WO2024046097A1 - Procédé de mesure de l'activité transférase terminale et kit - Google Patents

Procédé de mesure de l'activité transférase terminale et kit Download PDF

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WO2024046097A1
WO2024046097A1 PCT/CN2023/112693 CN2023112693W WO2024046097A1 WO 2024046097 A1 WO2024046097 A1 WO 2024046097A1 CN 2023112693 W CN2023112693 W CN 2023112693W WO 2024046097 A1 WO2024046097 A1 WO 2024046097A1
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primer
template
reverse
rna
cdna
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Chinese (zh)
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肖泽涛
钟淑瑶
章瑞程
王慧
张超
杨容青
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广东菲鹏生物有限公司
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    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • 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
    • C12Q1/6851—Quantitative amplification
    • 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]

Definitions

  • the present disclosure relates to the technical field of molecular biology, and relates to a method and a kit for detecting terminal transferase activity.
  • RNA reverse transcription reaction refers to that during the entire reverse transcription process, reverse transcriptase first uses RNA as a template to extend, and then uses its terminal deoxyribonucleotidyl transferase (Tdt) activity to switch the extension of RNA as a template. to DNA-templated extension.
  • Tdt terminal deoxyribonucleotidyl transferase
  • This process mainly utilizes the three major activities of reverse transcriptase: (1) DNA polymerization activity using RNA as template; (2) terminal deoxyribonucleotidyl transferase (Tdt) activity; (3) DNA using DNA as template polymerization activity.
  • Tdt activity is the most critical factor affecting the template switching effect.
  • Tdt activity refers to the ability to add several nucleotides to the 3′ end of a synthetic product without the need for a template.
  • reverse transcriptase can exhibit Tdt activity.
  • M-MLV reverse transcriptase will preferentially add cytosine to the 3' end of the amplicon.
  • reverse transcriptase first uses mRNA as a template, and after extending its 5' end, it uses the Tdt activity of reverse transcriptase to add several peptides to the 3' end of the extension product.
  • Cytidylic acid these few cytidylic acid can be annealed to a template-switching oligo (TSO) with a corresponding number of guanylic acid at the 3' end, so that the extension product can be a TSO molecule.
  • TSO template-switching oligo
  • this one-strand cDNA will have a TSO-binding region at the 3' end and a GSP primer-binding region at the 5' end due to the template switching effect.
  • primers can be designed for rapid amplification of cDNA using these two regions;
  • the TSO region can introduce unique molecular or cell tags (UMI, Cell barcode), which allows every cell or even every mRNA to be With a unique label, it is convenient to correct and trace the source of each read after sequencing. This method allows each cell to carry a unique identity tag, which facilitates the tracing of the sequencing information of each cell. Therefore, RACE based on the template switching effect helps reduce the difficulty of analysis based on high-throughput sequencing. .
  • the key to realizing RACE and applying it to ScRNA-seq is the template switching effect in the reverse transcription reaction.
  • the main factor affecting the template switching effect is the Tdt activity of reverse transcriptase.
  • the higher the Tdt activity the higher the transcription efficiency of the reverse transcription template. Therefore, the higher the concentration of the one-strand cDNA that successfully converted the template (successfully carrying TSO), which means The more efficiently cellular mRNA is captured, the greater the abundance and accuracy obtained after sequencing.
  • the Tdt activity is low and the amount of cellular mRNA captured is low, then the final sequencing result will have a low degree of recovery of the sequence information of the cell sample. Therefore, accurate and simple measurement of the template conversion efficiency of the reverse transcription reaction is a key point to ensure the success of ScRNA-seq and even to apply this sequencing technology to industrial production.
  • the mass spectrometry-coupled capillary electrophoresis method uses the reverse transcriptase to be tested and primers with fluorescent markers (FAM) to perform RACE of RNA of a specific length, and then uses mass spectrometry and capillary electrophoresis to analyze the obtained cDNA products. Finally, the template conversion efficiency was characterized based on the peak area of the cDNA that was successfully converted on the plate.
  • the qPCR method based on fluorescent dyes first uses an ordinary PCR instrument to perform reverse transcription and template conversion, and then uses the cDNA product as a template to perform a qPCR reaction based on the fluorescent dye method. Finally, the Ct value is used to characterize the successful template conversion of cDNA.
  • the sequencing method uses first-generation sequencing or second-generation sequencing to obtain the template conversion efficiency of reverse transcription by performing sequence analysis and statistics on cDNA samples.
  • these technologies have more or less defects and shortcomings, which are likely to hinder the application of this detection method in industrial production.
  • the purpose of this disclosure is to provide a method and kit for detecting terminal transferase activity to truly and accurately detect the terminal transferase activity of reverse transcriptase, more truly and accurately reflect the template conversion efficiency of the reverse transcription reaction, and further provide a method for cellular mRNA to be Capture efficiency provides scientifically accurate evaluation.
  • the proposal of this disclosure will help the success of ScRNA-seq and the application of this sequencing technology to industrial production.
  • the method provided by the present disclosure is suitable for debugging the reverse transcription system, has simple operation steps, low cost, and short detection cycle, and can meet the needs of large-scale detection.
  • the present disclosure provides a method for detecting terminal transferase activity, which includes the following steps:
  • (d) Set a reverse primer in the reverse transcription primer region, set a forward inner primer in the RNA template, set a forward outer primer in the TSO, use the product of the above step (c) as a template, and obtain the forward inner primer through qPCR reaction With the Ct1 value of the amplification product of the reverse primer, obtain the Ct2 value of the amplification product of the forward outer primer and the reverse primer through qPCR reaction, and calculate the difference ⁇ Ct between the Ct1 value and the Ct2 value to obtain the activity of terminal transferase; qPCR reaction Use the same probe.
  • the inventor discovered that reverse transcription primers (RT primer) and reverse transcriptase are used to perform reverse transcription based on the template switching effect.
  • the reverse transcription product can be directly used in qPCR reactions after dilution.
  • the qPCR reaction includes two reaction systems, the inner reaction system (Inner primer system) composed of forward inner primer and reverse primer, the inner reaction system (Outer primer system) composed of forward outer primer and reverse primer, and the forward inner primer It anneals and amplifies only the RNA template region of cDNA, and the forward outer primer anneals and amplifies only the TSO region of cDNA.
  • the obtained Inner system Ct value can characterize the amount of total cDNA in the reverse transcription product, while the Outer system Ct value is used to characterize the amount of cDNA that has been successfully converted from the template. Therefore, the difference ( ⁇ Ct) between the Ct value of the Outer system and the Ct value of the Inner system can accurately reflect the template conversion efficiency.
  • the smaller the difference ( ⁇ Ct) the higher the amount of cDNA that has been successfully converted to the template, that is, the higher the concentration of one-strand cDNA that has been successfully converted to the template (successfully carrying TSO), indicating that high Tdt activity indicates the template transcription efficiency of reverse transcription. high.
  • the difference ( ⁇ Ct) between the Ct value of the Outer system and the Ct value of the Inner system can accurately reflect the template conversion efficiency.
  • the position of the external primer has a certain impact on the Ct value. Theoretically, if ⁇ Ct is used to characterize the conversion efficiency of the template, the amplification efficiency of the outer primer and the inner primer should be as close as possible. Therefore, the closer the distance between the outer primer and the inner primer to each other, the better.
  • the probes involved in this disclosure should be close to the reverse primer, but the distance of the inner and outer primers from the probe does not have much impact.
  • the background technology mentioned above needs to degrade the RNA template and purify the cDNA product before measuring the template conversion efficiency. Otherwise, it will affect the accuracy of measuring the template conversion efficiency.
  • the RNA template used in the existing technology is only 20-30nt. , so the cDNA obtained by reverse transcription is shorter, which will increase the difficulty of cDNA purification.
  • the steps of degrading RNA templates and purifying cDNA are relatively cumbersome, which limits the application of the background technology to large-scale industrial detection.
  • the detection method provided by the present disclosure does not require the degradation of the RNA template and the purification of the cDNA product after the reverse transcription reaction, which greatly simplifies the entire operation process. It is more suitable for debugging the reverse transcription system of template switching effect and large-scale industrial detection.
  • the detection method provided by the present disclosure has the advantages of low cost and short detection cycle. Therefore, the present disclosure is suitable for debugging the reverse transcription system of template switching effect and large-scale industrial detection.
  • Both amplifications use the same reverse primer and use a Taqman probe to avoid differences in amplification efficiency due to different probes, reverse primer positions or sequences, which is conducive to more accurate Characterize template conversion efficiency.
  • the forward inner (or outer) primer and the reverse primer can also be appropriately diluted as needed to perform subsequent qPCR reactions.
  • the reverse transcription primer in step (a) is a stem-loop reverse transcription primer or a Poly T reverse transcription primer.
  • Poly T reverse transcription primers can be selected from universal Poly T reverse transcription primers. As shown in SEQ ID NO:8:
  • RNA-cDNA intermediate with sticky ends in step (b) adds multiple identical deoxynuclei to the 3′ end of the cDNA chain of the RNA-cDNA intermediate through the terminal transferase activity of the reverse transcriptase itself. Obtained by glucoside method.
  • reverse transcriptase In steps (a)-(c), reverse transcriptase, dNTPs, RNase inhibitors, template-switching oligonucleotides (TSO), and buffers are used to perform RNA template-dependent DNA extension.
  • the reverse transcription product can be directly used in qPCR reactions after dilution.
  • the reverse transcriptase buffer contains MnCl 2 , and the final concentration of MnCl 2 is 4-16mM; the inventor found that in the reverse transcription system, after the concentration of MnCl 2 is diluted to the above concentration, a better template is obtained conversion efficiency. For example, 8-16mM, 4-8mM, 4-6mM.
  • the final concentration of MnCl2 is 4-8mM.
  • the plurality of deoxynucleotides added at the end in step (b) are a plurality of cytosine deoxynucleotides (C).
  • three cytosine deoxynucleotides (C) are added to the end in step (b).
  • the 3' annealing region on the TSO contains three ribonucleotide residues.
  • the 3' annealing region contains three nuclear guanine (rG) ribonucleotides.
  • the 5' end region of the TSO further includes one or more of the following: barcode sequence, unique molecular identifier (UMI), amplification primer sequence, sequencing primer sequence, capture primer sequence, sequence-specific nucleic acid Enzyme cleavage sites, modified nucleotides, biotinylated nucleotides, and 5' modifications, etc.
  • barcode sequence unique molecular identifier
  • UMI unique molecular identifier
  • amplification primer sequence sequencing primer sequence
  • capture primer sequence sequence-specific nucleic acid Enzyme cleavage sites
  • modified nucleotides biotinylated nucleotides
  • 5' modifications etc.
  • the cDNA product obtained in the above step (c) needs to be diluted 10-100 times for use in the qPCR reaction of step (d).
  • Taqman-qPCR technology can be used to obtain accurate results of template conversion efficiency. This avoids the tedious steps of degrading the RNA template and purifying the cDNA product.
  • the dilution factor of the cDNA product obtained in step (c) is 50 times.
  • the inventor Since there are many factors that affect template conversion efficiency, such as the type of reverse transcriptase, buffer components, input amounts of templates and primers, etc., the inventor also screened the concentrations of reverse transcription primers and TSO in the reverse transcription system, as follows :
  • the concentration of TSO is 2-100 ⁇ M
  • the concentration of the reverse transcription primer is 2-100 ⁇ M
  • the concentration of TSO is 20-100 ⁇ M
  • the concentration of the stem-loop primer is 20-100 ⁇ M.
  • the concentration of TSO is 20-100 ⁇ M
  • the concentration of the stem-loop primer is 20-100 ⁇ M.
  • TSO concentration is 20-50 ⁇ M
  • stem loop primer concentration is 20-50 ⁇ M.
  • the final concentration of the forward outer primer is 0.3-0.5 ⁇ M
  • the final concentration of the forward inner primer is 0.3-0.5 ⁇ M
  • the final concentration of the reverse primer is 0.3 -0.5 ⁇ M
  • the final concentration of the probe is 0.1-0.3 ⁇ M.
  • the final concentration of the forward outer primer is 0.3 ⁇ M
  • the final concentration of the forward inner primer is 0.3 ⁇ M
  • the final concentration of the reverse primer is 0.3 ⁇ M
  • the final concentration of the probe is 0.1 ⁇ M.
  • the RNA template is selected from: mRNA, non-coding RNA, miRNA, siRNA, piRNA, lncRNA or ribosomal RNA.
  • the RNA template is selected from: miRNA.
  • the reverse transcriptase is M-MLV reverse transcriptase, HIV-1 reverse transcriptase, AMV reverse transcriptase or telomerase reverse transcriptase with reduced or eliminated RNase activity.
  • Different reverse transcriptases have a certain impact on template conversion efficiency. When testing, the same reverse transcriptase should be selected as much as possible to reduce the impact of different reverse transcriptases on the detection of terminal transferase activity.
  • the method for judging the activity of terminal transferase is as follows:
  • the annealing step in step (a) adopts gradient annealing. Gradient annealing helps improve binding efficiency.
  • the initial annealing temperature of gradient annealing in step (a) is 65°C, and decreases by 0.1°C per second until 4°C, and the annealing time is 15-20 minutes;
  • the annealing time is 16 minutes.
  • reaction temperature conditions of steps (b) and (c) are determined based on the optimal reaction temperature of reverse transcriptase, and the reaction time is 90-180 minutes;
  • reaction time of step (c) is 120 min.
  • the qPCR reaction in step (d) also includes a probe
  • the probe is a Taqman probe. Since Taqman-qPCR technology has higher reaction sensitivity than other technologies, the inventors optimized the amount of primers and the dilution factor of the reverse transcription product (for example, 10-100 times) before using Taqman-qPCR technology. Accurate results of template conversion efficiency can be obtained. This avoids the tedious steps of degrading the RNA template and purifying the cDNA product.
  • the present disclosure also provides a kit, which includes: qPCR polymerase, reverse primer, forward inner primer, forward outer primer, probe, reverse transcription primer, RNA template, TSO, the reverse primer, forward inner primer, forward outer primer,
  • the nucleotide sequences of the RNA template, TSO, and probe are shown in SEQ ID NO: 1-3 and SEQ ID NO: 5-7, and the nucleotide sequence of the reverse transcription primer is shown in SEQ ID NO: 4 or SEQ ID NO:8, see Table 1.
  • the above-mentioned kit also includes materials such as reverse transcription reaction buffer, dNTPs, qPCR buffer, and water.
  • the detection method provided by this disclosure divide the reverse transcription product into two parts, one part is used to obtain the Ct1 value of the forward inner primer and reverse primer amplification product through qPCR, and the other part is used to obtain the forward outer primer and reverse primer amplification product through qPCR
  • the Ct2 value where the Ct1 value can characterize the amount of total cDNA in the reverse transcription product, and the Ct2 value is used to characterize the amount of cDNA that has been successfully converted into a template. Therefore, the difference ⁇ Ct between the Ct1 value and the Ct2 value can accurately reflect the template conversion efficiency.
  • Both amplifications use the same reverse primer and a Taqman probe to avoid differences in reverse transcription efficiency due to different probes, reverse primer positions or sequences, which is conducive to more accurate Characterize template conversion efficiency.
  • the detection method provided by the present disclosure does not require degradation of the RNA template and purification of the cDNA product after the reverse transcription reaction, which greatly simplifies the entire operation process. It is more suitable for debugging the reverse transcription system of template switching effect and large-scale industrial detection.
  • the detection method provided by the present disclosure has the advantages of low cost and short detection cycle. Therefore, the present disclosure is suitable for debugging the reverse transcription system of template switching effect and large-scale industrial detection.
  • Figure 1 is a detection principle diagram of steps (a)-(c) of the detection method provided in Embodiment 1 of the present disclosure
  • Figure 2 is a detection principle diagram of step (d) of the detection method provided in Embodiment 1 of the present disclosure
  • Figure 3 shows the ⁇ Ct values of different reverse transcriptases at different primer concentrations in Experimental Example 1;
  • Figure 4 shows the ⁇ Ct values of different reverse transcriptases at higher primer concentrations in Experimental Example 1;
  • Figure 5 shows the ⁇ Ct values of different reverse transcriptases at different MnCl 2 concentrations in Experimental Example 2;
  • Figure 6 is a schematic diagram of the detection of linear primers.
  • This embodiment provides a method for detecting terminal transferase activity of reverse transcriptase, which specifically includes the following steps.
  • the Poly T reverse transcription primer shown in Figure 6 can also be used to perform gradient annealing. The principle is shown in Figure 6.
  • cytosine deoxynucleotide (C) will complementary pair with the guanine nucleotide (rG) on the TSO, thereby realizing the switching of the template from RNA to DNA (template conversion), and then using reverse transcriptase
  • the DNA-dependent DNA polymerase activity continues to extend using TSO as the DNA template, and finally amplifies a cDNA with Stem-loop primer, miRNA and TSO region, which is a cDNA with successful template conversion.
  • the cDNA generated by the reverse transcription reaction based on template switching includes both cDNA with successful template switching and cDNA with failed template switching.
  • the qPCR reaction includes two reaction systems, the inner reaction system composed of forward inner primer (Inner PCR-primer) and reverse primer (Inner primer system), and the inner reaction system composed of forward outer primer (Outer PCR-primer) and reverse primer.
  • the forward inner primer only anneals and amplifies the RNA template region of cDNA
  • the forward outer primer only anneals and amplifies the TSO region of cDNA.
  • the obtained Inner system Ct value can characterize the amount of total cDNA in the reverse transcription product, while the Outer system Ct value is used to characterize the amount of cDNA that has been successfully converted from the template. Therefore, the difference ( ⁇ Ct) between the Ct value of the Outer system and the Ct value of the Inner system can accurately reflect the template conversion efficiency, that is, the terminal transferase activity of reverse transcriptase.
  • cDNA products with successful template conversion can be annealed and amplified with the forward outer primer and forward inner primer, while cDNA products with failed template conversion can only be annealed and amplified with the forward inner primer.
  • Both amplifications used the same reverse primer and the same Taqman probe.
  • the 5′ ⁇ 3′ exonuclease activity of Taq enzyme can be used to cleave the probe, generating a fluorescent signal that is captured by the instrument, thereby generating the corresponding Ct value.
  • the detection method provided by the present disclosure does not require degradation of the RNA template and purification of the cDNA product after the reverse transcription reaction, greatly simplifying the entire operation process, and also has the advantages of low cost and short detection cycle. Therefore, the present disclosure is suitable for debugging the reverse transcription system of template switching effect and large-scale industrial detection.
  • This experimental example explores the effect of different concentrations of stem-loop reverse transcription primers and TSO on template conversion efficiency in a reverse transcription reaction system.
  • the concentrations of the stem-loop reverse transcription primer and TSO to: 26nM, 0.1 ⁇ M, 0.2 ⁇ M, 2 ⁇ M, 10 ⁇ M and 20 ⁇ M respectively.
  • the reverse transcriptases used are all MMLV enzymes, and their names are MM02 reverse transcriptase (Feipeng Biotech, product number MD311) and SDmmlv reverse transcriptase (Feipeng Biotechnology, product number MDAR013). Unless otherwise specified, the concentrations of the miRNA template and other primers are based on the standards in Example 1.
  • the results show that when the concentration of the stem-loop reverse transcription primer is 20 ⁇ M and the concentration of TSO is 20 ⁇ M, the ⁇ Ct value is the smallest, which means that the amount of cDNA with successful template conversion is higher, that is, the amount of cDNA with successful template conversion is higher.
  • the Tdt activity of MM02 reverse transcriptase is higher than SDmmlv.
  • the reverse transcriptases are MM02 reverse transcriptase and SDmmlv reverse transcriptase.
  • the results show that when the concentration of the stem-loop reverse transcription primer is 100 ⁇ M and the TSO primer concentration is 100 ⁇ M, the ⁇ Ct value is the smallest, and the template conversion efficiency of the reverse transcription reaction is the highest at this concentration.
  • the concentration of the stem-loop reverse transcription primer and TSO was increased from 20 ⁇ M to 100 ⁇ M, the ⁇ Ct did not decrease significantly, that is, the template conversion efficiency did not increase significantly. Therefore, from a cost perspective, the optimal concentration of stem-loop reverse transcription primer and TSO is 20 ⁇ M.
  • the results showed that the Tdt activity of MM02 reverse transcriptase was higher than that of SDmmlv.
  • This experimental example explores the effect of different MnCl 2 concentrations on template conversion efficiency in the reverse transcription reaction system.
  • MnCl 2 concentrations of 0mM, 4mM, 8mM, 16mM and 23.25mM were respectively used to perform reverse transcription reactions according to the detection method of Example 1 to explore the impact of MnCl 2 on template conversion efficiency, and different reverse transcriptases were used to participate in the reaction.
  • the reverse transcriptases are MM02 reverse transcriptase and SDmmlv reverse transcriptase. Unless otherwise specified, the concentrations of the miRNA template and related primers are based on the standards in Example 1.
  • the results show that when the MnCl concentration is 8mM and the reverse transcriptase is MM02, the ⁇ Ct value is the smallest, which means that the amount of cDNA with successful template conversion is higher, that is, one strand of cDNA with successful template conversion (successful The higher the concentration of TSO), which indicates the high Tdt activity of reverse transcriptase, the higher the transcription efficiency of the reverse transcription template. And when the MnCl 2 concentration is 8mM, the Tdt activity of MM02 reverse transcriptase is higher than that of SDmmlv reverse transcriptase.
  • the present disclosure provides a method and kit for detecting terminal transferase activity.
  • This detection method is suitable for debugging the reverse transcription system. It has simple operation steps, low cost and short detection cycle. It can meet the needs of large-scale detection and has broad application prospects and high market value.

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Abstract

La présente invention concerne un procédé de mesure de l'activité transférase terminale et un kit, et se rapporte au domaine technique de la biologie moléculaire. Selon le procédé de mesure de la présente invention, l'activité transférase terminale de la transcriptase inverse peut être mesurée de façon réelle et précise, de manière à ce que l'efficacité de conversion de matrice d'une réaction de transcription inverse puisse être reflétée de façon plus réelle et précise, fournissant ainsi une évaluation scientifiquement précise de l'efficacité de la capture de l'ARNm des cellules. Le procédé selon l'invention est approprié pour ajuster et tester des systèmes de transcription inverse. Le procédé est simple et peu coûteux, et la période de mesure est courte ; de plus, le procédé peut satisfaire aux exigences de mesure à grande échelle.
PCT/CN2023/112693 2022-08-29 2023-08-11 Procédé de mesure de l'activité transférase terminale et kit WO2024046097A1 (fr)

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