CN113463203B - Construction method of in-situ sequencing library for realizing in-situ detection of multiple RNAs - Google Patents
Construction method of in-situ sequencing library for realizing in-situ detection of multiple RNAs Download PDFInfo
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Abstract
The invention discloses a construction method of an in-situ sequencing library for realizing multiplex RNA in-situ detection, which constructs the in-situ sequencing library by utilizing a specially designed nucleic acid probe technology and realizes high-multiplex RNA in-situ detection by utilizing an in-situ sequencing technology and a fluorescence microscopic imaging technology; and compared with the whole label placed at one end, the whole label sequence is placed on two chains of the identification probe pair, so that the average distance between a sequencing position and a connecting position is shortened, and the signal intensity and the specificity are improved.
Description
Technical Field
The invention belongs to the technical field of RNA in-situ expression analysis, and particularly relates to a construction method of an in-situ sequencing library for realizing multiplex RNA in-situ detection.
Background
Spatial transcriptomics is an emerging gene expression analysis technique in recent years, which refers to a series of techniques that enable spatially localized and quantitative analysis of genes. Current spatial transcriptomics methods are largely divided into two major categories, methods based on microscopic imaging and methods based on sequencing. The former has in situ sequencing technology and single molecule fluorescence in situ hybridization-based coding technology, and the latter is a series of microarray-based primer coding addressing methods.
The in situ sequencing technology is a space transcriptomics technology which relies on a new generation sequencing chemical technology to realize in situ analysis of various gene expressions, and can be divided into full transcriptome in situ sequencing and targeted in situ sequencing, wherein the in situ sequencing library is constructed by using probes. The technology mainly uses a series of signals coded by different color fluorescence to detect different genes by sequencing signals from single RNA molecules in a cell or tissue in multiple rounds. Spatial transcriptomics techniques, represented by in situ sequencing, mainly include in situ sequencing techniques, fluorescence in situ sequencing techniques (FISSEQ) and STARmap (space-resolved transcript amplicon readout mapping) techniques. FISSEQ, instead of using probes for targeted detection, adopts a method similar to the next generation sequencing technology to directly reverse transcribe RNA into cDNA in situ and then cyclize and amplify the cDNA, and then directly performs in situ sequencing chemistry of longer fragments in cells, thereby obtaining a series of gene sequence fragments to realize multiplex RNA in situ detection. Both in-situ sequencing and STARmap technologies are based on capturing a target short sequence by using a lock probe or encoding different genes by using a lock probe with a label so as to realize simultaneous detection, and sequencing by the technology has a relatively limited target gene (N times of square labels of 4) because of a relatively short sequencing length (4-6 nt).
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a construction method of an in-situ sequencing library for realizing in-situ detection of multiple RNAs.
The technical scheme of the invention is as follows:
the construction method of the in-situ sequencing library for realizing the in-situ detection of the multiple RNAs comprises the following steps:
(1) Designing at least one recognition probe pair according to a target sequence on the target RNA; each identification probe pair consists of an upstream identification probe and a downstream identification probe, wherein the upstream identification probe comprises an upstream label sequence, the downstream identification probe comprises a downstream label sequence, and the upstream label sequence and the downstream label sequence form a complete label sequence for reading a corresponding target sequence;
(2) Delivering the at least one recognition probe pair into a cell to recognize and hybridize with a target sequence on the target RNA, wherein the 3 'end of the upstream recognition probe and the 5' end of the downstream recognition probe are adjacent to each other and are connected by a DNA ligase to form at least one chain DNA molecule;
(3) Hybridizing at least one chain DNA molecule obtained in the step (2) with a DNA splint sequence, enabling the 5 'end and the 3' end of the at least one chain DNA molecule to be close, and connecting the at least one chain DNA molecule with the DNA splint sequence through DNA ligase to form at least one circular DNA molecule;
(4) And performing rolling circle amplification by taking the at least one circular DNA molecule as a template to obtain amplified products, namely the in-situ sequencing library, decoding different labels in the in-situ sequencing library by an in-situ sequencing technology to obtain complete label sequences on each amplified product, and further obtaining the types of target RNAs detected by different amplified products, thereby obtaining in-situ expression information of different target RNAs and realizing in-situ detection of multiple RNAs.
In a preferred embodiment of the present invention, the target RNA includes RNA of the cell itself and RNA of the exogenous gene expressed inside the cell.
In a preferred embodiment of the invention, the DNA ligase is Splint R ligase.
In a preferred embodiment of the invention, the cells are free test cells or cells within a tissue.
In a preferred embodiment of the present invention, the target RNA includes RNA of the cell itself and RNA expressed in the cell by an exogenous gene, and the cell is a cell in a free test cell or tissue.
In a preferred embodiment of the present invention, the target RNA includes RNA of the cell itself and RNA expressed in the cell by an exogenous gene, and the DNA ligase is Splint R ligase.
In a preferred embodiment of the invention, the cell is a free test cell or a cell within a tissue and the DNA ligase is Splint R ligase.
In a preferred embodiment of the invention, the target RNA comprises RNA of the cell itself and RNA expressed in the cell by an exogenous gene, the cell is a cell in a free test cell or tissue, and the DNA ligase is Splint R ligase.
The beneficial effects of the invention are as follows:
1. The invention constructs an in-situ sequencing library by utilizing a specially designed nucleic acid probe technology, and realizes the in-situ detection of the RNA with high multiple by utilizing an in-situ sequencing technology and a fluorescence microscopic imaging technology.
2. In the invention, the complete tag sequence is placed on two chains of the identification probe pair, compared with the whole tag placed at one end, the average distance between a sequencing position and a connecting position is shortened, and the signal intensity and the specificity are improved.
3. In the invention, the complete tag sequence is placed on two chains of the identification probe pair, and compared with the situation that the complete tag is placed on one end, if the probe pair is mismatched, the generated error tag can be eliminated.
Drawings
FIG. 1 is a schematic diagram of the experiment of example 1 of the present invention.
FIG. 2 is a schematic diagram of in situ sequencing decoding of the in situ sequencing library generated in example 1 of the present invention.
FIG. 3 is a graph showing the experimental results of example 1 of the present invention.
Detailed Description
The technical scheme of the invention is further illustrated and described below by the specific embodiments in combination with the accompanying drawings.
Example 1 colorectal cancer Paraffin tissue section in situ analysis library method for in situ detection of 30 mRNA for experimental sample
The experimental principle of this embodiment is shown in fig. 1, and the specific steps are as follows:
Pretreatment of paraffin tissue:
Baking paraffin tissue slice in oven at 65deg.C for 30min, immediately soaking in xylene-containing staining jar for 10min, and replacing xylene for 10min. And taking out the glass slide from the second staining jar containing xylene, immediately placing the glass slide into the staining jar containing absolute ethyl alcohol for soaking for 2min, and replacing the glass slide with the absolute ethyl alcohol for soaking for 2min. Immediately placing the glass slide into a staining jar containing 95% ethanol for soaking for 2min, and replacing the glass slide with 95% ethanol for soaking for 2min. Immediately placing the glass slide into a staining jar containing 85% ethanol for soaking for 2min, and replacing the glass slide with 85% ethanol for soaking for 2min. Immediately placing the glass slide into a staining jar containing 70% ethanol for soaking for 2min, and replacing 70% ethanol for soaking for 2min.
The slide glass is taken out of the ethanol solution, soaked in DEPC-H 2 O for 5min, soaked in DEPC-PBS for 2min, and the residual ethanol is washed off. 4% PFA in DEPC-PBS was dropped onto the slide, covered completely with tissue samples, incubated at room temperature for 10min and rinsed with DEPC-PBS. 30mL of 0.1M HCl was placed in a staining jar and preheated to 37℃and 0.1mg/mL pepsin 30uL was added, immediately after mixing, the slide was immersed therein and incubated at 37℃for 30min. The slide glass was taken out and immersed in DEPC-H 2 O for 5min, and then immersed in DEPC-PBS for 2min for washing. Respectively passing through gradient ethanol: dehydrating 70%, 85% and 100% for 2min, and air drying. And finally, washing with DEPC-PBS-Tween20 for three times for later use.
(II) in situ nucleic acid detection:
(1) Hybridization of double ligation probes:
50. Mu.L of hybridization mixture containing 6 XSSC, 10% formamide and 0.1. Mu.M of each double-linked probe was added dropwise to the slide glass, and incubated at 37℃for 4 hours to allow each recognition probe pair to sufficiently hybridize with the target sequence of the corresponding gene.
The ligation probe sequences are shown in the following table:
(2) The above identification is directed to the first step connection:
After three washes with DEPC-PBS-Tween 20, 50. Mu.L of ligation reaction mixture containing 25% glycerol, 0.2. Mu.g/. Mu.L BSA, 1X SplintR buffer (NEB), 0.1U/. Mu. L SPLINTR LIGASE (NEB) and 1U/. Mu. L RiboLock RNA enzyme inhibitor (Thermo) was added to the samples and incubated at 37℃for 30min.
(3) Splint primer hybridization:
after three washes with DEPC-PBS-Tween 20, 50. Mu.L of hybridization mixture containing 6 XSSC, 10% formamide and 0.5. Mu.M splint primer at a final concentration was added to the sample, and incubated at 30℃for 30min.
The sequences of the splint primer are as follows: 5'-ggctccactaaatagacgca-3', SEQ ID NO.61.
(4) Probe-loop and rolling circle amplification:
After three washes with DEPC-PBS-Tween 20, 50. Mu.L of hybridization mixture containing 5% glycerol, 0.2. Mu.g/. Mu.L BSA, 1 XPhi 29 polymerase buffer (Thermo), 1mM dNTPs, 0.1U/. Mu. L T4 DNA LIGASE (Thermo) and 1U/. Mu.L Phi29 DNA polymerase (Thermo) was added to the sample and incubated at 37℃for 6 hours or 30℃overnight.
(5) Detection probe hybridization and image acquisition
Hybridization of the rolling circle amplification product with a detection probe (5'-tgcgtctatttagtggagcc-3', SEQ ID NO.62,5' end labeled with Cy 3), and finally nuclear staining with DAPI, detection, specifically: after washing the sample three times with DEPC-PBS-Tween 20, a reaction mixture containing 0.1. Mu.M detection probe, 20% formamide and 2 XSSC was added, and after incubation at 37℃for 30min, the samples were subjected to gradient ethanol: 70%, 85% and 100% dehydration treatment, each for 2min. And (5) airing in air. Finally adding DAPI with 0.5 mug/mLGold Antifade Mountan caplets (FERMANTAS) were incubated at room temperature for 10min, then examined by fluorescence microscopy and photographed.
(6) Elution of detection probes
The slide glass after photographing and imaging is placed in 70% ethanol for incubation, and the cover glass and the sealing tablet are washed off. Then, an elution buffer solution of 60% formamide and 0.1% Triton-X was added to the reaction zone, and the reaction was repeated 3 times at 37℃for 10 minutes. Finally, the samples were washed three times with DEPC-PBS-Tween 20.
(III) four rounds of in situ nucleic acid detection, the specific principle is shown in FIG. 2, and the specific principle is as follows:
(1) Anchored primer hybridization and fluorescent probe ligation
To the sample, 50. Mu.L of a sequencing reaction mixture containing 0.2. Mu.M anchor primer, 0.2. Mu.M each of fluorescent probes, 1 XT 4 DNA LIGASE buffer, 1mM ATP and 0.1 u/. Mu. L T4 DNA ligase was added, and incubated at 30℃for 1 hour. After three washes with DEPC-PBS-Tween 20, air dried. Finally, gold Antifade Mountan caplets (FERMANTAS) containing 0.5 μg/mL DAPI were added and incubated at room temperature for 10min before fluorescence microscopy and photography.
(2) Elution of fluorescent probes
The slide glass after photographing and imaging is placed in 70% ethanol for incubation, and the cover glass and the sealing tablet are washed off. Then, an elution buffer solution of 60% formamide and 0.1% Triton-X was added to the reaction zone, and the reaction was repeated 3 times at 37℃for 10 minutes. Finally, the samples were washed three times with DEPC-PBS-Tween 20.
(3) Repeating steps (1) and (2) three more times, and the above anchor primer and fluorescent probe sequences are shown in the following table:
| Sequence number | Name of the name | Sequence(s) | 5' Tag | 3' Tag |
| SEQ ID NO.63 | Anchor primer-L | tgcgtctatttagtg | P | / |
| SEQ ID NO.64 | Anchor primer-R | ctatttagtggagcc | / | / |
| SEQ ID NO.65 | Seq-base1-A | ctatcnnan | Cy5 | / |
| SEQ ID NO.66 | Seq-base1-T | ctatcnntn | AF488 | / |
| SEQ ID NO.67 | Seq-base1-C | ctatcnncn | TXR | / |
| SEQ ID NO.68 | Seq-base1-G | ctatcnngn | Cy3 | / |
| SEQ ID NO.69 | Seq-base2-A | ctatcnnna | Cy5 | / |
| SEQ ID NO.70 | Seq-base2-T | ctatcnnnt | AF488 | / |
| SEQ ID NO.71 | Seq-base2-C | ctatcnnnc | TXR | / |
| SEQ ID NO.72 | Seq-base2-G | ctatcnnng | Cy3 | / |
| SEQ ID NO.73 | Seq-base3-A | annnctatc | P | Cy5 |
| SEQ ID NO.74 | Seq-base3-T | tnnnctatc | P | AF488 |
| SEQ ID NO.75 | Seq-base3-C | cnnnctatc | P | TXR |
| SEQ ID NO.76 | Seq-base3-G | gnnnctatc | P | Cy3 |
| SEQ ID NO.77 | Seq-base4-A | nannctatc | P | Cy5 |
| SEQ ID NO.78 | Seq-base4-T | ntnnctatc | P | AF488 |
| SEQ ID NO.79 | Seq-base4-C | ncnnctatc | P | TXR |
| SEQ ID NO.80 | Seq-base4-G | ngnnctatc | P | Cy3 |
The above sequence n is any one of a, t, c, g.
(4) Analysis of Gene
By synthesizing four rounds of sequencing signals, each signal point obtains the color sequence of four rounds, and the base sequence on the corresponding probe designed before can know what kind of gene the signal is, as shown in figure 3.
The foregoing description is only illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the scope of the invention, i.e., the invention is not to be limited to the details of the invention.
Sequence listing
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<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 28
tagtggagcc acctctatcc tttgccttga cgatacag 38
<210> 29
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 29
tttgccatcc actatcttcc tatctcattg cgtctatt 38
<210> 30
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 30
tagtggagcc tcctctatcc tttggtctca gacaccac 38
<210> 31
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 31
ttgccatcca ctacttttcc tatcacagtg cgtctatt 38
<210> 32
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 32
tagtggagcc gcctctatcc ttttcagatg acacgacc 38
<210> 33
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 33
ttgggcagga cgtcagttcc tatcacgctg cgtctatt 38
<210> 34
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 34
tagtggagcc ggctctatcc tttgccgcag ctgttcac 38
<210> 35
<211> 39
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 35
aaagtcaaag aggtgctttc ctatctcagt gcgtctatt 39
<210> 36
<211> 39
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 36
tagtggagcc atctctatcc ttgccgagag gcgatgggc 39
<210> 37
<211> 37
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 37
aatattggag aggccttcct atctcactgc gtctatt 37
<210> 38
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 38
tagtggagcc tgctctatcc ttgatcctgg cctgaact 38
<210> 39
<211> 39
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 39
tcgaaggtga catagtgttc ctatctcgtt gcgtctatt 39
<210> 40
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 40
tagtggagcc cgctctatcc ttgctgtagt agagtccg 38
<210> 41
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 41
attctcctcg gtgtccttcc tatctctttg cgtctatt 38
<210> 42
<211> 39
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 42
tagtggagcc cactctatcc ttggtgttcg cctcttgac 39
<210> 43
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 43
gaatcactgc cagtcattcc tatctcgatg cgtctatt 38
<210> 44
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 44
tagtggagcc tcctctatcc ttggattttt aagaaaaa 38
<210> 45
<211> 39
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 45
agttctcgaa gtctgacttc ctatctcact gcgtctatt 39
<210> 46
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 46
tagtggagcc gcctctatcc ttgctccaaa ttccctgg 38
<210> 47
<211> 39
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 47
ttcttgctct atggtcgttc ctatctcagt gcgtctatt 39
<210> 48
<211> 39
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 48
tagtggagcc acctctatcc ttagttagca gaatcttga 39
<210> 49
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 49
cacactcaca ctcatattcg atagtcggtg cgtctatt 38
<210> 50
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 50
tagtggagcc agctctatcc ttccaacttc catgcaca 38
<210> 51
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 51
gatctccacg tagtccttcc tatctcgctg cgtctatt 38
<210> 52
<211> 37
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 52
tagtggagcc aactctatcc ttgtatttct ccccgtt 37
<210> 53
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 53
atgctggttg tacaggttcc tatctcgttg cgtctatt 38
<210> 54
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 54
tagtggagcc aactctatcc ttccgaggcg cccgggtt 38
<210> 55
<211> 39
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 55
cttaacaggt gctttggttc ctatctcact gcgtctatt 39
<210> 56
<211> 39
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 56
tagtggagcc agctctatcc ttacttgggg gtcaggagt 39
<210> 57
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 57
actccctgtc ctgaatttcc tatctcagtg cgtctatt 38
<210> 58
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 58
tagtggagcc agctctatcc tttctttgca gttggtca 38
<210> 59
<211> 39
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 59
atgtactcga tctcatcttc ctatctcact gcgtctatt 39
<210> 60
<211> 38
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 60
tagtggagcc ccctctatcc ttacaggatg gcttgaag 38
<210> 61
<211> 20
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 61
ggctccacta aatagacgca 20
<210> 62
<211> 20
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 62
tgcgtctatt tagtggagcc 20
<210> 63
<211> 15
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 63
tgcgtctatt tagtg 15
<210> 64
<211> 15
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 64
ctatttagtg gagcc 15
<210> 65
<211> 9
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 65
ctatcnnan 9
<210> 66
<211> 9
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 66
ctatcnntn 9
<210> 67
<211> 9
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 67
ctatcnncn 9
<210> 68
<211> 9
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 68
ctatcnngn 9
<210> 69
<211> 9
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 69
ctatcnnna 9
<210> 70
<211> 9
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 70
ctatcnnnt 9
<210> 71
<211> 9
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 71
ctatcnnnc 9
<210> 72
<211> 9
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 72
ctatcnnng 9
<210> 73
<211> 9
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 73
annnctatc 9
<210> 74
<211> 9
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 74
tnnnctatc 9
<210> 75
<211> 9
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 75
cnnnctatc 9
<210> 76
<211> 9
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 76
gnnnctatc 9
<210> 77
<211> 9
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 77
nannctatc 9
<210> 78
<211> 9
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 78
ntnnctatc 9
<210> 79
<211> 9
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 79
ncnnctatc 9
<210> 80
<211> 9
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 80
ngnnctatc 9
Claims (1)
1. A multiplex RNA in situ detection method for non-diagnostic or non-therapeutic purposes using colorectal cancer paraffin tissue sections as experimental samples and 30 target mrnas as subjects for RNA in situ detection, characterized in that: the method comprises the following steps:
(1) Designing thirty sets of recognition probe pairs according to target sequences on the 30 target mRNAs; each group of identification probe pairs consists of an upstream identification probe and a downstream identification probe, wherein the upstream identification probe comprises an upstream label sequence, the downstream identification probe comprises a downstream label sequence, the upstream label sequence and the downstream label sequence form a complete label sequence for reading the corresponding target sequences, and the information of the identification probe pairs is shown in the following table:
(2) Delivering thirty sets of recognition probe pairs obtained in the step (1) into cells of the colorectal cancer paraffin tissue section, and enabling the thirty sets of recognition probe pairs to recognize and hybridize with a target sequence on target RNA, wherein the 3 'end of an upstream recognition probe and the 5' end of a downstream recognition probe are close to each other, and are connected through DNA ligase capable of connecting DNA by taking RNA as a template to form corresponding chain DNA molecules, and the DNA ligase capable of connecting DNA by taking RNA as a template is Splint R ligase;
(3) Hybridizing the chain DNA molecule obtained in the step (2) with a DNA splint sequence shown as SEQ ID NO.61, enabling the 5 'end and the 3' end of the corresponding chain DNA molecule to be close, and then connecting the two ends through DNA ligase to form a corresponding circular DNA molecule;
(4) Performing rolling circle amplification by using the circular DNA molecule obtained in the step (3) as a template and using T4 DNA ligase and Phi29 DNA polymerase to obtain an amplification product, namely an in-situ sequencing library;
(5) Decoding different labels in the in-situ sequencing library through four rounds of in-situ nucleic acid detection to obtain complete label sequences on each amplified product, further obtaining the types of target RNAs detected by different amplified products, obtaining in-situ expression information of different target RNAs, and realizing multiple RNA in-situ detection; the four rounds of in situ nucleic acid detection include the steps of:
a) Anchor primer hybridization and fluorescent probe ligation; the information for the anchor primer and fluorescent probe is shown in the following table, where n is any one of a, t, c, g:
；
b) Eluting the fluorescent probe;
c) Repeating steps a and b three more times;
d) Analysis of genes: and synthesizing four rounds of sequencing signals, obtaining the color sequence of each signal point, and knowing the signal of the gene of the point according to the base sequence on the probe designed before.
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| US20020006617A1 (en) * | 2000-02-07 | 2002-01-17 | Jian-Bing Fan | Nucleic acid detection methods using universal priming |
| BR112013020773B1 (en) * | 2011-02-15 | 2023-01-24 | Leica Biosystems Newcastle Limited | METHOD FOR LOCALIZED IN SITU DETECTION OF RNA, METHOD FOR DETERMINING THE PRESENCE AND LOCATION OF A GENETIC SEQUENCE AND METHOD FOR IN SITU LOCATION AND/OR DETECTION OF A SPECIFIC RNA |
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