CN113005181B - Primer group for detecting non-coding small RNA (ribonucleic acid) by using multiplex fluorescent quantitative PCR (polymerase chain reaction) based on stem-loop method - Google Patents
Primer group for detecting non-coding small RNA (ribonucleic acid) by using multiplex fluorescent quantitative PCR (polymerase chain reaction) based on stem-loop method Download PDFInfo
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Abstract
The invention discloses a primer group for detecting non-coding small RNA by multiplex fluorescence quantitative PCR based on a stem-loop method, which comprises a primer group and a primer group. The invention establishes a method for detecting non-coding small RNA by multiplex fluorescence quantitative PCR based on a stem-loop method, and realizes PCR amplification of reverse transcription products of a plurality of targets by 1 pair of upstream and downstream universal primers. The reverse transcription extension primer is added in the reverse transcription process, the reverse transcription product can be prolonged, and the design of an upstream primer and a probe can be greatly facilitated due to the increase of the length of the amplification product; multiple target miRNAs can be subjected to reverse transcription in a single-tube reaction, and then multiple fluorescent quantitative PCR detection is carried out, so that multiple detection of miRNAs based on stem-loop reverse transcription primers is realized, the operation is simpler and more convenient, and the clinical diagnosis application requirements of multiple target miRNA combined expression analysis are met.
Description
Technical Field
The invention relates to the technical field of fluorescent quantitative PCR, in particular to a primer group for detecting non-coding small RNA by multiple fluorescent quantitative PCR based on a stem-loop method.
Background
Micrornas, mirnas, are a class of non-coding single-stranded RNA molecules of about 22 nucleotides in length encoded by endogenous genes. Research shows that miRNA is involved in disease occurrence and development. Therefore, the detection of the expression difference of the miRNA can provide ideas for disease screening and diagnosis, thereby realizing the application of the miRNA in clinical diagnosis. It is generally believed that effective diagnosis of disease is difficult to achieve using changes in expression of a single miRNA target. The application of miRNA in clinical diagnosis can be effectively realized only by combining a plurality of miRNA targets and referring to internal reference homogenization and quantitative detection. In addition, the operation method of clinical diagnosis generally reduces manual operation steps and operation errors, so that the method is simple, convenient and efficient.
Currently, methods for detecting miRNA are largely classified into a probe direct hybridization method that does not require sample amplification and a method based on amplification, such as: the northern blotting method, the microarray method, the cloning and sequencing method, the real-time fluorescent quantitative PCR method and the like.
The method for detecting miRNA by real-time fluorescence quantitative PCR has the advantages of high sensitivity, strong specificity, simplicity, rapidness, low cost and the like, and is the most common method for realizing the quantitative detection of miRNA. At present, real-time fluorescent quantitative PCR detection comprises two main methods, namely Tailing Reaction RT-PCR (labeling Reaction RT-PCR) and Stem-loop RT-PCR (Stem-loop RT-PCR), according to different reverse transcription methods. No matter the tailing method RT-PCR or the stem-loop method RT-PCR, reverse transcription and quantitative detection are generally step-by-step operations, the operation is more complicated, and the experiment error can be increased by manual pipetting and other operations. Although the operation of the stem-loop RT-PCR is relatively simple and convenient, and the detection of single miRNA is the most accurate, the realization of multiplex detection is basically difficult. Moreover, since the 3' end of the stem-loop reverse transcription primer is a specific sequence, in principle, to realize multiplex detection, a specific binding sequence on the stem-loop reverse transcription primer needs to be designed for different target mirnas, so that the cost is increased, and the deviation of the reverse transcription efficiency is caused by the difference of the length and the base of the specific binding sequence. Therefore, the stem-loop method RT-qPCR is not suitable for multiplex detection under the condition of a large number of target miRNAs.
When the number of the target miRNAs is proper, the characteristics of high specificity and accuracy of the stem-loop method can be considered through the design of the stem-loop method, and the requirement of effective multiple detection of a plurality of target miRNAs can be met. Based on the above, patent CN201510639411 discloses a miRNA multiplex detection method developed based on stem-loop RT-PCR, which includes the steps of reverse transcription, single-strand complementary extension, S1 nuclease digestion, solid-phase adsorption extraction and fluorescent quantitative detection, but the steps are complicated and the components are more complex. Compared with the patent CN201510639411, the invention has the following advantages: the technology principle is completely different, the invention only needs to add reverse transcription extension primer in the reverse transcription process, then obtains the needed long fragment cDNA through reverse transcription reaction, and the patent CN201510639411 obtains cDNA through reverse transcription, and then obtains the needed product through single-strand complementary extension. The method is simple to operate and process, and does not need the steps of single-strand complementary extension, S1 nuclease digestion, solid-phase adsorption extraction and the like. ③ CN201510639411, the reverse transcription primer used in the invention is of a linear structure, while the reverse transcription primer used in the invention is of a typical stem-loop structure, which can improve the specificity of the reverse transcription reaction. The invention has low cost, does not need nuclease digestion, solid phase adsorption extraction and other reagents, and can greatly reduce the use cost.
The invention has certain innovation in technology, can accurately detect the non-coding small RNA gene in a sample, and provides an accurate and sensitive multiple detection method with simpler operation, relatively low cost for the fluorescent quantitative detection of miRNA.
Disclosure of Invention
Based on the defects of the prior art, the invention provides a primer group for detecting non-coding small RNA by multiplex fluorescence quantitative PCR based on a stem-loop method. The invention can realize reverse transcription and fluorescent quantitative detection in a single-tube PCR tube. Based on the consistent characteristics of the length, the structure and the like of the mature non-coding small RNA, the invention is suitable for detecting various mature non-coding small RNAs including miRNA, such as piRNA, siRNA and the like.
The first purpose of the invention is to provide a reverse transcription extension primer for detecting non-coding small RNA by multiplex fluorescent quantitative PCR based on a stem-loop method.
The second purpose of the invention is to provide a reverse transcription primer for detecting non-coding small RNA by multiplex fluorescent quantitative PCR based on a stem-loop method.
The third purpose of the invention is to provide an upstream universal primer for detecting non-coding small RNA by multiplex fluorescent quantitative PCR based on a stem-loop method.
The fourth purpose of the invention is to provide a downstream universal primer for detecting non-coding small RNA by multiplex fluorescent quantitative PCR based on a stem-loop method.
The fifth purpose of the invention is to provide a specific probe for detecting non-coding small RNA by multiplex fluorescent quantitative PCR based on a stem-loop method.
The sixth purpose of the invention is to provide a primer group for quantitative fluorescent quantitative PCR detection of non-coding small RNA.
The seventh purpose of the invention is to provide a method for detecting non-coding small RNA by quantitative fluorescence quantitative PCR.
The eighth purpose of the invention is to provide the application of the primer group in the fluorescent quantitative PCR detection of non-coding small RNA or the application in the preparation of a kit for the fluorescent quantitative PCR detection of non-coding small RNA.
In order to achieve the purpose, the invention is realized by the following scheme:
the inventor finds a method capable of detecting multiple non-coding small RNAs simultaneously through extensive and intensive research, and experimental results show that the method has higher sensitivity and accuracy.
Specifically, the non-coding small RNA detected by the invention is short-sequence RNA comprising miRNA, piRNA and siRNA, and the invention can simultaneously detect 1-5 non-coding small RNAs.
The invention claims a reverse transcription extension primer for detecting non-coding small RNA by multiplex fluorescence quantitative PCR based on a stem-loop method, which comprises the following components in sequence from the 5 'end to the 3' end: an upstream universal primer sequence and a specific binding sequence 1; the specific binding sequence 1 is the same as 6-15 basic groups at the 5' end of the small non-coding RNA to be detected; the sequence of the upstream universal primer is 16-25 bases; the 3' end of the reverse transcription extension primer cannot be extended continuously; the sequence of the upstream universal primer is not synonymous with or complementary to a non-coding small RNA sequence to be detected, and the number of the basic groups of the upstream universal primer is 16-25.
Preferably, the reverse transcription primer is 22-60 bases in length.
Preferably, the reverse transcription extension primer is phosphorylated at the 3 'end, modified at the 3' MGB, or diluted with an amino group to enable continued extension.
And a reverse transcription primer for detecting the non-coding small RNA by the multiplex fluorescence quantitative PCR based on the stem-loop method, wherein the reverse transcription primer sequentially comprises the following components from the 5 'end to the 3' end: the kit comprises a stem-loop sequence and a specific binding sequence 2, wherein the specific binding sequence is 2 and 6-12 reverse complementary sequences of the 3' end of the non-coding small RNA to be detected.
Preferably, the reverse transcription primer is 40-60 bases in length.
And the upstream universal primer for detecting the non-coding small RNA by the multiplex fluorescence quantitative PCR based on the stem-loop method has the same sequence as that of the upstream universal primer in the reverse transcription extension primer, namely the reverse transcription extension primer sequence comprises the upstream universal primer sequence.
Preferably, the length of the upstream universal primer is 16-20 bases.
And the downstream universal primer for detecting the non-coding small RNA by the multiplex fluorescence quantitative PCR based on the stem-loop method has the same sequence as part of the stem-loop sequence in the reverse transcription primer, namely the reverse transcription primer comprises the sequence of the downstream universal primer, namely the downstream universal primer is arranged on the stem-loop sequence, and the number of the basic groups of the downstream universal primer is 16-25.
Preferably, the length of the downstream universal primer is 16-20 bases.
When a plurality of non-coding small RNAs are detected, the upstream universal primer sequence of each non-coding small RNA is the same as the downstream universal primer sequence, and multiple fluorescent quantitative PCR can be carried out for amplification detection at the same time.
And a specific probe for detecting the non-coding small RNA by multiplex fluorescence quantitative PCR based on a stem-loop method, wherein the specific probe is the same as a partial fragment of the sequence of the non-coding small RNA to be detected; the number of the basic groups of the specific probe is 17-30.
Preferably, the specific probe is a taqman probe or a taqman-MGB probe.
Preferably, the specific probe is 3' labeled with any one of a quencher group BHQ1, BHQ2 or TAMRA.
Therefore, the invention claims a primer group for detecting non-coding small RNA by quantitative fluorescence quantitative PCR, which comprises the reverse transcription extension primer, the reverse transcription primer, the upstream universal primer and/or the downstream universal primer.
Preferably, a primer group for quantitative fluorescent quantitative PCR detection of non-coding small RNA comprises the reverse transcription extension primer, the reverse transcription primer, the upstream universal primer and the downstream universal primer.
Therefore, the invention also claims a method for detecting non-coding small RNA by quantitative fluorescence quantitative PCR, which comprises the following steps:
s1, carrying out reverse transcription reaction on the reverse transcription extension primer, the reverse transcription primer and the small non-coding RNA to be detected in the same system to obtain a reverse transcription product;
s2, taking the reverse transcription product of the step S1 as a template, and carrying out fluorescent quantitative PCR reaction by using the upstream universal primer, the downstream universal primer and the specific probe.
The non-coding small RNA can be: piRNA, siRNA, or miRNA.
Preferably, multiplex quantitative fluorogenic quantitative PCR is performed to detect non-coding small RNAs, the 5' end of the specific probe is marked with different fluorescent reporter groups, and the small RNAs are 2 to 5 small RNAs.
More preferably, the plurality of small RNAs is 2 to 4 small RNAs.
More preferably, the fluorescent reporter group is selected from FAM, HEX, VIC, Texas-Red, and CY 5.
Preferably, the final concentration of the reverse transcription primers is 50-200 nM, the final concentration of the reverse transcription extension primers is 50-200 nM, the final concentration of the upstream universal primers is 50-200 nM, the final concentration of the downstream universal primers is 50-200 nM, and the final concentration of the amplification system of the specific probe is 50-200 nM.
And (2) carrying out reverse transcription on the small non-coding RNA to be detected by utilizing a stem-loop method to synthesize first strand cDNA, and simultaneously utilizing the reverse transcription extension primer to be complementarily paired and combined with the first strand cDNA so that the reverse transcription 3' end of the first strand cDNA further synthesizes a complementary sequence of an upstream universal primer of the reverse transcription extension primer, so as to introduce the complementary sequence of the universal upstream universal primer and provide a binding region of the upstream universal primer for subsequent amplification.
Preferably, the procedure for reverse transcription is: hold at 25 ℃ for 5min, 50 ℃ for 15min, 85 ℃ for 5min, 4 ℃.
Preferably, the system of reverse transcription is: 5 × RT buffer 4 μ L; dNTPs (10Mm each) 1. mu.L; 2 mul of reverse transcription extension primer of each non-coding small RNA to be detected; each stem-loop reverse transcription primer of the non-coding small RNA to be detected is 2 mu L; M-MLV (H-) RNA reverse transcriptase (200U/. mu.L) 1. mu.L; RNase inhibitor (40U/. mu.L) 1. mu.L; RNase free H2O was made up to 20. mu.L.
Preferably, the procedure for quantitative PCR is: 10min at 37 ℃; 5min at 95 ℃; 10s at 95 ℃ and 45s at 60 ℃ and the cycle number is 45 times; keeping the temperature constant at 4 ℃.
Preferably, the system for quantitative PCR is: 10 × Buffer (Mg)2+plus) 4. mu.L; dNTPs (10Mm each, containing dUTP) 1. mu.L; 2 mu L of universal upstream primer; 2 mu L of universal downstream primer; each specific probe of each non-coding small RNA to be detected is 0.5 mu L; 0.5. mu.L of DNA Polymerase (AceTaq DNA Polymerase (5U/. mu.l)); UDG enzyme (1U/. mu.l) 1. mu.L; 2 mu L of reverse transcription product; ddH2O make up to 50. mu.L.
UDG enzyme is added into a PCR system, so that product pollution can be effectively prevented, and the detection accuracy is improved.
The primer group is applied to the fluorescent quantitative PCR detection of the non-coding small RNA or the preparation of a kit for the fluorescent quantitative PCR detection of the non-coding small RNA. Also belong to the protection scope of the present invention.
Preferably, the fluorescent quantitative PCR is multiplex fluorescent quantitative PCR.
Compared with the prior art, the invention has the following beneficial effects:
the invention establishes a multiple fluorescence quantitative PCR detection method of non-coding small RNA based on a stem-loop method, and realizes the PCR detection of reverse transcription products of multiple targets by 1 pair of upstream and downstream universal primers. The reverse transcription extension primer is added in the reverse transcription process, the reverse transcription product can be prolonged, and the design of an upstream primer and a probe can be greatly facilitated due to the increase of the length of the amplification product; multiple target miRNAs can be subjected to reverse transcription in a single-tube reaction, and then multiple fluorescent quantitative PCR detection is carried out, so that multiple detection of miRNAs based on stem-loop reverse transcription primers is realized, the operation is simpler and more convenient, and the clinical diagnosis application requirements of multiple target miRNA combined expression analysis are met. Meanwhile, UDG enzyme is added into a PCR system, so that product pollution can be effectively prevented, and the detection accuracy is improved.
Drawings
Fig. 1 is a schematic diagram of the design principle and steps.
FIG. 2 shows the results of the detection by the common stem-loop method and the has-miR-21-5p probe method according to the method of the invention.
FIG. 3 is a monoclonal sequencing diagram of the products of the ordinary stem-loop method RT-qPCR and the RT-qPCR of the present invention.
FIG. 4 shows the results of multiplex detection of Has-miR-21-5p, Has-miR-143 and RUN48 by the RT-qPCR method of the invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
The sequence of the small non-coding RNA used in the following examples was based on the information described in miRbase.
Example 1 primer and Probe design
The design is mainly divided into 3 steps:
designing a reverse transcription extension primer aiming at a non-coding small RNA sequence to be detected.
Designing a reverse transcription primer by using a small RNA stem-loop reverse transcription method;
corresponding to the corresponding sequence, universal upstream and downstream primers and specific probes are designed, and then the generation detection non-coding small RNA is detected, and the specific design idea is shown in figure 1.
The specific design is as follows:
1. reverse transcription extension primer: the reverse transcription extension primer comprises the following components from the 5 'end to the 3' end in sequence: an upstream universal primer sequence and a specific binding sequence 1; the specific binding sequence 1 is the same as 6-15 basic groups at the 5' end of the small non-coding RNA to be detected; the sequence of the upstream universal primer is 16-25 bases; the 3' end of the reverse transcription extension primer cannot be extended continuously; the sequence of the upstream universal primer is not synonymous with or complementary to a non-coding small RNA sequence to be detected, and the number of basic groups of the upstream universal primer is 16-25; the reverse transcription primer is 22-60 bases in length; the reverse transcription extension primer is phosphorylated at the 3 'end, modified with 3' MGB or diluted with amino group to enable continued extension.
2. Reverse transcription primer: the reverse transcription primer comprises the following components from the 5 'end to the 3' end in sequence: the kit comprises a stem-loop sequence and a specific binding sequence 2, wherein the specific binding sequence is a reverse complementary sequence of 2 and 6-12 of the 3' end of the small non-coding RNA to be detected, and the length of a reverse transcription primer is 40-60 basic groups.
3. An upstream universal primer: the sequence of the reverse transcription extension primer is the same as the sequence of the upstream universal primer in the reverse transcription extension primer, namely the sequence of the reverse transcription extension primer comprises the sequence of the upstream universal primer, and the length of the upstream universal primer is 16-20 bases.
4. Specific probes: which is the same as a partial fragment of the non-coding small RNA sequence to be detected; the number of the basic groups of the specific probe is 17-30, and the specific probe is a taqman probe or a taqman-MGB probe; any one of a quencher group BHQ1, BHQ2 or TAMRA is labeled at 3 'and a reporter group is labeled at 5' and labeled with a fluorescent group such as FAM, HEX/VIC, Texas-Red or CY 5.
5. A downstream universal primer: the sequence of the reverse transcription primer is the same as a part of the stem-loop sequence in the reverse transcription primer, namely the reverse transcription primer comprises the sequence of the downstream universal primer, namely the downstream universal primer is 16-25 bases on the stem-loop sequence, and the length of the downstream universal primer is 16-20 bases.
Example 2 primer set design example
Corresponding primer and probe design is carried out on miR-21-5p (nucleotide sequence is shown as SEQ ID NO. 1: UAGCUUAAUCAGACUGAUGUUGA) by the method of example 1.
As shown in Table 1, the 3 'end of the reverse transcription extension primer is designed to have a length of 15 bases, the underlined sequence is consistent with the 5' end sequence of the Has-miR-21-5p sequence (namely, a specific binding or complementary sequence), and the nucleotide sequence is shown as SEQ ID NO. 2. Meanwhile, an upstream universal primer, a downstream universal primer and a specific probe are respectively designed aiming at the amplified fragments, and the nucleotide sequences of the upstream universal primer, the downstream universal primer and the specific probe are respectively shown in SEQ ID NO. 3-6. The underlined sequence of the reverse transcription primer (i.e., the RT primer) is the region complementary to the 3' end of the sequence of interest.
TABLE 1 primer and Probe sequence Listing
Example 3 detection method for detecting non-coding small RNA by multiplex fluorescence quantitative PCR
Firstly, extracting sample non-coding small RNA
Non-coding small RNA of the sample was extracted using miRcute kit, and the concentration and purity of RNA (OD260/0D280) were determined by UV spectrophotometry.
II, reverse transcription
Performing reverse transcription by using a reverse transcription extension primer and a stem-loop reverse transcription primer, preparing a reverse transcription system in a PCR tube, and then putting the PCR tube in an ABI2720 PCR instrument, incubating for 5min at 25 ℃, incubating for 15min at 50 ℃, incubating for 5min at 85 ℃ and hold at 4 ℃. The reverse product was immediately subjected to qPCR reaction or stored at-20 ℃.
Reverse transcription system: 5 × RT buffer 4 μ L; dNTPs (10mM each) 1. mu.L; 1 mu L of reverse transcription extension primer (2 mu M) of each non-coding small RNA to be detected; 1 muL of each stem-loop reverse transcription primer (2 muM) of the non-coding small RNA to be detected; M-MLV (H-) RNA reverse transcriptase (200U/. mu.L) 1.5. mu.L; RNase inhibitor (40U/. mu.L) 1. mu.L; RNase free H2Make up to 20. mu.L of O.
3. Quantitative PCR
Using upstream universal primers, universal primers and specific probes to perform quantitative PCR, preparing a quantitative PCR system in a PCR tube, placing the PCR tube in a PCR amplification instrument, and performing quantitative reaction according to the following conditions: the reaction is carried out for 10min at 37 ℃, for 5min at 95 ℃, then for 10s at 95 ℃ and for 45s at 60 ℃ with the cycle number of 45 times.
Quantitative PCR system: 10 × Buffer (Mg)2+plus) 4. mu.L; dNTPs (containing dUTP,10Mm each)1 μ L; 1 μ L of upstream universal primer (10 μ M); 1 μ L of downstream universal primer (10 μ M); 1 mu L of each specific probe (10 mu M) of each non-coding small RNA to be detected; AceTaq DNA polymerase (5U/. mu.l) 0.5. mu.L; reverse transcription of 2. mu.L; ddH2O make up to 50. mu.L.
Example 4 fluorescent quantitative PCR detection of Has-miR-21-5p
Instrument
PCR instruments, e.g. ABI2720, fluorescent quantitative PCR instruments, e.g. ABI7500, etc
II, reagent
miRcute kit、M-MLV(H-)Reverse Transcriptase、RNase inhibitor、AceTaq DNA Polymerase，dNTPs、5×RT buffer、10×Buffer(Mg2+plus)。
Third, primer probe
When the method of the embodiment 3 is used for detecting Has-miR-21-5p, the primer combination of the embodiment 2 is used, and specifically:
the nucleotide sequence of the reverse transcription extension primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the reverse transcription primer is shown as SEQ ID NO. 3;
the nucleotide sequences of the upstream and downstream universal primers of qPCR are shown as SEQ ID NO.4 and SEQ ID NO. 5;
the nucleotide sequence of the specific probe is shown in SEQ ID NO. 6.
Fourth, experiment method
1. RNA extraction
The concentration and purity (OD) of RNA were determined by UV spectrophotometry using miRcute kit for plasma miRNA in collected blood samples260/OD280) And then used for subsequent detection.
2. Reverse transcription
A reverse transcription system was prepared in PCR tubes as in Table 2, and then the PCR tubes were placed in an ABI2720 PCR instrument and incubated at 25 ℃ for 5min, 50 ℃ for 15min, 85 ℃ for 5min, 4 ℃ hold. The reverse product was immediately subjected to qPCR reaction or stored at-20 ℃.
Table 2: reverse transcription reaction system
| Components | Addition amount (μ L) |
| RNase free H2O | up to 20 |
| 5×RT buffer | 4 |
| dNTPs(10mM each) | 1 |
| Reverse transcription extension primer (2. mu.M) | 1 |
| Stem loop reverse transcription primer (2. mu.M) | 1 |
| RNA Reverse Transcriptase (M-MLV (H-) Reverse Transcriptase) (200U/. mu.L) | 1 |
| RNase inhibitor (RNase inhibitor) (40U/. mu.L) | 1 |
| Total RNA or miRNA (10 pg-1 ug) \ | 1～11 |
| |
20 |
3. Quantitative detection by taqman probe method
A reaction system of fluorescent quantitative PCR (taqman probe method) was prepared in a PCR tube according to table 3, and then the PCR tube was placed in an ABI7500 fluorescent quantitative PCR instrument for qPCR reaction: incubate at 95 ℃ for 10min, then at 90 ℃ for 15s, 60 ℃ for 45s, cycle 45 reactions, and finally maintain at 4 ℃.
Table 3: qPCR reaction system
| Components | Addition amount (μ L) |
| ddH2O | 38 |
| 10×Buffer(Mg2+plus) | 4 |
| dNTPs (containing dUTP,10Mm each) | 1 |
| Upstream general primer F-U (10. mu.M) (SEQ ID NO.4) | 2 |
| Downstream general primer R-U (10. mu.M) (SEQ ID NO.5) | 2 |
| Specific probe miR21-2P ( |
0.5 |
| DNA Polymerase (AceTaq DNA Polymerase (5U/. mu.l)) | 0.5 |
| |
2 |
| Total volume | 50 |
Fifth, experimental results
FIG. 2, panel a, shows the results of quantitative determination of RT-qPCR in this example. Amplification curves normally follow typical sigmoidal curves.
Comparative example 1 fluorescent quantitative PCR detection of Has-miR-21-5p by ordinary stem-loop method
First, experiment method
Designing corresponding reverse transcription primers, upstream primers, downstream primers and probes for the has-miR-21-5p sequence according to a common stem-loop method.
The sequence of the stem-loop reverse transcription primer miR21-NF is as follows: GGTCGTATGCAAAGCAGGGTCCGAGGTATCCTTAGCACGCATCGCACTGCATACGACCTCAACA (SEQ ID NO.7)
The sequence of an upstream primer miR21-F is as follows: GCGTAGCTTATCAGACTGATGTTG (SEQ ID NO.8)
The sequence of a downstream primer miR21-R is as follows: GTCGTATCCAGTGCAGGG (SEQ ID NO.9)
The sequence of a specific probe miR21-P is as follows: AGTCGTATCCAGTGCGAATACCTCGGA (SEQ ID NO.10)
A reverse transcription system was prepared in PCR tubes as per Table 4, and then the PCR tubes were placed in an ABI2720 PCR instrument and incubated at 25 ℃ for 5min, 50 ℃ for 15min, 85 ℃ for 5min, 4 ℃ hold. The reverse product was immediately subjected to qPCR reaction or stored at-20 ℃.
Table 4: reverse transcription reaction system
| Components | Addition amount (μ L) |
| RNase free H2O | up to 20 |
| 5×RT buffer | 4 |
| dNTPs(10mM each) | 1 |
| Stem-loop reverse transcription primer miR21-NF (2 mu M) (SEQ ID NO.7) | 1 |
| RNA Reverse Transcriptase (M-MLV (H-) Reverse Transcriptase) (200U/. mu.L) | 1 |
| RNase inhibitor (RNase inhibitor) (40U/. mu.L) | 1 |
| Total RNA or miRNA (10 pg-1 ug) \ | 1～11 |
| |
20 |
A reaction system of fluorescent quantitative PCR (taqman probe method) was prepared in a PCR tube according to table 5, and then the PCR tube was placed in an ABI7500 fluorescent quantitative PCR instrument for qPCR reaction: incubate at 95 ℃ for 10min, then at 90 ℃ for 15s, 60 ℃ for 45s, cycle 45 reactions, and finally maintain at 4 ℃.
Table 5 general stem-loop method qPCR reaction system:
| components | Addition amount (μ L) |
| ddH2O | 38 |
| 10×Buffer(Mg2+plus) | 4 |
| dNTPs (containing dUTP,10Mm each) | 1 |
| Upstream primer miR21-F (10. mu.M) (SEQ ID NO.8) | 2 |
| Downstream primer miR21-R (10. mu.M) (SEQ ID NO.9) | 2 |
| Specific probe miR21-P ( |
0.5 |
| DNA Polymerase (AceTaq DNA Polymerase (5U/. mu.l)) | 0.5 |
| |
2 |
| Total volume | 50 |
Second, experimental results
The b picture in the figure 2 is the result of the fluorescence quantitative PCR detection of the common stem-loop method Has-miR-21-5p in the comparative example 1.
Table 6 comparison of example 4 with comparative example 1:
according to the detection results shown in the attached figure 2 and shown in the table 6, the CT values of the fluorescence quantitative PCR detection results of the example 4 and the comparative example 1 are basically consistent, which shows that the performance of the miRNA detected by the present invention is equivalent to that of the normal stem-loop method RT-qPCR, and the miRNA can be used for subsequent multiplex detection.
Example 5 sequencing validation of the Sanger Generation of the qPCR product
First, experiment method
The fluorescent quantitative PCR products of example 4 and comparative example 1 were sent to Biotech (Shanghai) Ltd for monoclonal, one-generation sequencing analysis, respectively.
Second, Experimental methods
The Sanger sequencing results of the PCR products of the fluorescent quantitative PCR detection of Has-miR-21-5p of the control example 1 are shown in a graph in FIG. 3; the Sanger sequencing results for the PCR quantitative products of RT-qPCR of example 4 are shown in panel b of figure 3.
Sequencing results show that the RT-qPCR products of the ordinary stem-loop method and the PCR products of the method are correct, and the RT-qPCR of the method in the embodiment 3 can effectively carry out quantitative detection on miRNA.
Example 6 multiplex fluorescent PCR detection of Has-miR-21-5p, Has-miR-143, RUN48
First, experiment method
Has-miR-21-5p (nucleotide sequence shown in SEQ ID NO. 1), Has-miR-143 (nucleotide sequence shown in SEQ ID NO.11, UGAGAUGAAGCACUGUAGCUCA) and internal reference RUN48 (nucleotide sequence shown in SEQ ID NO.12, AGUGAUGAUGACCCCAGGUAACUCUUGAGUGUGUCGCUGAUGCCAUCACCGCAGCGCUCUGACC) are selected as small non-coding RNA to be detected, primers are designed according to the method of the embodiment 1, and multiple PCR detection is carried out according to the method of the embodiment 3.
miRNA extraction from blood samples using the method of example 3, followed by 10-fold gradient dilution to 3 gradients (i.e., 10)-1、10-2、10-3) And carrying out multiple detection of miRNA.
Specifically, the reverse transcription extension primer, the stem-loop reverse transcription primer and the specific probe of the Has-miR-21-5p are respectively SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 6;
the reverse transcription extension primer, the reverse transcription primer and the specific probe of the Has-miR-143 are respectively shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO. 15;
the reverse transcription extension primer, the stem-loop reverse transcription primer and the specific probe of RUN48 are respectively shown as SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO. 18;
the sequences of the upstream and downstream universal primers are shown as SEQ ID NO.4 and SEQ ID NO.5,
specific sequences and their nucleotide modifications are shown in table 7.
Table 7:
the reverse transcription reaction system is shown in Table 8, the components are added into the PCR tube according to the table 8, the PCR tube is placed in a PCR amplification instrument, and the reverse transcription reaction is carried out according to the following conditions: the reaction is carried out for 5min at 25 ℃, 20min at 55 ℃ and 5min at 85 ℃.
Table 8: reverse transcription system
The quantitative system is shown in table 9, the components are added into the PCR tube according to table 9, the PCR tube is placed in the PCR amplificator, and the quantitative reaction is performed according to the following conditions: the reaction is carried out for 10min at 37 ℃, for 5min at 95 ℃, then for 10s at 95 ℃ and for 45s at 60 ℃ with the cycle number of 45 times.
Table 9: qPCR system
| Components | Volume (μ L) |
| ddH2O | 36 |
| 10×Buffer(Mg2+plus) | 4 |
| dNTPs (containing dUTP,10mM each) | 1 |
| F-U(10μM)(SEQ ID NO.4) | 2 |
| R-U(10μM)(SEQ ID NO.5) | 2 |
| miR21-2P(10μM)(SEQ ID NO.6) | 0.5 |
| miR142-2P(10μM)(SEQ ID NO.15) | 0.5 |
| RUN48-2P(10μM)(SEQ ID NO.18) | 0.5 |
| DNA Polymerase (AceTaq DNA Polymerase (5U/. mu.l)) | 0.5 |
| UDG enzyme (1U/. mu.l) | 1 |
| |
2 |
| Total volume | 50 |
Second, experimental results
The results of RT-qPCR multiple detection of Has-miR-21-5p, Has-miR-143 and RUN48 are shown in figure 4, wherein a is a quantification result of Has-miR-21-5p, b is a quantification result of Has-miR-143 and c is a quantification result of RUN48, and as can be seen from figure 4, the method can realize detection of a plurality of non-coding small RNAs by a single tube.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
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Claims (5)
1. A primer group for detecting non-coding small RNA by fluorescent quantitative PCR is characterized by comprising a reverse transcription extension primer, a reverse transcription primer, an upstream universal primer, a downstream universal primer and a specific probe;
the reverse transcription extension primer comprises the following components from the 5 'end to the 3' end in sequence: an upstream universal primer sequence and a specific binding sequence 1; the specific binding sequence 1 is the same as 6-15 basic groups of the 5' end of the small non-coding RNA to be detected; the sequence of the upstream universal primer is 16-25 bases; the 3' end of the reverse transcription extension primer cannot be extended continuously; the sequence of the upstream universal primer is not synonymous with or complementary to a non-coding small RNA sequence to be detected, and the number of basic groups of the upstream universal primer is 16-25;
the reverse transcription primer comprises the following components from the 5 'end to the 3' end in sequence: a stem-loop sequence and a specific binding sequence 2, wherein the specific binding sequence 2 is a reverse complementary sequence of 6-12 bases at the 3' end of the non-coding small RNA to be detected;
the sequence of the upstream universal primer is the same as that of the upstream universal primer in the reverse transcription extension primer;
the sequence of the downstream universal primer is the same as part of the stem-loop sequence in the reverse transcription primer, and the number of the basic groups of the downstream universal primer is 16-25;
the specific probe is the same as a part of fragments of the non-coding small RNA sequence to be detected, and the number of basic groups of the specific probe is 17-30.
2. A method for detecting non-coding small RNA by fluorescent quantitative PCR for non-disease treatment and diagnosis purposes is characterized by comprising the following steps:
s1, carrying out reverse transcription reaction on the reverse transcription extension primer, the reverse transcription primer and the small non-coding RNA to be detected in the claim 1 in the same system to obtain a reverse transcription product;
s2, performing fluorescent quantitative PCR reaction by using the reverse transcription product of the step S1 as a template and using the upstream universal primer, the downstream universal primer and the specific probe of claim 1.
3. The method of claim 2, wherein multiple fluorescent quantitative PCR is performed to detect a plurality of small non-coding RNAs, the 5' end of the specific probe is labeled with different luminescent groups, and the plurality of small non-coding RNAs is 2-5 small non-coding RNAs.
4. The method according to claim 2 or 3, wherein the final concentration of the reverse transcription primer, the reverse transcription extension primer, the upstream universal primer, the downstream universal primer and the specific probe in the system according to claim 1 is 50-200 nM.
5. The primer group of claim 1 is applied to the fluorescent quantitative PCR detection of non-coding small RNA for non-disease treatment and diagnosis purposes, or is applied to the preparation of a kit for the fluorescent quantitative PCR detection of non-coding small RNA.
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