CN113373140A - Method and kit for generating and amplifying cDNA (complementary deoxyribonucleic acid) from single cell or trace RNA (ribonucleic acid) - Google Patents
Method and kit for generating and amplifying cDNA (complementary deoxyribonucleic acid) from single cell or trace RNA (ribonucleic acid) Download PDFInfo
- Publication number
- CN113373140A CN113373140A CN202110743091.8A CN202110743091A CN113373140A CN 113373140 A CN113373140 A CN 113373140A CN 202110743091 A CN202110743091 A CN 202110743091A CN 113373140 A CN113373140 A CN 113373140A
- Authority
- CN
- China
- Prior art keywords
- primer
- race
- template
- full
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention provides a method and a kit for generating and amplifying cDNA (complementary deoxyribonucleic acid) from single cell or trace RNA (ribonucleic acid) and application of the method and the kit.
Description
Technical Field
The invention belongs to the technical field of molecular biology. In particular, the present invention provides a method and kit for generating and amplifying cDNA from single cells or trace amounts of RNA. The invention also relates to the use of the methods and kits of the invention, e.g., 3 'and 5' bidirectional RACE, conventional sequencing, next generation sequencing, transcriptome banking, etc., on the resulting cDNA, e.g., for fetal detection in prenatal and postnatal care, disease (e.g., cancer) screening, cell screening in assisted reproduction and cell therapy, etc.
Background
RACE (Rapid amplification of cDNA Ends) is a technique for rapid amplification of the cDNA ends from transcripts based on reverse transcription PCR. RACE includes 3'RACE and 5' RACE. The primer containing polyT is used as a reverse transcription primer, a gene specific primer is designed according to the known sequence of a gene, a 3'RACE is used for obtaining a 3' end sequence, a 5'RACE is used for obtaining a 5' end sequence, and finally, a complete cDNA sequence is obtained through assembly.
The RACE process mainly comprises the following steps: RNA extraction, reverse transcription, 3 'or 5' end amplification, clone conversion and sequencing analysis. At present, the traditional RACE is separately carried out by a 3'RACE process and a 5' RACE process, different oligonucleotides containing polyT are respectively used as reverse transcription primers to carry out reverse transcription in the reverse transcription process, and then the 3'RACE process and the 5' RACE process are separately carried out.
Conventional RACE requires an initial amount of total RNA, typically 10ng to 1. mu.g. Moreover, single cell RACE is currently not achievable because the total RNA content of single cells does not meet the requirements of traditional RACE.
Therefore, the method for carrying out RACE on single cells or trace RNA is provided, and has important significance for the development of the transcriptome sequencing technology.
Summary of The Invention
The present invention provides a method and kit for generating and amplifying cDNA from single cell or trace RNA. The invention also relates to 5'RACE and 3' RACE of the resulting cDNA.
The invention can solve the problem that the traditional RACE requires high RNA starting amount (unidirectional RACE requires at least 1ng of total RNA), and can perform bidirectional RACE by using lysate of single cells or total RNA as a starting material as low as 5 pg. The present invention may start with 1 to 5000 cell lysates or 5pg to 5 μ g total RNA. The invention may also start with a small number of cells, even a single cell. This overcomes the problems of low RNA content and difficult extraction of single cells.
The invention can solve the problems that the traditional 5'RACE and 3' RACE need to be subjected to reverse transcription twice and the process is complex, can support the subsequent simultaneous 5'RACE and 3' RACE by one-time reverse transcription, and has simple operation process.
The invention can also solve the defect that the traditional transcriptome sequencing can not obtain the complete transcript end.
The whole process of the method only needs 3-5 hours, provides a rapid 5 'and 3' RACE mode for analyzing single cells, a few cells or trace RNA, and has important application prospect in the aspects of tumor detection, new gene cognition and the like.
In one aspect, the present invention provides a method for generating cDNA from a microrna sample, comprising:
(1) taking mRNA in a sample as a template, taking a reverse transcription primer as a primer, and carrying out reverse transcription by utilizing reverse transcription activity to obtain a cDNA chain;
(2) adding oligoC to the 3' end of one strand of cDNA using end transfer activity; and
(3) and further extending one strand of the cDNA by using the conversion template as a template and utilizing the conversion activity of the template to obtain the full-length cDNA.
In another aspect, the present invention provides a method for generating and amplifying cDNA from a trace amount of an RNA sample, comprising:
(1) taking mRNA in a sample as a template, taking a reverse transcription primer as a primer, and carrying out reverse transcription by utilizing reverse transcription activity to obtain a cDNA chain;
(2) adding oligoC to the 3' end of one strand of cDNA using end transfer activity;
(3) further extending cDNA chain to obtain full-length cDNA by using the conversion template as template and utilizing the template conversion activity; and
(4) the full-length cDNA is amplified by using DNA polymerase activity, with the full-length cDNA as a template and the 5 'full-length amplification primer and the 3' full-length amplification primer as primers.
In yet another aspect, the invention provides a method of performing 5'RACE and 3' RACE on a microrna sample comprising:
(1) taking mRNA in a sample as a template, taking a reverse transcription primer as a primer, and carrying out reverse transcription by utilizing reverse transcription activity to obtain a cDNA chain;
(2) adding oligoC to the 3' end of one strand of cDNA using end transfer activity;
(3) further extending cDNA chain to obtain full-length cDNA by using the conversion template as template and utilizing the template conversion activity;
(4) optionally, amplifying the full-length cDNA using DNA polymerase activity using the full-length cDNA as a template and the 5 'full-length amplification primer and the 3' full-length amplification primer as primers; and
(5) and/or, using the full-length cDNA as a template, using a 3' RACE universal primer and a 3' RACE gene specific primer as primers, and using the DNA polymerase activity to amplify the 5' end sequence of the full-length cDNA.
In yet another aspect, the invention provides a kit for performing the method of the invention. In one embodiment, the kit comprises:
(1) a reverse transcriptase having a terminal transfer activity, a template switching activity and a reverse transcription activity;
(2) high fidelity DNA polymerase;
(3) a reverse transcription primer;
(4) converting the template;
(5) 5' full-length amplification primers;
(6) 3' full-length amplification primers;
(7) a 5'RACE universal long primer and optionally a 5' RACE universal short primer;
(8) a 3'RACE universal long primer and optionally a 3' RACE universal short primer; and/or
(9) Optionally a cell lysis agent, an rnase inhibitor, a reverse transcription reaction buffer, dntps, an amplification buffer, a RACE dilution buffer, and double distilled water that is sterilized and/or filtered.
In one embodiment, the reverse transcription primer consists of, from 5' to 3', a 3' linker sequence, a polyT and an anchor base VN; the switch template consists of a 5' linker sequence and oligoG from 5' end to 3' end; the 5 'full-length amplification primer consists of a 5' adaptor sequence; the 3 'full-length amplification primer consists of a 3' adaptor sequence; the 5' RACE universal long primer consists of a 5' adaptor sequence and a 5' adaptor sequence from a 5' end to a 3' end, and the 5' RACE universal short primer consists of a 5' adaptor sequence; the 3' RACE universal long primer consists of a 3' adaptor sequence and a 3' adaptor sequence from a 5' end to a 3' end, and the 3' RACE universal short primer consists of a 3' adaptor sequence.
Drawings
FIG. 1 is a schematic flow chart of the present invention for simultaneously performing 3'RACE and 5' RACE on a single cell or a micro sample.
FIG. 2 is a graph showing the results of electrophoresis of the 3'RACE product and the 5' RACE product in example 1.
FIG. 3 is a graph showing the results of electrophoresis of the 3'RACE product and the 5' RACE product in example 2.
FIG. 4 is a graph showing the results of electrophoresis of the 3'RACE product and the 5' RACE product in example 3.
Detailed Description
Embodiments of the invention
In a first aspect, the present invention provides a method of generating cDNA from a microrna sample, comprising:
(1) performing reverse transcription by using mRNA in a sample as a template and a reverse transcription primer containing polyT as a primer to obtain a cDNA strand by utilizing the reverse transcription activity, wherein the reverse transcription primer contains or consists of polyT (for example, at least 15T, for example, 30T);
(2) adding oligoC to the 3' end of one strand of cDNA using end transfer activity; and
(3) further extending one strand of cDNA to obtain full-length cDNA using a template transfer activity, wherein the template transfer activity is a transfer template containing or consisting of oligoG.
In a second aspect, the present invention provides a method for generating and amplifying cDNA from a microrna sample, comprising:
(1) taking mRNA in a sample as a template, taking a reverse transcription primer containing or consisting of polyT as a primer, and carrying out reverse transcription by utilizing the reverse transcription activity to obtain a cDNA chain, wherein the reverse transcription primer also contains a 3 'joint sequence positioned at the 5' end of the polyT;
(2) adding oligoC to the 3' end of one strand of cDNA using end transfer activity;
(3) further extending a cDNA chain to obtain a full-length cDNA by using a conversion template with oligoG as a template and utilizing the template conversion activity, wherein the conversion template also contains a 5 'joint sequence positioned at the 5' end of the oligoG; and
(4) amplifying the full-length cDNA by using DNA polymerase activity by using the full-length cDNA as a template and a 5 'full-length amplification primer and a 3' full-length amplification primer as primers, wherein the 5 'full-length amplification primer comprises or consists of a 5' adaptor sequence; the 3 'full length amplification primer comprises or consists of a 3' adaptor sequence; optionally, the 5 'linker sequence is the same or different from the 3' linker sequence.
In a third aspect, the present invention provides a method of performing 5'RACE and 3' RACE on a microrna sample comprising:
(1) taking mRNA in a sample as a template, taking a reverse transcription primer containing or consisting of polyT as a primer, and carrying out reverse transcription by utilizing the reverse transcription activity to obtain a cDNA chain, wherein the reverse transcription primer also contains a 3 'joint sequence positioned at the 5' end of the polyT;
(2) adding oligoC to the 3' end of one strand of cDNA using end transfer activity;
(3) further extending a cDNA chain to obtain a full-length cDNA by using a conversion template with oligoG as a template and utilizing the template conversion activity, wherein the conversion template also contains a 5 'joint sequence positioned at the 5' end of the oligoG;
(4) optionally, amplifying the full-length cDNA using DNA polymerase activity using the full-length cDNA as a template and a 5 'full-length amplification primer and a 3' full-length amplification primer, wherein the 5 'full-length amplification primer comprises or consists of a 5' linker sequence; the 3 'full length amplification primer comprises or consists of a 3' adaptor sequence; optionally, the 5 'linker sequence is the same or different from the 3' linker sequence; and
(5) amplifying the 5 'end sequence of the full-length cDNA by using DNA polymerase activity with the full-length cDNA as a template and the 5' RACE universal primer and the 5'RACE gene specific primer as primers, and/or amplifying the 3' end sequence of the full-length cDNA by using the DNA polymerase activity with the full-length cDNA as a template and the 3'RACE universal primer and the 3' RACE gene specific primer as primers,
wherein said 5'RACE universal primer comprises or consists of a 5' linker sequence and said 3'RACE universal primer comprises or consists of a 3' linker sequence; optionally, the 5'RACE universal primer further comprises a 5' adaptor sequence located 5 'of the 5' adaptor sequence, the 3'RACE universal primer further comprises a 3' adaptor sequence located 5 'of the 3' adaptor sequence, optionally the 5 'adaptor sequence is the same as or different from the 3' adaptor sequence,
further optionally, the 5'RACE universal primer is used in combination with a 5' RACE universal short primer, the 5'RACE universal short primer comprising or consisting of a 5' adaptor sequence, the 3'RACE universal short primer is used in combination with a 3' RACE universal short primer, the 3'RACE universal short primer comprising or consisting of a 3' adaptor sequence.
In a fourth aspect, the present invention provides a kit for generating cDNA from a microrna sample for use in carrying out the method of the first aspect.
In a fifth aspect, the present invention provides a kit for generating and amplifying cDNA from a microrna sample for use in performing the method of the second aspect.
In a sixth aspect, the present invention provides a kit for performing 5'RACE and 3' RACE on a trace RNA sample for use in performing the method of the third aspect.
In one embodiment, the reverse transcription primer comprises or consists of a polyT and a 3 'linker sequence located 5' to the polyT; and/or, the switch template comprises or consists of an oligoG and a 5 'linker sequence located at the 5' end of the oligoG.
In one embodiment, the polyT in the reverse transcription primer is 3-50, 4-40, 5-30 or 6-20, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 ts.
In one embodiment, the reverse transcription primer contains an anchor base at the 3' end.
In one embodiment, the anchor base is V, which represents A, C and G.
In one embodiment, the anchor base is VN, V stands for A, C and G, and N stands for A, C, T and G.
In one embodiment, the oligogs in the conversion template are 3-6, such as 3, 4, 5, or 6G.
In one embodiment, the number of G in the oligoG in the conversion template is the same as the number of C in the oligoC.
In one embodiment, the number of G's in the oligoG in the conversion template is greater than the number of C's in the oligoC. In one embodiment, the first G at the 3' end of the oligoG is an LNA modified dG.
In one embodiment, the second and third G at the 3' end of the oligoG is rG.
In one embodiment, the 5 'full length amplification primer is partially or completely identical to the conversion template portion and the 3' full length amplification primer is partially or completely identical to the reverse transcription primer.
In one embodiment, the 5'RACE universal primer is partially or completely identical to the 5' full length amplification primer and/or the conversion template, and the 3'RACE universal primer is partially or completely identical to the 3' full length amplification primer and the reverse transcription primer.
In one embodiment, wherein the 5 'full length amplification primer is partially or completely identical to the 5' adaptor sequence and/or the 3 'full length amplification primer is partially or completely identical to the 3' adaptor sequence.
In one embodiment, said 5'RACE universal primer is partially or completely identical to said 5' adaptor sequence and/or said 3'RACE universal primer is partially or completely identical to said 3' adaptor sequence.
In one embodiment, the 5'RACE universal long primer is a mixture of, or consists of, a 5' RACE universal long primer and a 5'RACE universal short primer, the 5' RACE universal long primer comprises, or consists of, a 5 'adaptor sequence at the 5' end and a 5 'adaptor sequence at the 3' end, and the 5'RACE universal short primer comprises, or consists of, a 5' adaptor sequence, and/or the 3'RACE universal primer is a mixture of, or consists of, a 3' RACE universal long primer and a 3'RACE universal short primer, the 3' RACE universal long primer comprises, or consists of, a 3 'adaptor sequence at the 5' end and a 3 'adaptor sequence at the 3' end, and the 3'RACE universal short primer comprises, or consists of, a 3' adaptor sequence.
In one embodiment, said 5'RACE universal long primer and said 5' RACE universal short primer are used in a ratio of 1:10 to 1:1, and/or said 3'RACE universal long primer and said 3' RACE universal short primer are used in a ratio of 1:10 to 1:1
In one embodiment, the 5 'adaptor sequence is the same or different from the 3' adaptor sequence.
In one embodiment, the 3 '/5' linker sequence preferably does not specifically bind to the template, preferably the sequence itself does not form a hairpin structure, and preferably the 3 'and 5' linker sequences do not complementarily hybridize to form a primer dimer.
In one embodiment, the 5 'and 3' RACE gene specific primers for RACE amplification are 15-30nt in length and 50-70% in GC content, said specific primers have a Tm of 65 ℃ or more, preferably a Tm of more than 70 ℃, and the design site of the specific primers should be located within 5kb of the template end, preferably within 5kb to 1kb of the end.
In one embodiment, any one, any two, or all three of the reverse transcriptase activity, the terminal transfer activity, and the template switching activity are from a reverse transcriptase.
In one embodiment, the reverse transcriptase is MMLV.
In one embodiment, the DNA polymerase activity is from a high fidelity DNA polymerase.
In one embodiment, the high fidelity DNA polymerase is at least one of Pfu, vent, KOD 1.
In one embodiment, said steps (1) to (3) are performed together at a temperature of 37 ℃ or 42 ℃ for 5-120, 10-100, 15-90, 20-80, 25-70 or 30-60, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or 120 minutes, such as 42 ℃, 90 minutes.
In one embodiment, said step (1) comprises a prior denaturation, e.g. at a temperature of 70-100 ℃, 75-95 ℃ or 80-90 ℃, e.g. 70 ℃, 72 ℃ or 85 ℃ for 1 second-30 minutes, e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 seconds or 2, 3, 4, 5, 10, 15, 20, 25 or 30 minutes, e.g. 72 ℃ denaturation for 3 minutes, and/or said step (3) comprises a subsequent inactivation, e.g. at a temperature of 70-100 ℃, 75-95 ℃ or 80-90 ℃, e.g. 70 ℃, 72 ℃ or 85 ℃ for 1 second-30 minutes, e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 seconds or 2, 3, 4, 5, 10, 15, 20, 25 or 30 minutes, for example, inactivation at 70 ℃ for 15 minutes.
In one embodiment, the step (4) is performed as follows: pre-denaturation at 98 ℃ for 1 min; 5-30, 7-25 or 10-20, e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 cycles, denaturation at 98 ℃ for 10 seconds, annealing at 65 ℃ for 15 seconds, extension at 72 ℃ for 1-30, 2-20 or 5-10 minutes, e.g., 5, 6, 7, 8, 9 or 10 minutes; the extension was carried out at 72 ℃ for 5 minutes.
In one embodiment, especially when the Tm of the GSP is >70 ℃, said step (5) is performed as follows: pre-denaturation at 98 ℃ for 1 min; 5 cycles, denaturation at 98 ℃ for 10 seconds, extension at 72 ℃ for 3 minutes; 5 cycles, denaturation at 98 ℃ for 10 seconds, annealing at 70 ℃ for 15 seconds, and extension at 72 ℃ for 3 minutes; 10-30 or 20-25, e.g., 20 (for mRNA samples) or 25 (for total RNA samples) cycles, denaturation at 98 ℃ for 10 seconds, annealing at 68 ℃ for 15 seconds, and extension at 72 ℃ for 3 minutes; the extension was carried out at 72 ℃ for 5 minutes.
In one embodiment, especially when the Tm of the GSP is from 60 to 70 ℃, said step (5) is performed as follows: pre-denaturation at 98 ℃ for 1 min; 10-30 or 20-25, e.g., 20 (for mRNA samples) or 25 (for total RNA samples) cycles, denaturation at 98 ℃ for 10 seconds, annealing at 68 ℃ for 15 seconds, and extension at 72 ℃ for 3 minutes; the extension was carried out at 72 ℃ for 5 minutes.
In one embodiment, when the amplified fragment is >3kb, the extension time can be extended, for example 30 seconds for every 1kb increase.
In one embodiment, the micro RNA sample contains 5pg to 5 μ g, 10pg to 1 μ g, 50pg to 100ng, 100pg to 10ng, or 500pg to 1ng, e.g. 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000pg total RNA or mRNA.
In one embodiment, the microrna sample is from 1-5000, 2-1000, 5-200, or 10-50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 cells.
In one embodiment, the target gene, e.g., the target gene to which the RACE is directed, has an fpkm (fragments Per base of exon model Per Million mapped fragments) of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100.
In one embodiment, the trace RNA sample is from a single cell.
In one embodiment, the single cell is lysed and subjected to a reverse transcription reaction, wherein the cell lysis agent may be selected from at least one of Tween 20, Triton X100, NP 40. Any cell lysis agent that does not completely inhibit the subsequent reverse transcription and PCR reactions can be used.
In one embodiment, the cell lysis agent is used at a concentration ranging from 0.02% to 0.2%.
In one embodiment, the cell is a mammalian cell, such as a germ cell, an embryonic cell, an amniotic fluid cell, a stem cell, a neural cell, a lymphocyte, a T cell, a B cell, a CAR-T cell, or a tumor cell.
Method of the invention
The process for simultaneously generating and amplifying cDNA from single cell or trace RNA and performing 3'RACE and 5' RACE on the obtained cDNA is shown in FIG. 1.
The method comprises the following steps: after isolation of single cells, the single cells were lysed using cell lysates to release mRNA with a polyA tail (indicated by 4A in the figure, but not limited to 4A, the same below).
Step two: using mRNA with polyA tail released from a single cell as a template, a reverse transcription primer having a 3 'linker sequence and a polyT sequence (which may further have a 3' anchor base VN), reverse transcription was performed by the reverse transcriptase activity of the reverse transcriptase, and a cDNA single strand was synthesized.
Step three: when the reverse transcriptase reaches the 5 '-end of the template mRNA, 3 to 5 dC (oligoC, shown as 4C in the figure, the same applies hereinafter) is added to the 3' -end of one strand of cDNA, independently of the template mRNA, by utilizing the terminal transferase activity of the reverse transcriptase.
Step four: the 3 'terminal oligoC sequence of one strand of cDNA is annealed to a template having a 5' linker sequence and an oligoG sequence, and one strand of cDNA is extended using the template-converting activity of reverse transcriptase, using the template-converting activity of the reverse transcriptase as a template, to produce an extended cDNA strand having a 5 'terminal 3' linker sequence and a sequence complementary to the 3 'terminal 5' linker sequence.
Step five: since the amount of the extended cDNA single strand product produced in step four is very small, the extended cDNA single strand is subjected to full-length amplification, and the amount of the product is amplified so that there is enough extended cDNA single strand as a template for subsequent experiments. The 5 'full-length amplification primer is designed according to the 5' adaptor sequence, and the 3 'full-length amplification primer is designed according to the 3' adaptor sequence. Sufficient cDNA double-stranded product was obtained by PCR amplification. (this step is an optional step depending on the initial amount of sample and abundance of the gene to be amplified.)
Step six: taking a certain amount of the full-length amplification product of the step five as a template to carry out 3'RACE and 5' RACE separately or together, wherein: the primers used in 3'RACE are respectively a 3' RACE gene specific primer and a universal primer R, the 3'RACE gene specific primer is designed according to a known sequence in the middle of cDNA, and the universal primer R is designed according to a 3' joint sequence; the primers used in 5'RACE are respectively a 5' RACE gene specific primer and a universal primer F, wherein the 5'RACE gene specific primer is designed according to a known sequence in the middle of cDNA, and the universal primer F is designed according to a 5' joint sequence. RACE was performed using two pairs of primers to finally obtain DNA products with the 5 'and 3' terminal sequences of the mRNA template, respectively.
Products of the invention
1. Cell lysis unit: cell lysis agents and RNase inhibitors
Cell lysis agent, 1% Tween 20;
RNase inhibitor, Murine RNase inhibitor (cat # R301) from Nanjing Novozam Biotechnology Ltd;
2. a reverse transcription unit: reverse transcriptase, reverse transcription reaction buffer, reverse transcription primer, conversion template, dNTP Mix and nuclease-free water
The reverse transcriptase is 10 Xenzyme Mix in HiScript-TS 5'/3' RACE Kit (cat No. RA101) of Nanjing Novozam Biotechnology GmbH;
the reverse transcription reaction Buffer solution is 5 XFS Buffer V2 in a Discover-sc WTA Kit V2 (cat No. N711) of Nanjing Novowed Biotechnology GmbH;
reverse transcription primers, oligonucleotides with a 3' linker sequence, a polyT sequence and an anchor base VN, as shown in table 1 SEQ ID NO: 1 (the single underlined part is a 3 'linker sequence), wherein V is any one of dA, dG and dC, and N is any one of dA, dG, dT and dC (wherein, the 3' linker sequence may have other 18-25nt sequences, and the number of T may vary, for example, 20-40, as long as it does not specifically bind to the template);
template was converted to an oligonucleotide with a 5' linker sequence and oligoG, as shown in table 1 SEQ ID NO: 2 (the double-underlined part is a 5 'linker sequence), wherein the first G base at the 3' end is an LNA modified deoxyribonucleotide, and the second and third G bases are ribonucleotides (wherein, the 5 'linker sequence may have other 18-25nt sequences, and the number of G's may vary, for example, 4, 5 or more, as long as they do not specifically bind to the template);
dNTP Mix, is a mixture of dNTP Mix (P031) of Biotechnology GmbH of Nanjing Novowed, dATP/dTTP/dCTP/dGGP, each at a concentration of 10 mM;
nuclease-free water: RNase-free ddH in HiScript-TS 5'/3' RACE Kit of Nanjing Novozam Biotechnology corporation2O；
3. Transcriptome amplification unit: full-length amplification reagent, 5 'full-length amplification primer and 3' full-length amplification primer
The full-length amplification reagent (containing high-fidelity DNA polymerase) is 2 XDiscover-sc PCR Mix in a Discover-sc WTA Kit V2 Kit of Nanjing Novowed Biotechnology GmbH;
the 5 'full-length amplification primer is an upstream primer designed according to a 5' adaptor sequence, and is shown as SEQ ID NO: 3 (double underlined 5' linker sequence);
the 3 'full-length amplification primer is a downstream primer designed according to a 3' adaptor sequence, and is shown as SEQ ID NO: 4 (single underlined section is the 3' linker sequence);
4. RACE unit: RACE reagent, 5'RACE universal primer, 3' RACE universal primer, 5'RACE gene specific primer, 3' RACE gene specific primer and dilution buffer
RACE reagent is 2 multiplied Amplification Mix in a Discover-sc WTA Kit V2 Kit of Nanjing Novowed Toxico corporation;
the 5' RACE universal primer is a mixture of a universal long primer designed from a 5' adaptor sequence and a short primer designed from a 5' adaptor sequence, as shown in table 1 SEQ ID NO: 5 and 6 (double underlined 5' linker sequence);
3' RACE Universal primer is a mixture of a universal long primer designed from 3' adaptor sequence and a short primer designed from 3' adaptor sequence as shown in Table 1 SEQ ID NO: 7 and 8 (single underlined 3' linker sequence);
the 5'RACE gene specific primers and 3' RACE gene specific primers, for β -actin (NM — 001101.5) exemplified in the examples, were designed based on the gene sequence of β -actin, as shown in table 1 in SEQ ID NO: 9 and 10;
the Dilution Buffer was a Dilution Buffer in HiScript-TS 5'/3' RACE Kit, Inc., Biotech, Nanjing Novozam.
Detailed Description
The technical scheme of the invention is further explained by the specific embodiment in combination with the attached drawings. However, the following examples are merely illustrative of the present invention and do not represent or limit the scope of the present invention. The protection scope of the invention is subject to the claims.
In the following examples, reagents and consumables are purchased from suppliers commonly used in the art, and experimental methods and technical means are conventional in the art, unless otherwise specified.
Material
1. Cell lysis unit: cell lysis agents and RNase inhibitors
Cell lysis agent, 1% Tween 20;
RNase inhibitor, Murine RNase inhibitor (cat # R301) from Nanjing Novozam Biotechnology Ltd;
2. a reverse transcription unit: reverse transcriptase, reverse transcription reaction buffer, reverse transcription primer, conversion template, dNTP Mix and nuclease-free water
The reverse transcriptase is 10 Xenzyme Mix in HiScript-TS 5'/3' RACE Kit (cat No. RA101) of Nanjing Novozam Biotechnology GmbH;
the reverse transcription reaction Buffer solution is 5 XFS Buffer V2 in a Discover-sc WTA Kit V2 (cat No. N711) of Nanjing Novowed Biotechnology GmbH;
reverse transcription primers, oligonucleotides with a 3' linker sequence, a polyT sequence and an anchor base VN, as shown in table 1 SEQ ID NO: 1 (the single underlined part is a 3' linker sequence), wherein V is any one of dA, dG and dC, and N is any one of dA, dG, dT and dC;
template was converted to an oligonucleotide with a 5' linker sequence and oligoG, as shown in table 1 SEQ ID NO: 2 (double-underlined 5 'linker sequence), wherein the first G base at the 3' end is an LNA-modified deoxyribonucleotide, and the second and third G bases are ribonucleotides;
dNTP Mix, is a mixture of dNTP Mix (P031) of Biotechnology GmbH of Nanjing Novowed, dATP/dTTP/dCTP/dGGP, each at a concentration of 10 mM;
nuclease-free water: limited stock for Nanjing NuoWeizan BiotechnologyRNase-free ddH in the HiScript-TS 5'/3' RACE Kit2O；
3. Transcriptome amplification unit: full-length amplification reagent, 5 'full-length amplification primer and 3' full-length amplification primer
The full-length amplification reagent (containing high-fidelity DNA polymerase) is 2 XDiscover-sc PCR Mix in a Discover-sc WTA Kit V2 Kit of Nanjing Novowed Biotechnology GmbH;
the 5 'full-length amplification primer is an upstream primer designed according to a 5' adaptor sequence, and is shown as SEQ ID NO: 3 (double underlined 5' linker sequence);
the 3 'full-length amplification primer is a downstream primer designed according to a 3' adaptor sequence, and is shown as SEQ ID NO: 4 (single underlined section is the 3' linker sequence);
4. RACE unit: RACE reagent, 5'RACE universal primer, 3' RACE universal primer, 5'RACE gene specific primer, 3' RACE gene specific primer and dilution buffer
RACE reagent is 2 multiplied Amplification Mix in a Discover-sc WTA Kit V2 Kit of Nanjing Novowed Toxico corporation;
the 5' RACE universal primer is a mixture of a universal long primer designed from a 5' adaptor sequence and a short primer designed from a 5' adaptor sequence, as shown in table 1 SEQ ID NO: 5 and 6 (double underlined 5' linker sequence);
3' RACE Universal primer is a mixture of a universal long primer designed from 3' adaptor sequence and a short primer designed from 3' adaptor sequence as shown in Table 1 SEQ ID NO: 7 and 8 (single underlined 3' linker sequence);
the 5'RACE gene specific primers and 3' RACE gene specific primers, for β -actin (NM — 001101.5) exemplified in the examples, were designed based on the gene sequence of β -actin, as shown in table 1 in SEQ ID NO: 9 and 10;
the Dilution Buffer was a Dilution Buffer in HiScript-TS 5'/3' RACE Kit, Inc., Biotech, Nanjing Novozam.
TABLE 1
The single underlined portion is the 3' linker sequence; the double underlined section is the 5' linker sequence.
Example 1
In this example, 5'RACE and 3' RACE were performed simultaneously on a single HEK293 cell as a single cell sample or 10pg total RNA extracted from HEK293 cells as a trace RNA sample (single cells typically contain about 10pg total RNA) against housekeeping gene β -actin (NM — 001101.5) (FPKM ≈ 200) using reverse transcriptase having terminal transfer activity and template conversion activity, as follows.
1. Sample processing (cell lysis)
(1) The 9 × cell lysate was prepared according to table 2 below, gently mixed by pipette and collected by brief centrifugation, avoiding the appearance of air bubbles during mixing.
TABLE 2
(2) Single cell samples and micro RNA samples were prepared according to Table 3 below and immediately placed on ice.
TABLE 3
| Components | Positive control | Single cell sample | Negative control |
| 9X cell lysate | 1μL | 1μL | 1μL |
| Nuclease-free H2O | 7μL | 7μL | 8μL |
| 10pg Total RNA | 1μL | - | - |
| 1 cell | - | 1μL | - |
| Total volume | 9μL | 9μL | 9μL |
2. Reverse transcription reaction
(1) An RNA denaturation reaction system was prepared according to Table 4 below, gently mixed using a pipette, collected by brief centrifugation, and placed on ice.
TABLE 4
| Components | Volume of |
| Product of the last step | 9μL |
| Reverse transcription primer (10. mu.M) | 2μL |
| dNTP Mix(10 mM) | 2μL |
| Total volume | 13μL |
The following program was run in a PCR instrument: 72 ℃ for 3 min; immediately placed on ice for 2 min.
(2) The reverse transcription reaction system was prepared as in table 5 below, gently mixed using a pipette, collected by brief centrifugation, and placed on ice.
TABLE 5
| Components | Volume of | |
| Product of the | 13μL | |
| 5×FS Buffer V2 | 4μL | |
| 10×Enzyme Mix | 2μL | |
| Conversion template (10 mu M) | 1μL | |
| Total volume | 20μL |
(3) Reverse transcription reaction conditions: at 42 ℃ for 90 min; 15 min at 70 ℃; and keeping at 4 ℃.
3. Full Length cDNA amplification
(1) The reaction system was prepared in a clean bench according to table 6 below, gently mixed by a pipette, centrifuged briefly and collected, and then placed on ice.
TABLE 6
| Components | Volume of |
| Product of the last step | 20μL |
| ddH2O | 4μL |
| 2×Discover-sc PCR Mix | 25μL |
| 5' full Length amplification primers and R (10. mu.M each) | 1μL |
| Total volume | 50μL |
(2) The reaction procedure shown in table 7 below was run in a PCR instrument.
TABLE 7
4. Rapid Amplification of CDNA Ends (RACE)
(1) Template dilution: mu.l of the full-length cDNA amplification product was added to 10. mu.l of Dilution Buffer to dilute the product and obtain 5'/3' RACE-Ready cDNA.
(2) The 5' RACE reaction system was prepared according to Table 8 below, gently mixed using a pipette, collected by brief centrifugation, and placed on ice.
TABLE 8
| Components | Volume of |
| 5'/3'RACE-Ready cDNA | 2.5μL |
| 5'GSP(β-actin)(10μM) | 1μL |
| 5' Universal primer mixture (Long and short primers 10. mu.M each) | 5μL |
| 2×PCR Mix | 25μL |
| ddH2O | 16.5μL |
| Total volume | 50μL |
The 3' RACE reaction system was prepared according to Table 9 below, gently mixed using a pipette, collected by brief centrifugation, and placed on ice.
TABLE 9
| Components | Volume of |
| 5'/3'RACE-Ready cDNA | 2.5μL |
| 3'GSP(β-actin)(10μM) | 1μL |
| 3' Universal primer mixture (Long and short primers 10. mu.M each) | 5μL |
| 2×PCR Mix | 25μL |
| ddH2O | 16.5μL |
| Total volume | 50μL |
(3) The reaction procedure shown in table 10 below was run in a PCR instrument.
Watch 10
5. Electrophoretic detection
mu.L of 5'RACE product and 3' RACE product were subjected to 1.5% agarose electrophoresis. The results are shown in FIG. 2, which shows that both 5'RACE and 3' RACE amplified specific bands, and the sizes of the bands are in accordance with expectations, indicating that the scheme can realize 5'RACE and 3' RACE on single cells or 10pg total RNA micro-samples simultaneously.
Example 2
In this example, 5'RACE and 3' RACE were performed simultaneously for UNC5B (NM — 001244889.2) (FPKM ≈ 1) with low-abundance expression. See example 1 for a specific experimental procedure except that the thermal cycling of the PCR amplification step was increased from 18 to 22 cycles in the full-length cDNA amplification segment and the template dilution step was omitted in the RACE segment.
The results are shown in FIG. 3, which shows that both 5'RACE and 3' RACE amplified specific bands, and the sizes of the bands are in accordance with expectations, indicating that the scheme can realize 5'RACE and 3' RACE on single cells or 10pg total RNA micro-samples simultaneously.
Example 3
In this example, 5'RACE and 3' RACE were performed simultaneously against β -actin on 1 μ g of total RNA extracted from HEK293 cells. See example 1 for a detailed experimental procedure, except that the cell lysis step and the full-length cDNA amplification step are omitted. The results are shown in FIG. 4.
The foregoing is considered as illustrative only of the embodiments of the invention. However, the scope of the present invention is not limited thereto. It should be understood by those skilled in the art that any changes or substitutions that can be easily made by those skilled in the art within the technical scope of the present disclosure fall within the protective scope and the disclosure of the present invention.
Claims (12)
1. A method of generating cDNA from a trace RNA sample, comprising the steps of:
(1) taking mRNA in a sample as a template, taking a reverse transcription primer as a primer, carrying out reverse transcription by utilizing reverse transcription activity to obtain a cDNA chain,
wherein the reverse transcription primer contains or consists of polyT,
optionally, the reverse transcription primer further comprises an anchor base V or VN at the 3' end of the polyT, V representing A, C and G, N representing A, C, T and G;
(2) adding oligoC to the 3' end of one strand of cDNA using end transfer activity; and
(3) further extending cDNA chain to obtain full-length cDNA by using the conversion template as template and utilizing the template conversion activity,
wherein the conversion template comprises or consists of oligoG,
optionally, the first G of the 3' end of the oligoG is LNA modified dG and the second and third G are rG.
2. A method of generating and amplifying cDNA from a trace RNA sample, comprising the steps of:
(1) the step (1) according to claim 1, wherein the reverse transcription primer further comprises a 3 'linker sequence located at the 5' end of the polyT;
(2) a step (2) as set forth in claim 1;
(3) step (3) as claimed in claim 1, wherein the conversion template further comprises a 5 'linker sequence located at the 5' end of the oligoG; and
(4) using the full-length cDNA as a template, using a 5 'full-length amplification primer and a 3' full-length amplification primer as primers, amplifying the full-length cDNA by using the activity of DNA polymerase,
wherein the 5 'full length amplification primer comprises or consists of a 5' adaptor sequence; the 3 'full length amplification primer comprises or consists of a 3' adaptor sequence; optionally, the 5 'linker sequence is the same or different from the 3' linker sequence.
3. A method of performing 5'RACE and 3' RACE on a trace RNA sample comprising the steps of:
(1) step (1) as claimed in claim 2;
(2) a step (2) as set forth in claim 2;
(3) a step (3) as set forth in claim 2;
(4) optionally, step (4) as recited in claim 2; and
(5) amplifying the 5 'end sequence of the full-length cDNA by using DNA polymerase activity with the full-length cDNA as a template and the 5' RACE universal primer and the 5'RACE gene specific primer as primers, and/or amplifying the 3' end sequence of the full-length cDNA by using the DNA polymerase activity with the full-length cDNA as a template and the 3'RACE universal primer and the 3' RACE gene specific primer as primers,
wherein said 5'RACE universal primer comprises or consists of a 5' linker sequence and said 3'RACE universal primer comprises or consists of a 3' linker sequence,
optionally, the 5'RACE universal primer further comprises a 5' adaptor sequence located 5 'of the 5' adaptor sequence, the 3'RACE universal primer further comprises a 3' adaptor sequence located 5 'of the 3' adaptor sequence, optionally the 5 'adaptor sequence is the same as or different from the 3' adaptor sequence,
further optionally, the 5'RACE universal primer is used in combination with a 5' RACE universal short primer, the 5'RACE universal short primer comprising or consisting of a 5' adaptor sequence, the 3'RACE universal short primer is used in combination with a 3' RACE universal short primer, the 3'RACE universal short primer comprising or consisting of a 3' adaptor sequence.
4. A method according to any one of claims 1 to 3, wherein the reverse transcription activity, the terminal transfer activity and the template switching activity are all derived from a reverse transcriptase, such as MMLV, and/or the DNA polymerase activity is derived from a high fidelity DNA polymerase, such as Pfu.
5. The method of any one of claims 1-3, wherein steps (1) to (3) are performed together at 42 ℃ for 90 minutes, optionally step (1) comprises a prior denaturation at 72 ℃ for 3 minutes, and/or step (3) comprises a subsequent inactivation at 70 ℃ for 15 minutes.
6. The method of claim 2 or 3, wherein the step (4) is performed as follows: pre-denaturation at 98 ℃ for 1 min; 5-30 cycles, denaturation at 98 ℃ for 10 seconds, annealing at 65 ℃ for 15 seconds, and extension at 72 ℃ for 1-30 minutes; the extension was carried out at 72 ℃ for 5 minutes.
7. The method of claim 3, wherein the step (5) is performed by: pre-denaturation at 98 ℃ for 1 min; 5 cycles, denaturation at 98 ℃ for 10 seconds, extension at 72 ℃ for 3 minutes; 5 cycles, denaturation at 98 ℃ for 10 seconds, annealing at 70 ℃ for 15 seconds, and extension at 72 ℃ for 3 minutes; 20 or 25 cycles, denaturation at 98 ℃ for 10 seconds, annealing at 68 ℃ for 15 seconds, and extension at 72 ℃ for 3 minutes; the extension was carried out at 72 ℃ for 5 minutes.
8. The method of claim 3, wherein the step (5) is performed by: pre-denaturation at 98 ℃ for 1 min; 20 or 25 cycles, denaturation at 98 ℃ for 10 seconds, annealing at 68 ℃ for 15 seconds, and extension at 72 ℃ for 3 minutes; the extension was carried out at 72 ℃ for 5 minutes.
9. The method of any one of claims 1-8, wherein the microRNA sample contains at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000pg total RNA or mRNA and/or the microRNA sample is from at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 cells.
10. The method of claim 3, wherein said RACE is directed against a target gene having an FPKM (fragments Per Kibase of exon model Per Million mapped fragments) of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100.
11. A kit for carrying out the method of any one of claims 1-10.
12. The kit of claim 11, comprising:
(1) a reverse transcriptase having a terminal transfer activity, a template switching activity and a reverse transcription activity;
(2) high fidelity DNA polymerase;
(3) a reverse transcription primer consisting of a 3' linker sequence, polyT and VN from the 5' end to the 3' end;
(4) a switch template consisting of a 5' linker sequence and oligoG from 5' end to 3' end;
(5) a 5 'full length amplification primer consisting of a 5' adaptor sequence;
(6) a 3 'full length amplification primer consisting of a 3' adaptor sequence;
(7) a 5'RACE universal long primer consisting of a 5' adaptor sequence and optionally a 5'RACE universal short primer consisting of a 5' adaptor sequence from the 5 'end to the 3' end;
(8) a 3' RACE universal long primer consisting of a 3' adaptor sequence and a 3' adaptor sequence from 5' end to 3' end and optionally a 3' RACE universal short primer consisting of a 3' adaptor sequence; and/or
(9) Optionally a cell lysis agent, an rnase inhibitor, a reverse transcription reaction buffer, dntps, an amplification buffer, a RACE dilution buffer, and double distilled water that is sterilized and/or filtered.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110743091.8A CN113373140A (en) | 2021-07-01 | 2021-07-01 | Method and kit for generating and amplifying cDNA (complementary deoxyribonucleic acid) from single cell or trace RNA (ribonucleic acid) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110743091.8A CN113373140A (en) | 2021-07-01 | 2021-07-01 | Method and kit for generating and amplifying cDNA (complementary deoxyribonucleic acid) from single cell or trace RNA (ribonucleic acid) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113373140A true CN113373140A (en) | 2021-09-10 |
Family
ID=77580453
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110743091.8A Pending CN113373140A (en) | 2021-07-01 | 2021-07-01 | Method and kit for generating and amplifying cDNA (complementary deoxyribonucleic acid) from single cell or trace RNA (ribonucleic acid) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113373140A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024046097A1 (en) * | 2022-08-29 | 2024-03-07 | 广东菲鹏生物有限公司 | Method for measuring terminal transferase activity and kit |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107893100A (en) * | 2017-11-16 | 2018-04-10 | 序康医疗科技（苏州）有限公司 | A kind of unicellular mRNA reverse transcriptions and the method for amplification |
| CN108103055A (en) * | 2018-01-09 | 2018-06-01 | 上海亿康医学检验所有限公司 | A kind of method of unicellular RNA reverse transcriptions and library construction |
| US20200181606A1 (en) * | 2017-05-29 | 2020-06-11 | President And Fellows Of Harvard College | A Method of Amplifying Single Cell Transcriptome |
| US20200248175A1 (en) * | 2017-10-23 | 2020-08-06 | Massachusetts Institute Of Technology | Calling genetic variation from single-cell transcriptomes |
| CN111549025A (en) * | 2020-06-08 | 2020-08-18 | 郑州大学第一附属医院 | Strand displacement primer and cell transcriptome library construction method |
-
2021
- 2021-07-01 CN CN202110743091.8A patent/CN113373140A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200181606A1 (en) * | 2017-05-29 | 2020-06-11 | President And Fellows Of Harvard College | A Method of Amplifying Single Cell Transcriptome |
| US20200248175A1 (en) * | 2017-10-23 | 2020-08-06 | Massachusetts Institute Of Technology | Calling genetic variation from single-cell transcriptomes |
| CN107893100A (en) * | 2017-11-16 | 2018-04-10 | 序康医疗科技（苏州）有限公司 | A kind of unicellular mRNA reverse transcriptions and the method for amplification |
| CN108103055A (en) * | 2018-01-09 | 2018-06-01 | 上海亿康医学检验所有限公司 | A kind of method of unicellular RNA reverse transcriptions and library construction |
| CN111549025A (en) * | 2020-06-08 | 2020-08-18 | 郑州大学第一附属医院 | Strand displacement primer and cell transcriptome library construction method |
Non-Patent Citations (2)
| Title |
|---|
| VAZYME: "HiScript-TS 5’/3’ RACE kit", 《HISCRIPT-TS 5’/3’ RACE KIT》 * |
| 贺斌等: "cDNA末端快速扩增技术及其方法的改进", 《浙江农业科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024046097A1 (en) * | 2022-08-29 | 2024-03-07 | 广东菲鹏生物有限公司 | Method for measuring terminal transferase activity and kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110050067B (en) | Methods of producing amplified double-stranded deoxyribonucleic acid, and compositions and kits for use in the methods | |
| JP5118025B2 (en) | Methods and kits for sense RNA synthesis | |
| EP2794926B1 (en) | Methods of constructing small rna libraries and their use for expression profiling of target rnas | |
| US20090325183A1 (en) | Sequencing methods | |
| Che et al. | Amplification of RNA transcripts using terminal continuation | |
| EP3394292A1 (en) | Methods of library construction for polynucleotide sequencing | |
| EP2272976A1 (en) | Method for differentiation of polynucleotide strands | |
| JP2009505663A (en) | Methods and kits for sense RNA synthesis | |
| US20200123606A1 (en) | Rt-qpcr method for direct quantitative detection of circulating mirna | |
| US8153401B2 (en) | Method for direct amplification from crude nucleic acid samples | |
| CN113373140A (en) | Method and kit for generating and amplifying cDNA (complementary deoxyribonucleic acid) from single cell or trace RNA (ribonucleic acid) | |
| CN113322523B (en) | RNA rapid library construction method and application thereof | |
| US20080233587A1 (en) | Method for direct amplification from crude nucleic acid samples | |
| US20110159548A1 (en) | Method for amplifying monomorphic-tailed nucleic acids | |
| JP2024515076A (en) | Amplification of single-stranded DNA | |
| JP5997407B1 (en) | Multi-item amplification method | |
| JP2022508834A (en) | Nucleic acid barcode | |
| EP3851542A1 (en) | Depletion of abundant uninformative sequences | |
| CN113862394B (en) | RPA detection method for tomato infertility virus | |
| CN110468179A (en) | The method of selective amplification nucleic acid sequence | |
| CN114250276B (en) | MicroRNA detection system and method based on exponential amplification reaction and Argonaute nuclease | |
| WO2021226917A1 (en) | Novel method of one-step whole transcriptome amplification | |
| CA2458297A1 (en) | Reproduction of ribonucleic acids | |
| Byrne et al. | RNA editing in Physarum mitochondria: assays and biochemical approaches | |
| Arneson et al. | Whole-genome amplification by Single-cell COMParative genomic hybridization PCR (SCOMP) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210910 |
|
| RJ01 | Rejection of invention patent application after publication |