Stem-loop method RT-qPCR kit for efficiently detecting exosome source microRNA

Abstract

The invention provides a method for detecting plasma exosome source miRNA (micro Ribonucleic Acid) and a detection kit. According to the invention, a Taqman probe real-time fluorescent quantitative PCR (Polymerase Chain Reaction) system is established to overcome the difficult-to-detect characteristics of low plasma content, short miRNAs sequence, high homology and the like by virtue of a method for identifying miRNA by screening different detection systems and designing specific primer probes through systematic research and repeated use of different samples for verification; the method and the kit provided by the invention can be used for screening and identifying an early biomarker miRNA, and have the advantages of wide detection spectrum, high sensitivity, good specificity, low detection cost, convenience in material taking and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical testing, and is mainly a detection technology for plasma exosome miRNA. Specifically, it relates to a stem-loop primer, a qPCR primer probe design method and a detection method for detecting EV-derived biomarker miRNA. Background Art

[0002] Extracellular vesicles (EV) are a general term for various membrane-structured vesicles released by cells. They have a nanoscale lipid bilayer membrane structure and are involved in the process of transporting proteins, lipids, RNA and DNA fragments. They are widely found in serum, plasma, urine, saliva, cerebrospinal fluid and breast milk. As key messengers of intercellular communication, exosomes deliver bioactive molecules to recipient cells, thereby participating in a variety of pathological and physiological processes, including tumors, cardiovascular diseases and neurodegenerative diseases. Exosomes contain a variety of small non-coding RNA species, among which microRNAs (miRNAs) are a type of small non-coding RNA (sncRNA) that is usually expressed in all eukaryotic cells. They regulate post-transcriptional gene expression by inhibiting specific target mRNAs and can be used as ideal biomarkers for diagnosing various diseases. The EV membrane effectively prevents the degradation of miRNA by ribonucleases that are abundant in biological fluids. Compared with free miRNA in plasma that is not coated by the EV membrane, miRNA transported by EV is more reliable as a biomarker of disease, making it more potential as a biomarker for diagnosis, treatment and prognosis.

[0003] Li et al. performed NGS sequencing and analysis on EV-derived miRNAs from normal people and patients with Parkinson's disease, and found 15 miRNAs that differed between normal people and patients with PD. Previous studies have reported that salivary miR-153 and miR-223 can be used as biomarkers for idiopathic PD, while serum miR-221 is a potential predictor of PD. In addition, miRNAs enriched in brain circulation have been used to distinguish between idiopathic and hereditary PD. Therefore, EV-derived miRNAs may be candidate biomarkers for PD, and accurately quantifying the expression levels of miRNAs in exosomes from different individuals is extremely important for improving the accuracy and reliability of biomarker research.

[0004] However, due to the lack of gold standard methods, the clinical application of miRNA is still limited to the diagnosis of a few diseases. At present, the methods used to detect exosomal miRNA mainly include Northern blot, microarray, NGS and RT-qPCR. The advantage of NGS sequencing is that it can discover new miRNAs or find differentially expressed miRNAs related to diseases. It is suitable for high-throughput miRNA analysis, but the detection cost is high and the cycle is long, which is not suitable for routine detection. Among them, RT-qPCR is currently the most commonly used detection method for detecting miRNA. It has a small sample input, fast detection time and significantly improves the dynamic range and sensitivity. It is also often used to verify the results of miRNA expression profiles identified and screened by the first two methods. Therefore, for the field of diagnosis, RT-qPCR is still the most potential, convenient and effective method. A major challenge for miRNA detection is the impact of its short sequence length (about 22nt) on its detection accuracy. Its extremely short sequence length makes it impossible to detect it using conventional RT-qPCR methods, and it is necessary to artificially design and extend the length of the miRNA sequence to be tested as a template for subsequent qPCR detection. Therefore, it is necessary to further develop a technology that can provide high-quality miRNA sample extraction technology, as well as to develop an efficient and sensitive miRNA qPCR detection method. Summary of the invention

[0005] In view of this, the present invention provides a method for detecting plasma exosome miRNA, aiming to provide a universal stem-loop primer, a specific primer probe, a kit and its application for detecting plasma exosome miRNA, so as to solve the problem of low reverse transcription and detection efficiency of miRNA stem-loop method.

[0006] One of the purposes of the present invention is to provide a method and a detection kit for detecting plasma exosome miRNA. By improving the conventional RT-qPCR technology, designing corresponding primer probe sets for each target miRNA, screening different miRNA extraction systems, reverse transcription systems and qPCR systems, the sensitivity and amplification efficiency of miRNA detection are improved, and low-abundance miRNA can be quantitatively detected by qPCR, laying a solid foundation for the application of exosome miRNA in clinical diagnosis, biomedical research and other aspects.

[0007] Reverse transcription stem-loop primers, qPCR primers and probe sequences were designed for three target miRNAs with different expression abundances: miR-191-5p>miR-30d-5p>miR-222-3p.

[0008] Target miRNA: Primer probe sequence of miR-30d-5p

[0009]

[0010] Target miRNA: Primer probe sequences of miR-222-3p

[0011]

[0012] Target miRNA: Primer probe sequences of miR-191-5p

[0013]

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) High specificity: By designing specific primers for each target miRNA, including stem-loop primers and specific MGB probes, high specificity of the reverse transcription process and qPCR detection process is ensured.

[0016] (2) Fast detection speed: Compared with the NGS library construction and sequencing process, the detection time is greatly shortened

[0017] (3) Intuitive interpretation of results: The expression levels of different targets can be judged by the CT value

[0018] (4) Wide applicability: Exosomes are widely found in serum, plasma, urine, saliva, cerebrospinal fluid, and breast milk, so the sample types tested are not limited to exosomes in plasma samples but can be diverse.

[0019] (5) The required PCR instrument and PCR system are essential for ordinary PCR laboratories, with low cost and simple and fast operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :C1-CNS, C1 first extraction kit Magen HiPure Exosome RNAKit extracts plasma samples (CNS) from patients with brain tumors as templates to detect the qPCR curve of target miRNA (miR-30d-5p) (Patent Table 11. qPCR test results of different extraction systems C1-CNS);

[0021] Figure 2 :C1-HP, C1-CP: C1 first extraction kit Magen HiPure Exosome RNAKit extracts healthy human plasma (HP), and blood disease patient plasma (CP) is used as a template to detect the qPCR curve of the target miRNA (miR-30d-5p) (Patent Table 11. qPCR test results of different extraction systems C1-HP, C1-CP);

[0022] Figure 3:C2 second extraction kit Qiagen exoRNeasy Midi Kit extracts healthy human plasma (HP), blood disease patient plasma and brain tumor patient plasma samples (CNS) as templates to detect the qPCR curve of target miRNA (miR-30d-5p) (Patent Table 11. qPCR test results of different extraction systems C2-HP, C2-CP, C2-CNS);

[0023] Figure 4 :C3 third extraction kit TIANGEN miRcute Serum / Plasma miRNA IsolationKit extracts healthy human plasma (HP), blood disease patient plasma and brain tumor patient plasma samples (CNS) as templates to detect the qPCR curve of target miRNA (miR-30d-5p) (Patent Table 11. qPCR test results of different extraction systems C3-HP, C3-CP, C3-CNS);

[0024] Figure 5 : R1, qPCR curves of the expression levels of three target miRNAs (miR-30d-5p, miR-222-3p, miR-191-5p) under RT reaction system 1 (patent Table 14. qPCR detection results of different reverse transcription systems R1);

[0025] Figure 6 : R2, qPCR curves of the expression levels of three target miRNAs (miR-30d-5p, miR-222-3p, miR-191-5p) under RT reaction system 2 (patent Table 14. qPCR detection results of different reverse transcription systems R2);

[0026] Figure 7 : R3, qPCR curves of the expression levels of three target miRNAs (miR-30d-5p, miR-222-3p, miR-191-5p) under RT reaction system 3 (patent Table 14. qPCR detection results of different reverse transcription systems R3);

[0027] Figure 8 : Q1, qPCR curves of the expression levels of three target miRNAs (miR-30d-5p, miR-222-3p, miR-191-5p) under qPCR system 1 (Patent Table 18. Detection results of different qPCR systems Q1);

[0028] Fig. 9 : Q2, qPCR curves of the expression levels of three target miRNAs (miR-30d-5p, miR-222-3p, and miR-191-5p) under qPCR system 2 (Patent Table 18. Detection results of different qPCR systems Q2);

[0029] Fig.10 : Q3, qPCR curves of the expression levels of three target miRNAs (miR-30d-5p, miR-222-3p, miR-191-5p) under qPCR system 3 (Patent Table 18. Detection results of different qPCR systems Q3);

[0030] Fig.11 :Q4-30d: qPCR curve of the expression of target miRNA (miR-30d-5p) under qPCR system 4 (Patent Table 18. Detection results of different qPCR systems Q4: miR-30d-5p);

[0031] Fig.12 : Q4-191, 222: qPCR curves of the expression levels of target miRNAs (miR-222-3p, miR-191-5p) under qPCR system 4 (Patent Table Table 18. Detection results of different qPCR systems Q4: miR-222-3p, miR-191-5p). DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention 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 invention and are not intended to limit the present invention. The reagents not described separately in detail in the present invention are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0033] Example 1

[0034] Reverse transcription stem-loop primers, qPCR primers and probe sequences were designed for three target miRNAs with different expression abundances: miR-191-5p>miR-30d-5p>miR-222-3p.

[0035] Table 1. Primer and probe sequences of target miRNA: miR-30d-5p

[0036]

[0037] Table 2. Primer and probe sequences of target miRNA: miR-222-3p

[0038]

[0039] Table 3. Primer and probe sequences of target miRNA: miR-191-5p

[0040]

[0041]

[0042] Embodiment 2:

[0043] Targeting three miRNAs (miR-191-5p, miR-30d-5p, miR-222-3p),

[0044] A total of three types of samples were selected for extraction system screening tests, namely healthy human plasma (HP), blood disease patient plasma (CP), and brain tumor patient plasma samples (CNS).

[0045] 1. Plasma Sample Collection

[0046] Blood was collected in EDTA anticoagulation tubes, and the blood samples were centrifuged at 4°C, 2000g for 20 min to separate plasma. The supernatant was aspirated into a new 1.5ml centrifuge tube, and centrifuged at 4°C, 16000×g for 20 min. The supernatant was aspirated and divided into 1.5ml centrifuge tubes at 1ml per tube. Samples with hemolysis level less than 4 were used for subsequent research, and plasma was frozen at -80°C.

[0047] Extraction system:

[0048] Table 4: Extraction system kit

[0049]

[0050] Using Taqman probe qPCR technology, the specific process of RT-qPCR reaction is divided into two steps:

[0051] Table 5. RT reaction system 1

[0052] Reagents Volume (10ul) 5×gDNAWiperMix 2ul Total RNA 2ul RNase-free ddH2O 6ul

[0053] After the reaction system is mixed, the genomic DNA removal reaction is carried out at 42°C for 2 minutes.

[0054] Table 6. RT reaction system 2

[0055] Reagents Volume (20ul) The mixture from the previous step 10μl Stem-loop primer (2 μM) 1μl 10×RTMix 2μl HiScriptIIEnzymeMix 2μl Nucleasefreewater 5μl

[0056] Table 7. RT reaction conditions

[0057] temperature time 25℃ 5min 52℃ 15min 85℃ 5min

[0058] 3.2qPCR reaction system and conditions:

[0059] Table 8. qPCR reaction system

[0060] Reagents Volume (20ul) Forward Primer (10 μM) 0.5ul Reverse Primer (10 μM) 0.5ul Specific Probe (10 μM) 0.5ul TemplateDNA 2ul 4×qPCRProbeMasterMix 5ul Nucleasefreewater 11.5ul

[0061] Table 9. qPCR reaction conditions

[0062]

[0063] The primer and probe sequences used for the three target miRNAs refer to Example 1.

[0064] After the PCR reaction was completed, the original experimental file was exported, and the single-plex detection effect of the three target miRNAs was analyzed with a fixed threshold of 0.05 and automatic baseline on the Jindian MC-1000 analysis software.

[0065] Experimental results and analysis:

[0066] Table 10. Results of different extraction systems

[0067]

[0068] Table 11. qPCR test results of different extraction systems

[0069]

[0070] Conclusion: ① As shown in Table 10, RNA concentrations were measured for the three types of samples tested by the three extraction kits. The RNA yields of C2 and C3 were relatively uniform. The RNA yields of C1 varied according to the sample type. The plasma of healthy subjects (HP) > plasma samples of brain tumor patients (CNS) > plasma of patients with blood diseases (CP). Healthy human plasma was selected as the test sample.

[0071] ② As shown in Table 11, the same target miRNA (miR-30d-5p) was detected for different sample types of products from the three extraction kits, and their CT values ​​were relatively consistent (CV < 2%). The CT values ​​of the C1 extraction products were all lower, indicating that the target miRNA content of the C1 extraction product accounted for the highest proportion, and C1 was selected as the extraction system.

[0072] Example 3

[0073] Referring to the results of Example 2, different reverse transcription systems were screened.

[0074] Reverse transcription system 1: R1 refers to the reverse transcription system and conditions in Example 1

[0075] Table 12. Reverse transcription system and conditions 2: R2

[0076]

[0077] Table 13. Reverse transcription system and conditions 3: R3

[0078]

[0079] The qPCR reaction system and conditions refer to Example 2. The result derivation refers to Example 2.

[0080] Table 14. qPCR test results of different reverse transcription systems

[0081]

[0082]

[0083] Conclusion: As shown in Table 14, the expression levels of three target miRNAs (miR-30d-5p, miR-222-3p, and miR-191-5p) under three reverse transcription systems were tested. The target miRNAs of the three reverse transcription systems were all detected, and the CT values ​​of R1 and R3 were relatively consistent (CV < 0.5%). The trend of the CT value detected by R1 was consistent with the expression level: miR-191-5p > miR-30d-5p > miR-222-3p. R1 was selected as the reverse transcription system.

[0084] Embodiment 4:

[0085] Referring to the results of Example 3, different qPCR systems were screened.

[0086] qPCR system 1: Q1 refers to the qPCR system and conditions in Example 1

[0087] Table 15. qPCR system and conditions 2: Q2

[0088]

[0089]

[0090] Table 16. qPCR system and conditions 3: Q3

[0091]

[0092] Table 17. qPCR system and condition 4: Q4

[0093]

[0094]

[0095] The results were derived with reference to Example 2.

[0096] Table 18. Detection results of different qPCR systems

[0097]

[0098] Conclusion: As shown in Table 18, the expression levels of three target miRNAs (miR-30d-5p, miR-222-3p, and miR-191-5p) were tested under three qPCR systems. All target miRNAs of the three qPCR systems were detected, and their CT values ​​were relatively consistent (CV < 2%). The CT values ​​detected by Q1 and Q2 were lower, while the detection results of Q1 were more stable (CV < 0.5%). Q1 was selected as the qPCR system.

[0099] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention 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 by the present invention should be covered within the protection scope of the present invention.

Claims

1. A method and a detection kit for detecting plasma exosome miRNA, characterized in that: By improving the conventional RT-qPCR technology, corresponding primer probe sets were designed for each target miRNA, and different miRNA extraction systems, reverse transcription systems and qPCR systems were screened to improve the sensitivity and amplification efficiency of miRNA detection.

2. The kit according to claim 1, characterized in that It targets three target miRNAs, namely miR-191-5p, miR-30d-5p, and miR-222-3p.

3. The kit according to claim 2, characterized in that The reaction system of miR-30d-5p is:

4. The kit according to claim 2, characterized in that The reaction system of miR-222-3p is:

5. The kit according to claim 2, characterized in that The reaction system of miR-191-5p is:

6. Use of the kit according to any one of claims 1 to 5.

Images