Volume (μ L) |
| LM buffer | 2 |
| TN5 swivel base complexs | 2 |
| Upper step product | <6 |
| ddH2O is complemented to | 10 |
(6) swivel base product is purified after step (5), specifically according to QIAquick PCR Purification
Kit (250) (QIAGEN, 28106) kit specification is purified；
(7) PCR amplification system is added afterwards in step (6), carries out the amplification in library, specifically：
5 PCR reaction systems of table
PCR amplification program：
4℃forever；
(8) magnetic bead is added afterwards in step (7), it is pure according to XP Beads are carried out to the library after amplification the step of step (4)
Change, product after purification is upper machine library.
Quality inspection and sequencing are carried out to upper machine library, specifically：
According to KAPA Library Quantifcation Kit (Kapabiosystems, Cat No.KK4854) explanation
Book is operated, and is detected using 480 real-time fluorescence quantitative PCR instrument of Roche Light Cycler, with reference to the standard in kit
Product carry out the absolute quantitation of library concentration, and the results are shown in Table 6.
6 qPCR testing results of table
| Sample | QPCR concentration (nM) |
| Experimental group | 115 |
| Control group | 95 |
The results show that the library of structure reaches confidential and seeks concentration, it can be used for machine sequencing.
Library denaturation, dilution and sequencing are carried out according to NextSeq sequenator operating processes, sequencing result is as shown in table 7.
7 sequencing data result of table
| Sample | Detect gene | It counts |
| Experimental group | HEPN1 | 1220 |
| Control group | HEPN1 | 6 |
As a result show that the HEPN1 gene expression amounts of patient's cancerous issue cell are substantially less than normal tissue cell.
Library is detected using 2% agarose gel electrophoresis, according to electrophoretic analysis result (Fig. 3), library master tape understands and leads
It is distributed in 500bp or so, band brightness is bright, consistent with expected results.
Embodiment 2：
The present embodiment examines patient with breast cancer's sample nucleic acid by real-time fluorescence quantitative PCR-TaqMan probe method
It surveys, to verify the sequencing result of embodiment 1.
(1) in embodiment 1 patient with breast cancer's normal structure (experimental group) and cancerous issue (control group) carry out respectively
RNA is extracted.
(2) RevertAid First Strand cDNA Synthesis Kit (ThermoFisher, goods is respectively adopted
Number：) and TaqManUniversal MasterMix II with UNG (Applied Biosystems, article No. K1622：
1605043) cDNA synthesis and PCR reactions are carried out.
(3) amplification and fluoroscopic examination are carried out using 480 real-time fluorescence quantitative PCR instrument of Roche Light Cycler.
Using 2-△△CtResult is analyzed
8 Q-PCR data results of table
As a result show that the HEPN1 gene expression amounts of patient's cancerous issue cell are substantially less than normal tissue cell, result
It is consistent with the unicellular transcript profile library sequencing result of structure.
Claims (10)
1. a kind of construction method in unicellular gene expression amount detection library, it is characterised in that follow the steps below：
(1) individual cells are sorted out by flow cytometer, be placed in Lysis Buffer buffer solutions；
(2) reverse transcription component needed for the synthesis of the first chain is directly added into containing single celled Lysis Buffer buffer solutions, it is inverse
Transcription synthesis First-Strand cDNA；
(3) amplification system directly is added in upper step system afterwards in step (2)；
(4) magnetic bead is added afterwards in step (3) and carries out PCR product purifying；
(5) swivel base system is added afterwards in step (4), carries out swivel base and adjunction head；
(6) swivel base product is purified after step (5)；
(7) PCR amplification system is added afterwards in step (6), carries out the amplification in library；
(8) magnetic bead is added afterwards in step (7) to purify the library after amplification, product after purification is upper machine library.
2. a kind of construction method in unicellular gene expression amount detection library according to claim 1, it is characterised in that institute
The cell stated includes cultured cell in vitro, and the source of species of the free cell in various histocytes and body fluid, cell includes
The mankind, rat, mouse.
3. a kind of construction method in unicellular gene expression amount detection library according to claim 1, it is characterised in that institute
The RNA amounts stated are down to unicellular grade or pg ranks.
4. a kind of construction method in unicellular gene expression amount detection library according to claim 1, it is characterised in that institute
The main component for the Lysis Buffer buffer solutions stated includes：SDS, NP-40, PMSF, NaCl, EDTA, Tris-HCl.
5. a kind of construction method in unicellular gene expression amount detection library according to claim 1, it is characterised in that institute
The reverse transcription component stated includes：Buffer solution, dNTPs, reverse transcriptase, RNase inhibitor and primer, wherein primer include reverse transcription
Primer and template redirect primer, if reverse transcriptase primer SC-RT-T30 and template redirect primer SC-TSO-GC3G, exist before addition
72 DEG C of denaturation 3min, the end of reverse transcriptase primer 5 ' are sequencing primer sequence, and 3 ' ends are oligo dT primer sequences, oligo dT primers
Sequence length is 20-60nt；It is sequencing primer sequence that template, which redirects the end of primer 5 ', and 3 ' ends redirect primer to redirect primer sequence
Sequence 3 ' holds the sequence containing 3-10nt length, redirects the Qian He that series are DNA, RNA or DNA and RNA, redirects sequence alkali
Base needs to modify, and the modification is repaiied including the modification that methylates, amination modification, phosphorylation modification, thio-modification, C3Spacer
Decorations.
6. a kind of construction method in unicellular gene expression amount detection library according to claim 1, it is characterised in that institute
The amplification system stated includes buffer solution, archaeal dna polymerase, dNTPs and a pair of of upstream and downstream primer.
7. a kind of construction method in unicellular gene expression amount detection library according to claim 1, it is characterised in that institute
The swivel base system stated includes LM Buffer, TN5 swivel bases complex and cDNA, and condition is 55 DEG C needed for swivel base, 15min, wherein
TN5 swivel base complexs include TN5 transposases, primer SC-TN5-ADT2 and SC-TN5-CORE.
8. a kind of construction method in unicellular gene expression amount detection library according to claim 1, it is characterised in that institute
The PCR amplification system stated includes buffer solution, archaeal dna polymerase, dNTPs and a pair of of upstream and downstream primer, wherein sense primer TN5-
INDEX, 5 ' ends are P7 joint sequences, and 3 ' ends are specific primer sequence, and downstream primer TS-Primer1,5 ' ends are P5 connector sequences
Row, 3 ' ends are specific primer sequence.
9. a kind of construction method in unicellular gene expression amount detection library according to claim 1, it is characterised in that institute
The upper machine library size stated is in 400-700bp.
10. a kind of construction method in unicellular gene expression amount detection library according to claim 1, it is characterised in that institute
The upper machine library stated, for Roche, Illumina, ThermoFisher, Pacific Biosciences, Hua Da gene,
Oxford Nanopore Technologies, Hua Yinkang, big desert gene high-flux sequence platform.
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Cited By (2)
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Application publication date: 20181002 |