Method for quantitatively detecting length of mRNA 3 'UTR

Abstract

The invention relates to the technical field of mRNA detection, in particular to a method for quantitatively detecting the length of mRNA 3 'UTR. The invention provides a CDS probe combined with a CDS region, a 3 'UTR probe combined with a 3' UTR region, a first fluorescent probe for identifying the CDS probe and a second fluorescent probe for identifying the 3 'UTR region, fluorescent groups carried by the first fluorescent probe and the second fluorescent probe are different, and standard DNA or mRNA containing 3' UTR sequences with different lengths are coupled to magnetic beads, so that the detection sensitivity of the CDS probe and the 3 'UTR probe is improved, and the detection sensitivity of the CDS probe and the 3' UTR probe is improved. Obtaining a magnetic bead standard product containing 3 'UTR regions with different lengths, and making a standard curve according to the magnetic bead standard product to obtain a standard equation; and determining the length of the 3 'UTR in the to-be-detected sample by using the fluorescence value of the to-be-detected sample and the standard equation so as to realize quantitative detection.

Description

Technical Field

[0001] The present application relates to the technical field of mRNA detection, and in particular to a method for quantitatively detecting the length of mRNA 3'UTR. Background Art

[0002] mRNA, also known as messenger RNA, is an RNA that transmits genetic information in cells. The 3' untranslated region of mRNA (abbreviated as 3'UTR) contains many binding sites for trans-acting elements such as miRNA (microRNA) and RNA-binding proteins, thereby regulating the stability, localization and translation efficiency of mRNA. The length of mRNA's 3'UTR depends on the use of the polyadenylation cleavage site (abbreviated as PAS) during the maturation of the precursor mRNA. More than 70% of the genes in the mammalian genome contain multiple PASs, which can cause these genes to have multiple mutants with different lengths of 3'UTR in the transcribed mRNA. The coding region sequences of these isomers are the same.

[0003] Therefore, detecting the length of the 3'UTR of mRNA is of great significance for analyzing trans-acting elements such as gene miRNA binding sites and RNA binding proteins. At present, traditional methods for detecting the length of 3'UTR include Northern blot hybridization, PCR and high-throughput sequencing. These methods can qualitatively analyze the length changes of the 3'UTR of mRNA, but cannot quantitatively analyze its length. In addition, these traditional methods require the sample or cell to be lysed before detection, resulting in the loss of the localization information of mRNA. Summary of the invention

[0004] The present application provides a CDS probe that binds to the CDS region and a 3'UTR probe that binds to the 3'UTR region, respectively. The CDS probe and the 3'UTR probe each carry a fluorescent group, and are hybridized with magnetic beads coupled to standard DNA or mRNA by single fluorescence in situ hybridization, and the fluorescent signal is collected. The length of the 3'UTR of the mRNA is quantitatively detected based on the fluorescent signal.

[0005] To this end, the embodiments of the present application disclose at least the following technical solutions:

[0006] The embodiment provides a method for quantitatively detecting the length of mRNA 3'UTR. The method includes: preparing a plurality of magnetic beads coupled with single-stranded nucleic acids, the single-stranded nucleic acids comprising a target gene CDS sequence, or comprising a target gene CDS sequence and 3'UTR sequences of different lengths; preparing a CDS probe binding to a CDS region and a 3'UTR probe binding to a 3'UTR region, the CDS probe having a first fluorescent group, the 3'UTR probe having a second fluorescent group, and the first fluorescent gene being different from the second fluorescent gene; performing a first mixed single-stranded hybridization reaction on each magnetic bead coupled with a single-stranded nucleic acid with the CDS probe and the 3'UTR probe at the same time; reading a first fluorescent value of the first mixed single-stranded hybridization reaction; constructing a standard curve according to the length of the 3'UTR sequence in the single-stranded nucleic acid and the first fluorescent value; preparing magnetic beads coupled with an mRNA to be tested; performing a second mixed single-stranded hybridization reaction on the magnetic beads coupled with the mRNA to be tested with the CDS probe and the 3'UTR probe at the same time; reading a second fluorescent value of the second mixed single-stranded hybridization reaction; determining the 3'UTR length of the mRNA to be tested according to the fluorescence curve and the second fluorescent value.

[0007] The method provided in the embodiment can hybridize with target DNA of different 3'UTR sequence lengths by combining CDS probes in the CDS region and 3'UTR probes in the 3'UTR region, and establish a connection between the fluorescent signal generated during the hybridization and the 3'UTR sequence length. Based on this connection, the fluorescent signal generated by hybridization with the 3'UTR sequence of the unknown mRNA can be calculated to obtain the 3'UTR sequence length. The method can accurately detect the length of the mRNA 3'UTR sequence, has high detection repeatability, and is widely practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 This is a schematic diagram of the method for quantitatively detecting the length of mRNA 3'UTR provided in the examples.

[0009] Figure 2 A schematic diagram of the process of quantitatively detecting the length of mRNA 3'UTR provided in the embodiment.

[0010] Figure 3 This is a schematic diagram of the method flow of steps S701 to S706 provided in an embodiment.

[0011] Figure 4 This is a schematic diagram of the method flow of step S701 provided in an embodiment.

[0012] Figure 5 This is a schematic diagram of the method flow of step S100 provided in an embodiment.

[0013] Figure 6 The standard curve and standard equation for detecting the length of HeLa cell mRNA by the method provided in the embodiment. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0015] like Figure 1 As shown, the present application provides a CDS probe and a 3'UTR probe that can perform base pairing and hybridization with the CDS region of the target gene and all or part of the sequence of the downstream 3'UTR region thereof, as well as a first fluorescent probe that identifies the CDS probe and a second fluorescent probe that identifies the 3'UTR probe, wherein the first fluorescent probe carries a first fluorescent group, and the second fluorescent probe carries a second fluorescent group that is different from the first fluorescent group. The present application first performs base pairing and hybridization with the CDS region and the 3'UTR region of the target sequence respectively through the CDS probe and the 3'UTR probe, captures the target sequence from the sample to be tested, then performs base pairing and hybridization with the CDS probe through the first fluorescent probe, and generates fluorescent information at the same time, performs base pairing and hybridization with the 3'UTR probe through the second fluorescent probe, and produces different fluorescent information at the same time, and then determines the length of the 3'UTR of the target sequence by collecting these fluorescent information, and realizes quantitative detection.

[0016] like Figure 2 As shown, the embodiment provides a method for quantitatively detecting the length of mRNA 3'UTR. The method comprises:

[0017] S100: preparing magnetic beads coupled with a single-stranded nucleic acid to be tested, wherein the single-stranded nucleic acid to be tested comprises a CDS sequence and a 3'UTR sequence of a target gene;

[0018] S200: providing a CDS probe that binds to the CDS region and a 3'UTR probe that binds to the 3'UTR region, as well as a first fluorescent probe that recognizes the CDS probe and a second fluorescent probe that recognizes the 3'UTR probe, wherein the first fluorescent probe carries a first fluorescent group, and the second fluorescent probe carries a second fluorescent group that is different from the first fluorescent group;

[0019] S300: performing a first mixed single-strand hybridization reaction on the magnetic beads coupled with the single-stranded nucleic acid to be detected, the CDS probe and the 3'UTR probe at the same time;

[0020] S400: performing a second mixed single-strand hybridization reaction on the product of the first mixed single-strand hybridization reaction with the first fluorescent probe and the second fluorescent probe at the same time;

[0021] S500: reading first fluorescence information generated by the reaction of a product of the first mixed single-stranded hybridization reaction with a first fluorescent probe according to the first fluorescent group, and reading second fluorescence information generated by the reaction of a product of the first mixed single-stranded hybridization reaction with a second fluorescent probe according to the second fluorescent group;

[0022] S600: Determine a fluorescence value to be detected corresponding to the single-stranded nucleic acid to be detected according to the first fluorescence information and the second fluorescence information;

[0023] S700: Determine the length of the 3'UTR sequence according to the standard equation and the fluorescence value to be measured.

[0024] like Figure 3 As shown, in the method provided in some embodiments, before step S700, the following steps are further included:

[0025] S701: preparing a plurality of magnetic beads coupled with a standard single-stranded nucleic acid, wherein the standard single-stranded nucleic acid comprises a target gene CDS sequence, or comprises a target gene CDS sequence and 3'UTR sequences of different lengths;

[0026] S702: performing a first mixed single-strand hybridization reaction on each magnetic bead coupled with a single-stranded nucleic acid and the CDS probe and the 3'UTR probe at the same time;

[0027] S703: performing a second mixed single-stranded hybridization reaction on the product of the first mixed single-stranded hybridization reaction and the first fluorescent probe and the second fluorescent probe;

[0028] S704: reading first fluorescence information generated by the reaction of the product of the first mixed single-stranded hybridization reaction with the first fluorescent probe according to the first fluorescent group, and reading second fluorescence information generated by the reaction of the product of the first mixed single-stranded hybridization reaction with the second fluorescent probe according to the second fluorescent group;

[0029] S705: determining a plurality of standard fluorescence values ​​corresponding to the single-stranded nucleic acid without a 3'UTR sequence and the single-stranded nucleic acid with 3'UTR sequences of different lengths, respectively, according to the first fluorescence information and the second fluorescence information;

[0030] S706: Determine a standard equation according to the plurality of standard fluorescence values ​​and the length of the 3'UTR sequence in the single-stranded nucleic acid, wherein the standard equation represents the relationship between the standard fluorescence value and the length of the 3'UTR sequence in the single-stranded nucleic acid.

[0031] The method provided in the embodiment can hybridize with target DNA of different 3'UTR sequence lengths through CDS probe and 3'UTR probe, and further hybridize with the first fluorescent probe and the second fluorescent probe to generate a fluorescent signal and perform signal amplification, and construct the connection between the fluorescent signal generated during the hybridization and the 3'UTR sequence length. And based on this connection, the fluorescent signal generated by hybridization with the 3'UTR sequence of the unknown mRNA can be calculated to obtain its 3'UTR sequence length. The method can accurately detect the length of the mRNA 3'UTR sequence, with high detection repeatability and wide practicality.

[0032] like Figure 4 As shown, in the method provided in some embodiments, step S701 includes:

[0033] S711: clone the first double-stranded DNA containing the CDS sequence of the target gene and the second double-stranded DNA containing the CDS sequence of the target gene and a 3'UTR sequence of a determined length into the basic plasmid to obtain a first vector and a second vector;

[0034] S721: synthesizing a biotin-labeled forward primer and a reverse primer, and using the first vector and the second vector as templates, respectively, to perform PCR amplification to obtain a biotin-labeled first double-stranded DNA and a biotin-labeled second double-stranded DNA;

[0035] S731: incubating the first double-stranded DNA labeled with biotin and the second double-stranded DNA labeled with biotin with magnetic beads carrying avidin, respectively, to obtain magnetic beads coupled with the first double-stranded DNA and magnetic beads coupled with the second double-stranded DNA;

[0036] S741: treating the magnetic beads coupled to the first double-stranded DNA and the magnetic beads coupled to the second double-stranded DNA with alkaline solution to obtain magnetic beads coupled to the first single-stranded DNA and magnetic beads coupled to the second single-stranded DNA, respectively.

[0037] In some embodiments, the length of the 3'UTR sequence is determined, and its length can be 10bp, 20bp, 30bp, 40bp, 50bp, 100bp, 300bp, 400bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 1000bp, 1200bp, 1300bp, 1400bp, 1500bp, 1600bp, 1800bp, 2000bp, 3000bp, 4000bp. Specifically, there are multiple second double-stranded DNAs, and the length of the 3'UTR sequence of each second double-stranded DNA and the first double-stranded DNA is an arithmetic progression. For example, one first double-stranded DNA and three second double-stranded DNAs (corresponding to three second single-stranded DNAs and three second double-stranded DNAs) are synthesized, and the three second double-stranded DNAs are 1000bp, 2000bp, and 3000bp longer than the first double-stranded DNA sequence, respectively.

[0038] In some embodiments, the amplified products in step S721 are also separated by agarose gel electrophoresis, and the biotin-labeled first double-stranded DNA and the biotin-labeled second double-stranded DNA are obtained after gel recovery.

[0039] In some embodiments, step S741 further comprises, after treating with alkaline solution (eg, 20 mM sodium hydroxide solution) for 1 hour, separating and removing the supernatant with a magnet, and washing the separated magnetic beads with TE buffer.

[0040] like Figure 5 As shown, step S100 provided in some embodiments specifically includes:

[0041] S101: Clone the single-stranded nucleic acid to be tested containing the CDS sequence and 3'UTR sequence of the target gene into a basic plasmid (pBluescript II-KS(+), Nanjing GenScript Biotechnology Co., Ltd.) to obtain a cloning vector;

[0042] S102: synthesizing a biotin-labeled forward primer and a reverse primer, and using the cloning vector as a template, performing PCR amplification to obtain a biotin-labeled double-stranded DNA to be tested;

[0043] S103: incubating the biotin-labeled double-stranded DNA to be tested with magnetic beads carrying avidin to obtain magnetic beads coupled with the double-stranded DNA to be tested;

[0044] S104: treating the magnetic beads coupled to the double-stranded DNA to be detected with an alkaline solution to obtain magnetic beads coupled to the single-stranded DNA to be detected.

[0045] In the step S200 provided in some embodiments, the CDS probe has a nucleotide sequence that is identical or complementary to a partial sequence of the CDS of the target gene, and the 3'UTR probe has a nucleotide sequence that is identical or complementary to a partial sequence of the 3'UTR of the target gene. Optionally, the CDS probe has multiple 3'UTR probes. For example, in a 3'UTR length detection process for human NUDT21, there are 12 CDS probes (as shown in SEQ ID NOs: 1 to 12, respectively), and 20 3'UTR probes (as shown in SEQ ID NOs: 13 to 32, respectively). In step S300, the magnetic beads coupled with the single-stranded nucleic acid to be detected are simultaneously subjected to a first mixed single-stranded hybridization reaction with the 12 CDS probes and 20 3'UTR probes.

[0046] In step S600 provided in some embodiments, the first fluorescence information is the fluorescence value generated by the reaction of the product of the first mixed single-stranded hybridization reaction with the first fluorescent probe, and the second fluorescence information is the fluorescence value generated by the reaction of the product of the first mixed single-stranded hybridization reaction with the first fluorescent probe, and the difference between the first fluorescence value and the second fluorescence value is used as the fluorescence value to be tested corresponding to the single-stranded nucleic acid to be tested.

[0047] In the above embodiment, the first fluorescent group is selected from FAM (carboxyfluorescein), TET (tetrachloro-6-carboxyfluorescein, JOE (2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein), TAMRA (carboxytetramethylrhodamine), ROX (carboxy-X-rhodamine), Cy3, Cy5, HEX (hexachlorofluorescein), TexasRed, VIC, Alexa594, Alexa488. The second fluorescent group is selected from FAM (carboxyfluorescein), TET (tetrachloro-6-carboxyfluorescein, JOE (2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein), TAMRA (carboxytetramethylrhodamine), ROX (carboxy-X-rhodamine), Cy3, Cy5, HEX (hexachlorofluorescein), TexasRed, VIC, Alexa594, Alexa488.

[0048] In step S700 provided in some embodiments, the standard equation represents the mathematical relationship between the difference between the first fluorescence value and the second fluorescence value and the 3'UTR probe. In some embodiments, the standard equation is a linear mathematical relationship.

[0049] The present application will be described below in conjunction with more specific embodiments, but this does not constitute a limitation on the implementation methods of the present application.

[0050] 1. Design the first double-stranded DNA, the second double-stranded DNA, the CDS probe, the 3'UTR probe, the first fluorescent probe and the second fluorescent probe

[0051] Taking the human NUDT21 gene as an example, based on the coding region (CDS, 684 bp) and 3' untranslated region (3'UTR, 3579 bp) sequences of its mRNA (NCBI sequence number: NM_007006.3), an Alexa594-labeled CDS probe and an Alexa488-labeled 3'UTR probe were designed, respectively.

[0052] Sequences of the first single-stranded DNA and the first double-stranded DNA: CDS sequence (NM_007006.3)

[0053] The sequences of the second single-stranded DNA and the second double-stranded DNA are: CDS+1000 bp of 3'UTR, SEQ ID NO:33; or CDS+2000 bp of 3'UTR, SEQ ID NO:34; or CDS+3000 bp of 3'UTR, SEQ ID NO:35.

[0054] The nucleotide sequences of the CDS probe set are shown in SEQ ID NOs: 1-12.

[0055] The nucleotide sequences of the 3'UTR probe set are shown in SEQ ID NOs: 13-32.

[0056] The first fluorescent probe: Alexa594 labeled at the 5' end, used to identify the CDS probe set, Molecular Instruments.

[0057] The second fluorescent probe: Alexa488 labeled at the 5' end, used to identify the 3'UTR probe set, Molecular Instruments.

[0058] 2. Step S701

[0059] A specific step S701 includes:

[0060] The first double-stranded DNA containing the CDS sequence of the target gene and the second double-stranded DNA containing the CDS sequence of the target gene and a 3'UTR sequence of a determined length were cloned into a basic plasmid (pBluescript II-KS(+), Nanjing GenScript Biotechnology Co., Ltd.) to obtain a first vector carrying the CDS sequence (NM_007006.3) and three second vectors carrying SEQ ID NOs: 33 to 35, respectively.

[0061] A forward primer and a reverse primer labeled with biotin are synthesized, and the first vector and the second vector are used as templates respectively, and PCR amplification is performed to obtain a first double-stranded DNA labeled with biotin and a second double-stranded DNA labeled with biotin.

[0062] The primer pair used to amplify the first vector includes:

[0063] The forward primer labeled with biotin at the 5' end is: 5'-ATGTCTGTGGTACCGCCCAA-3', SEQ ID NO: 36; the reverse primer (3'UTR-0bp): 5'-TCAAACAATAGAAAGGTGGC-3', SEQ ID NO: 37, and the product is the first double-stranded DNA.

[0064] The primer pairs used to amplify the second vector include:

[0065] Reverse primer (3'UTR-1000bp): 5'-ACATCTGGCAAGATTCCTTT-3', SEQ ID NO: 38. The product is a second double-stranded DNA comprising CDS and 1000bp 3'UTR sequence.

[0066] Reverse primer (3'UTR-2000bp): 5'-AAACAGTGCCCTTATACCCT-3', SEQ ID NO: 39. The product is a second double-stranded DNA, which includes CDS and 2000bp 3'UTR sequence.

[0067] Reverse primer (3'UTR-3000bp): 5'-CAACAACCACTAAGCCTTCT-3', SEQ ID NO: 40. The product is a second double-stranded DNA, which includes CDS and 3000bp 3'UTR sequence.

[0068] The PCR reaction system is shown in Table 1, and the PCR reaction program is shown in Table 2.

[0069] Table 1

[0070] Volume (μl) 2×Taq Plus Master Mix (purchased from Novozymes Biotech Co., Ltd.) 25 Forward primer (10 μM) 2 Reverse primer (10 μM) 2 template 1 Sterile water 20 Total volume 50

[0071] Table 2

[0072] Temperature(℃) time 95 3 minutes 95 15 seconds 60 20 seconds 35 cycles 72*(*Extension time depends on the length of the product and can be 60 seconds / kb) 45 seconds to 3 minutes 40 seconds 72 10 minutes

[0073] The recovered biotin-labeled first double-stranded DNA and the second double-stranded DNA were used as standards and mixed with avidin-coupled Dynabead magnetic beads (purchased from Thermo) at a ratio of 20 μg DNA / 1 mg magnetic beads, incubated at room temperature for 1 hour, and coupled to the magnetic beads. Washed twice with 100 μL TE buffer (10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA), washed twice with 100 μL double distilled water, mixed the double-stranded DNA standard-magnetic bead complex with 100 μL NaOH (20 mM) solution, placed on a rotating mixer, and incubated at room temperature for 1 hour; the test tube was placed on a magnet to separate the magnetic beads from the supernatant, the supernatant was discarded, and washed 3 times with TE buffer to obtain magnetic beads coupled to the first single-stranded DNA and magnetic beads coupled to the second single-stranded DNA.

[0074] 3. Steps S702 to S706

[0075] In some embodiments, steps S702 to S706 specifically include:

[0076] S702: performing a first mixed single-strand hybridization reaction on the magnetic beads coupled to the first single-stranded DNA and the CDS probe and the 3'UTR probe at the same time, and performing a first mixed single-strand hybridization reaction on the magnetic beads coupled to the second single-stranded DNA and the CDS probe and the 3'UTR probe at the same time;

[0077] S703: reacting the magnetic beads coupled to the first single-stranded DNA to obtain a first mixed single-stranded hybridization reaction product, and simultaneously performing a second mixed single-stranded hybridization reaction on the first fluorescent probe and the second fluorescent probe; reacting the magnetic beads coupled to the second single-stranded DNA to obtain a first mixed single-stranded hybridization reaction product, and simultaneously performing a second mixed single-stranded hybridization reaction on the first fluorescent probe and the second fluorescent probe;

[0078] S704: reading first fluorescence information generated by a magnetic bead reaction coupled to the first single-stranded DNA according to Alexa594 of the first fluorescent probe, and reading second fluorescence information generated by three magnetic bead reactions coupled to the second single-stranded DNA according to Alexa488 of the second fluorescent probe;

[0079] S705: obtaining a first standard fluorescence value of the magnetic beads coupled to the first single-stranded DNA and three second standard fluorescence values ​​of the magnetic beads coupled to the second single-stranded DNA;

[0080] S706: Determine a standard equation according to the four standard fluorescence values ​​and the length of the 3'UTR sequence in the single-stranded nucleic acid, wherein the standard equation represents the relationship between the standard fluorescence value and the length of the 3'UTR sequence in the single-stranded nucleic acid.

[0081] The conditions for the first mixed single-strand hybridization reaction and the second mixed single-strand hybridization reaction are as follows: wash the magnetic beads twice with 2×SSC buffer, add hybridization buffer, place on a rotating mixer, pre-hybridize at 37°C for 30 minutes, resuspend the magnetic beads with hybridization buffer containing CDS probe and 3'UTR probe, place on a rotating mixer, and incubate at 37°C for 12 hours. Wash the magnetic beads 4 times with washing buffer, wash the magnetic beads once with 5×SSCT buffer, resuspend the magnetic beads with amplification buffer, place on a rotating mixer, and pre-amplify at room temperature for 30 minutes. Take an equal volume of the second fluorescent probe labeled with Alexa488 (for identifying the 3'UTR probe group, Molecular Instruments) and the first fluorescent probe labeled with Alexa594 (for identifying the CDS probe group, Molecular Instruments), mix them evenly, heat shock at 95°C for 90s, and cool at room temperature in the dark for more than 30min; add 50μL amplification buffer to the Hairpin B1 and B2 probe mixture, resuspend the magnetic beads, place on a rotating mixer, incubate at room temperature overnight in the dark for signal amplification; discard the amplification buffer, wash the magnetic beads 5 times with 5×SSCT; resuspend the magnetic beads with TE buffer and add them to a 96-well plate.

[0082] The fluorescence intensity of each well was read by Synergy H1 multifunctional microplate reader (BioTek, USA), and the excitation / emission light was set as follows: excitation 485nm / emission 528nm (detection of 3'UTR probe), excitation 594nm / emission 650nm (detection of CDS probe), and endpoint detection was used. The 528nm fluorescence reading of the calibrated single-stranded DNA standard and the 3'UTR length (0, 1000bp, 2000bp, 3000bp) were plotted as scatter plots, and the standard curve was drawn to obtain the regression equation.

[0083] Among them, 2×SSC buffer is an aqueous solution containing 3M sodium chloride and 0.3M sodium citrate. 5×SSCT buffer is a 5×SSC buffer containing 0.1% Tween-20. Hybridization buffer is a 5×SSC buffer containing 30% formamide, 9mM citric acid, 0.1% Tween-20, 50μg / ml heparin, 1×Denhardt's solution and 1% dextran sulfate. Washing buffer is a 5×SSC buffer containing 30% formamide, 9mM citric acid, 0.1% Tween-20, 50μg / ml heparin, 1×Denhardt's solution. Amplification buffer is a 5×SSC buffer containing 0.1% Tween-20 and 2.5% dextran sulfate.

[0084] Figure 6 An example is shown in which the standard equation provided is Y = 0.1956 x X - 11.44, R 2 =0.9662.

[0085] 3. Step S100

[0086] In one embodiment, the culture dish is placed on ice, the HeLa cells are scraped off, collected in a 1.5 ml centrifuge tube, and centrifuged to obtain a cell pellet; the cells are resuspended with a lysis solution, and the supernatant is retained after centrifugation; oligodT magnetic beads (purchased from Thermo) are added to the cell lysate, placed on a rotating mixer, and incubated at room temperature for 30 minutes; the test tube is placed on a magnet to separate the magnetic beads from the supernatant, and the supernatant is discarded to obtain magnetic beads coupled to the mRNA to be tested.

[0087] 4. Steps S200 to S700

[0088] In some embodiments, steps S200 to S700 specifically include:

[0089] S200: providing CDS probes as shown in SEQ ID NOs: 1 to 12 and 3'UTR probes as shown in SEQ ID NOs: 13 to 32, a first fluorescent probe labeled with Alexa594, and a second fluorescent probe labeled with Alexa488;

[0090] S300: performing a first mixed single-stranded hybridization reaction on the magnetic beads coupled to the mRNA to be tested and the CDS probes shown in SEQ ID NOs: 1 to 12 and the 3'UTR probes shown in SEQ ID NOs: 13 to 32 at the same time;

[0091] S400: performing a second mixed single-strand hybridization reaction on the product of the first mixed single-strand hybridization reaction with the first fluorescent probe and the second fluorescent probe at the same time;

[0092] S500: reading the fluorescence information to be measured generated by the second mixed single-strand hybrid reaction according to Alexa594 of the first fluorescent probe;

[0093] S600: Calculating the fluorescence value to be measured according to the fluorescence information to be measured;

[0094] S700: Input the fluorescence value to be measured into a standard equation to determine the length of the 3'UTR sequence.

[0095] The specific steps S200 to S700 of the first mixed single-stranded hybridization reaction, the second mixed single-stranded hybridization reaction and the fluorescence value reading refer to the above embodiment.

[0096] In one embodiment, the results of the 3'UTR length detection of the NUDT21 mRNA of the sample to be tested are shown in Table 3. The 3'UTR length detection was performed on the two extracted samples, and the lengths thereof were 1421 bp and 1124 bp, respectively.

[0097] Table 3

[0098]

[0099] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A method for quantitatively detecting the length of mRNA 3'UTR, comprising: Prepare magnetic beads coupled with a single-stranded nucleic acid to be tested, wherein the single-stranded nucleic acid to be tested comprises a CDS sequence and a 3'UTR sequence of a target gene; Providing a CDS probe that binds to the CDS region and a 3'UTR probe that binds to the 3'UTR region, as well as a first fluorescent probe that recognizes the CDS probe and a second fluorescent probe that recognizes the 3'UTR probe, wherein the first fluorescent probe carries a first fluorescent group, and the second fluorescent probe carries a second fluorescent group that is different from the first fluorescent group; Performing a first mixed single-strand hybridization reaction on the magnetic beads coupled with the single-stranded nucleic acid to be detected, the CDS probe and the 3'UTR probe at the same time; Performing a second mixed single-strand hybridization reaction on the product of the first mixed single-strand hybridization reaction with the first fluorescent probe and the second fluorescent probe at the same time; Reading first fluorescence information generated by the reaction of the product of the first mixed single-stranded hybridization reaction with the first fluorescent probe according to the first fluorescent group, and reading second fluorescence information generated by the reaction of the product of the first mixed single-stranded hybridization reaction with the second fluorescent probe according to the second fluorescent group; Determine the fluorescence value to be tested corresponding to the single-stranded nucleic acid to be tested according to the first fluorescence information and the second fluorescence information; Determine the length of the 3'UTR sequence according to the standard equation and the fluorescence value to be measured.

2. The method according to claim 1, before determining the length of the 3'UTR sequence according to the standard equation and the fluorescence value to be measured, further comprising: Prepare a plurality of magnetic beads coupled with a standard single-stranded nucleic acid, wherein the standard single-stranded nucleic acid comprises a target gene CDS sequence, or comprises a target gene CDS sequence and 3'UTR sequences of different lengths; Performing a first mixed single-strand hybridization reaction on each magnetic bead coupled with a single-stranded nucleic acid and the CDS probe and the 3'UTR probe at the same time; Performing a second mixed single-strand hybridization reaction on the product of the first mixed single-strand hybridization reaction and the first fluorescent probe and the second fluorescent probe; Reading first fluorescence information generated by the reaction of the product of the first mixed single-stranded hybridization reaction with the first fluorescent probe according to the first fluorescent group, and reading second fluorescence information generated by the reaction of the product of the first mixed single-stranded hybridization reaction with the second fluorescent probe according to the second fluorescent group; Determine a plurality of standard fluorescence values ​​corresponding to the single-stranded nucleic acid without the 3'UTR sequence and the single-stranded nucleic acid with the 3'UTR sequence of different lengths according to the first fluorescence information and the second fluorescence information; A standard equation is determined according to a plurality of standard fluorescence values ​​and the length of the 3'UTR sequence in the single-stranded nucleic acid, and the standard equation characterizes the relationship between the standard fluorescence value and the length of the 3'UTR sequence in the single-stranded nucleic acid.

3. According to the method of claim 1, a plurality of magnetic beads coupled with standard single-stranded nucleic acids are prepared, wherein the standard single-stranded nucleic acids contain the target gene CDS sequence, or contain the target gene CDS sequence and 3'UTR sequences of different lengths, comprising: The first double-stranded DNA containing the CDS sequence of the target gene and the second double-stranded DNA containing the CDS sequence of the target gene and a 3'UTR sequence of a determined length are cloned into the basic plasmid to obtain a first vector and a second vector; Synthesizing a biotin-labeled forward primer and a reverse primer, and using the first vector and the second vector as templates, respectively, to perform PCR amplification to obtain a biotin-labeled first double-stranded DNA and a biotin-labeled second double-stranded DNA; The first double-stranded DNA labeled with biotin and the second double-stranded DNA labeled with biotin are incubated with magnetic beads carrying avidin, respectively, to obtain magnetic beads coupled with the first double-stranded DNA and magnetic beads coupled with the second double-stranded DNA; The magnetic beads coupled to the first double-stranded DNA and the magnetic beads coupled to the second double-stranded DNA are treated with alkaline solution to obtain magnetic beads coupled to the first single-stranded DNA and magnetic beads coupled to the second single-stranded DNA, respectively.

4. According to the method of claim 3, the length of the 3'UTR sequence is determined to be 10bp, 20bp, 30bp, 40bp, 50bp, 100bp, 300bp, 400bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 1000bp, 1200bp, 1300bp, 1400bp, 1500bp, 1600bp, 1800bp, 2000bp, 3000bp, and 4000bp.

5. The method according to claim 3, wherein there are a plurality of second double-stranded DNAs, and the length of the extra 3'UTR sequence of each second double-stranded DNA is in an arithmetic progression compared to the first double-stranded DNA.

6. According to the method of claim 1, preparing magnetic beads coupled with a single-stranded nucleic acid to be detected, wherein the single-stranded nucleic acid to be detected comprises a CDS sequence and a 3'UTR sequence of a target gene, comprising: The single-stranded nucleic acid to be tested, which includes the CDS sequence and 3'UTR sequence of the target gene, is cloned into the basic plasmid to obtain a cloning vector; Synthesize biotin-labeled forward primers and reverse primers, use the cloning vector as a template, and perform PCR amplification to obtain biotin-labeled double-stranded DNA to be tested; Incubating the biotin-labeled double-stranded DNA to be tested with magnetic beads carrying avidin to obtain magnetic beads coupled with the double-stranded DNA to be tested; The magnetic beads coupled to the double-stranded DNA to be tested are treated with an alkali solution to obtain magnetic beads coupled to the single-stranded DNA to be tested.

7. The method according to claim 1, wherein a CDS probe that binds to the CDS region and a 3'UTR probe that binds to the 3'UTR region are provided, and a first fluorescent probe that recognizes the CDS probe and a second fluorescent probe that recognizes the 3'UTR probe are provided, wherein the first fluorescent probe carries a first fluorescent group and the second fluorescent probe carries a second fluorescent group that is different from the first fluorescent group. The CDS probe has a nucleotide sequence that is identical or complementary to a portion of the CDS sequence of the target gene, and the 3'UTR probe has a nucleotide sequence that is identical or complementary to a portion of the 3'UTR sequence of the target gene; In the step of performing a first mixed single-strand hybridization reaction on the magnetic beads coupled with the single-stranded nucleic acid to be detected, the CDS probe and the 3'UTR probe are simultaneously reacted. The magnetic beads coupled with the single-stranded nucleic acid to be detected are simultaneously subjected to a first mixed single-stranded hybridization reaction with the 12 CDS probes and the 20 3'UTR probes.

8. The method according to claim 1, in the step of determining the fluorescence value to be detected corresponding to the single-stranded nucleic acid to be detected according to the first fluorescence information and the second fluorescence information, The first fluorescence information is the fluorescence value generated by the reaction of the product of the first mixed single-stranded hybridization reaction with the first fluorescent probe, and the second fluorescence information is the fluorescence value generated by the reaction of the product of the first mixed single-stranded hybridization reaction with the first fluorescent probe. The difference between the first fluorescence value and the second fluorescence value is used as the fluorescence value to be tested corresponding to the single-stranded nucleic acid to be tested.

9. According to the method of claim 1, the first fluorescent group is selected from FAM (carboxyfluorescein), TET (tetrachloro-6-carboxyfluorescein, JOE (2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein), TAMRA (carboxytetramethylrhodamine), ROX (carboxy-X-rhodamine), Cy3, Cy5, HEX (hexachlorofluorescein), TexasRed, VIC, Alexa594, Alexa488, and the second fluorescent group is selected from FAM (carboxyfluorescein), TET (tetrachloro-6-carboxyfluorescein, JOE (2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein), TAMRA (carboxytetramethylrhodamine), ROX (carboxy-X-rhodamine), Cy3, Cy5, HEX (hexachlorofluorescein), TexasRed, VIC, Alexa594, Alexa488.

10. A method for quantitatively detecting the length of mRNA 3'UTR, comprising: Prepare a first single-stranded DNA magnetic bead containing a human NUDT21 gene CDS sequence and three second single-stranded DNA magnetic beads containing SEQ ID NOs: 33 to 35; Providing CDS probes as shown in SEQ ID NOs: 1 to 12 and 3'UTR probes as shown in SEQ ID NOs: 13 to 32, performing a first mixed single-strand hybridization reaction on magnetic beads coupled to the first single-stranded DNA, the CDS probe and the 3'UTR probe, and performing a first mixed single-strand hybridization reaction on magnetic beads coupled to the second single-stranded DNA, the CDS probe and the 3'UTR probe; A first fluorescent probe labeled with Alexa594 and a second fluorescent probe labeled with Alexa488 are provided, and a magnetic bead coupled with the first single-stranded DNA is reacted to obtain a first mixed single-stranded hybridization reaction product, and the first fluorescent probe and the second fluorescent probe are subjected to a second mixed single-stranded hybridization reaction; a magnetic bead coupled with the second single-stranded DNA is reacted to obtain a first mixed single-stranded hybridization reaction product, and the first fluorescent probe and the second fluorescent probe are subjected to a second mixed single-stranded hybridization reaction; Read the first fluorescence value generated by the reaction of the magnetic beads coupled to the first single-stranded DNA according to Alexa594 of the first fluorescent probe, and read the second fluorescence values ​​generated by the reaction of three magnetic beads coupled to the second single-stranded DNA according to Alexa488 of the second fluorescent probe; Calculate the difference between the second fluorescence value and the first fluorescence value of each pair of magnetic beads coupled to the second single-stranded DNA to obtain three standard fluorescence values; A standard equation is determined according to the three standard fluorescence values ​​and the length of the 3'UTR sequence in the single-stranded nucleic acid, and the standard equation represents the relationship between the standard fluorescence value and the length of the 3'UTR sequence in the single-stranded nucleic acid; preparing magnetic beads coupled to the mRNA to be tested; The magnetic beads coupled to the mRNA to be tested are simultaneously subjected to a first mixed single-stranded hybridization reaction with the CDS probes shown in SEQ ID NOs: 1 to 12 and the 3'UTR probes shown in SEQ ID NOs: 13 to 32; Performing a second mixed single-strand hybridization reaction on the product of the first mixed single-strand hybridization reaction with the first fluorescent probe and the second fluorescent probe at the same time; Reading the fluorescence information to be measured generated by the second mixed single-stranded hybrid reaction according to Alexa594 of the first fluorescent probe; Calculating the fluorescence value to be measured according to the fluorescence information to be measured; The fluorescence value to be measured is input into the standard equation to determine the length of the 3'UTR sequence.

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