An internal reference for RT-qPCR quantification and its preparation method and application

Abstract

The present invention provides an internal reference for RT-qPCR quantification, as well as its preparation and application, belonging to the field of genetic engineering technology. The internal reference is selected from at least one of miR-423-3p, miR-128-3p, miR-30d-5p, miR-339-3p, miR-625-3p, miR-140-3p, let-7g-5p, miR-148b-3p, miR-191-5p, and miR-24-3p. The present invention uses small RNA sequencing technology to quantify the expression of small extracellular vesicle miRNAs in the plasma of patients with pancreatitis and pancreatic cancer, and screens the miRNAs with the most stable expression across different individuals as candidate internal references. The stability of the selected candidate internal references is verified using small RNA sequencing technology in another independent group of patients with pancreatitis and pancreatic cancer.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to an internal reference for RT-qPCR quantification, a preparation method thereof, and an application thereof. Background Art

[0002] Pancreatic cancer is widely considered the king of cancers due to its difficulty in early diagnosis, low surgical resection rates, and high recurrence and metastasis after surgery. The median survival for untreated pancreatic cancer patients is only approximately four months, and most die within a year of diagnosis. This is because the vast majority of patients are diagnosed in the advanced stages, excluding surgical treatment options. The overall five-year survival rate is less than 10%. Currently, surgery is the only effective treatment for pancreatic cancer, and early diagnosis increases the survival rate to 60%. Therefore, establishing an effective screening mechanism and striving for early detection, diagnosis, and treatment are key to improving the prognosis of pancreatic cancer.

[0003] Extracellular vesicles (EVs), also known as exosomes, are tiny membrane vesicles secreted by most cells and serve as an important liquid biopsy medium. Their role in early cancer diagnosis, prognosis monitoring, and immunotherapy has garnered widespread attention and in-depth research. Exosomes contain a rich array of proteins, lipids, mRNA, lncRNA, and miRNA. Recent studies have demonstrated that exosomes secreted by tumor cells specifically overexpress miRNAs, which may be associated with tumor growth and metastasis. Several exosomal miRNAs have been implicated in pancreatic cancer. Xu et al. found that plasma miR-938 expression levels have diagnostic value in distinguishing pancreatic cancer from chronic pancreatitis and other pancreatic tumors, while miR-486-5p has diagnostic value in distinguishing pancreatic cancer from healthy controls or chronic pancreatitis (PMID: 26114496). Based on miRNA microarray analysis, Frampton et al. identified a biomarker panel in the blood that can distinguish pancreatic cancer patients from healthy controls. The panel included seven upregulated miRNAs (miR-21, miR-23a, miR-31, miR-100, miR-143, miR-155, and miR-221) and three downregulated miRNAs (miR-148a, miR-217, and miR-375). They also verified that overexpression of miR-21 and miR-31 and downregulation of miR-375 in tumor tissues were associated with lower overall survival in patients (PMID: 24575833). Multiple studies have shown that miRNA expression profiles can distinguish between cancer patients and non-cancer patients. Therefore, accurately quantifying miRNA expression levels in exosomes from different individuals is extremely important for improving the accuracy and reliability of biomarker research.

[0004] Real-time quantitative PCR is the most commonly used method for measuring miRNA expression levels. However, there is currently no standardized, stable internal reference for quantifying exosomal miRNAs across all sample types. Currently, researchers typically employ two methods for quantifying exosomal miRNAs: maintaining a consistent starting volume of biological fluids or incorporating synthetic, non-human miRNA sequences (known as the external reference method). However, both methods have significant drawbacks. They require high technical expertise and, even if the procedure is error-free, they cannot correct for inherent variability between individuals. External reference methods cannot even correct for exosome isolation and enrichment. The lack of a stable, endogenous reference for exosomal miRNAs significantly limits the potential for translation of biomarkers from clinical research to clinical application. Currently, the majority of exosomal miRNA studies in pancreatic cancer utilize classic tissue / cell internal references, such as U6. However, U6 is localized exclusively to the nucleus and not in the cytoplasm where RNA is loaded into exosomes. Therefore, the detection of U6 expression may be due to insufficient purity of the exosome isolation method. MiR-30a-5p and miR-30e-5p have been reported to be used as internal controls for the quantification of plasma exosomal miRNAs in pancreatic cancer. However, the stability of their expression in pancreatic cancer patient populations has not been verified. Blindly using unverified internal controls may lead to erroneous or even contrary conclusions.

[0005] Due to the difficulty in standardization in exosomal miRNA quantification studies, on the one hand, sequencing data has a poor agreement with RT-qPCR results, reducing the possibility of biomarker discovery, and on the other hand, it also limits the application of exosomal miRNA biomarkers from research to clinical practice. In Chinese patent application CN109536502A, based on a small number of samples, a miRNA stably expressed in the plasma exosomes of patients with gestational trophoblastic tumor (GTC) was identified. This miRNA was used as a candidate internal control and validated in another cohort, thereby identifying an internal control suitable for RT-qPCR quantification of exosomal miRNA in the plasma of patients with GTC. However, the sample size of this study was too small, making the analysis results difficult to represent the entire GTC patient population. Moreover, the internal control identified in this study cannot be directly used for RT-qPCR quantification of small extracellular vesicle miRNA in the plasma of pancreatic cancer patients. Currently, there are no reports on the use of internal controls for RT-qPCR quantification of small extracellular vesicle miRNA in the plasma of pancreatic cancer patients.

[0006] Based on this background, the present invention used plasma from patients with pancreatic cancer and pancreatitis as research samples. The researchers used the L3525 small extracellular vesicle (exosome) extraction reagent, independently developed by Shanghai Silidi Biomedical Technology Co., Ltd., to isolate plasma exosomes. They then used small RNA sequencing to quantify the expression of miRNAs in these plasma exosomes. They found that miR-423-3p had the most stable expression across individuals. Subsequently, validation in independent cohorts of patients with pancreatitis and pancreatic cancer confirmed that miR-423-3p had the most stability and high expression abundance among several candidate internal references, thereby identifying a suitable internal reference for quantitative analysis of small extracellular vesicle miRNAs in pancreatic cancer plasma by RT-qPCR. Summary of the Invention

[0007] The present invention aims to propose and validate an internal reference for RT-qPCR quantification of exosomal miRNA in pancreatic cancer patients, which can be used to correct experimental deviations between different samples, better standardize quantitative data, and improve the accuracy and reliability of exosomal miRNA as a biomarker for the diagnosis or treatment of pancreatic cancer.

[0008] The technical solution of the present invention is achieved as follows:

[0009] The present invention provides an internal reference for RT-qPCR quantification, wherein the internal reference is selected from at least one of miR-423-3p, miR-128-3p, miR-30d-5p, miR-339-3p, miR-625-3p, miR-140-3p, let-7g-5p, miR-148b-3p, miR-191-5p and miR-24-3p; the nucleotide sequence of the miR-423-3p is shown in SEQ ID NO.1; the nucleotide sequence of the miR-128-3p is shown in SEQ ID NO.2; the nucleotide sequence of the miR-30d-5p is shown in SEQ ID NO.3; the nucleotide sequence of the miR-339-3p is shown in SEQ ID NO.4; the nucleotide sequence of the miR-625-3p is shown in SEQ ID NO.5; the nucleotide sequence of the miR-140-3p is shown in SEQ ID NO. NO.6; the nucleotide sequence of the let-7g-5p is shown in SEQ ID NO.7; the nucleotide sequence of the miR-148b-3p is shown in SEQ ID NO.8; the nucleotide sequence of the miR-191-5p is shown in SEQ ID NO.9; the nucleotide sequence of the miR-24-3p is shown in SEQ ID NO.10.

[0010] As a further improvement of the present invention, the internal reference is miR-423-3p.

[0011] The present invention further protects the use of the above-mentioned internal reference gene in preparing a biomarker or kit for diagnosing chronic pancreatitis and pancreatic cancer for quantitative detection kit of plasma small extracellular vesicle miRNA RT-qPCR or a kit.

[0012] The present invention further protects a method for preparing the above-mentioned internal reference for RT-qPCR quantification, comprising the following steps:

[0013] S1. Extraction of plasma small extracellular vesicles;

[0014] S2. Extraction and expression detection of plasma small extracellular vesicle miRNAs;

[0015] S3. Sequencing, annotation, filtering, and expression normalization are performed on the extracted miRNAs to obtain the normalized miRNA expression levels;

[0016] S4: Screen miRNAs according to the screening criteria and select stable miRNAs as candidate internal controls to be verified;

[0017] S5. Perform stability verification on the candidate internal references to be verified and select the best one to be the final internal reference.

[0018] As a further improvement of the present invention, the method for extracting plasma small extracellular vesicles in step S1 includes: at least one of: reagent extraction method, ultracentrifugation extraction method, density gradient centrifugation extraction method, ultrafiltration centrifugation extraction method, and magnetic bead immunoextraction method.

[0019] As a further improvement of the present invention, the specific method for extracting plasma small extracellular vesicle miRNA in step S2 is: using a miRNeasy Serum / Plasma Kit to extract plasma small extracellular vesicle miRNA.

[0020] As a further improvement of the present invention, the screening criteria in step S4 are:

[0021] 1) The expression level is high in patients with chronic pancreatitis and pancreatic cancer, and the normalized expression level is greater than 8;

[0022] 2) The coefficient of variation is small and the expression is stable.

[0023] As a further improvement of the present invention, step S4 specifically includes:

[0024] S41: miRNAs with expression levels less than 8 after normalization were eliminated;

[0025] S42: Calculate the coefficient of variation of each remaining miRNA and sort the coefficients of variation from small to large;

[0026] S43: Select the miRNAs ranked in the top N by coefficient of variation, and perform stability analysis on the expression levels of the selected miRNAs;

[0027] S44: Select the top M miRNAs with the highest comprehensive stability as candidate internal controls to be verified;

[0028] Wherein, N and M are natural numbers, and N is greater than M.

[0029] As a further improvement of the present invention, the value of N is 60, and the value of M is 10.

[0030] As a further improvement of the present invention, the candidate internal references to be verified include miR-423-3p, miR-128-3p, miR-30d-5p, miR-339-3p, miR-625-3p, miR-140-3p, let-7g-5p, miR-148b-3p, miR-191-5p and miR-24-3p.

[0031] The present invention further protects a detection reagent comprising the above-mentioned internal reference for RT-qPCR quantification.

[0032] The present invention further protects a detection kit comprising the above-mentioned detection reagent.

[0033] The present invention further protects the use of the above detection kit in RT-qPCR quantification of chronic pancreatitis and pancreatic cancer.

[0034] The present invention has the following beneficial effects:

[0035] (1) Based on the data of second-generation sequencing, the present invention discovered and published for the first time 10 potential internal reference proteins, miR-423-3p, miR-128-3p, miR-30d-5p, miR-339-3p, miR-625-3p, miR-140-3p, let-7g-5p, miR-148b-3p, miR-191-5p, and miR-24-3p, that can be used for RT-qPCR quantification of small extracellular vesicle miRNAs in the plasma of pancreatic cancer patients.

[0036] (2) The present invention discovered and published for the first time a new internal reference miR-423-3p suitable for RT-qPCR quantification of plasma small extracellular vesicle miRNA in pancreatic cancer patients.

[0037] (3) The present invention uses small RNA sequencing technology to quantify the expression of plasma small extracellular vesicle miRNAs in patients with pancreatitis and pancreatic cancer, and screens the miRNAs with the most stable expression among different individuals as candidate internal controls; small RNA sequencing technology is used to verify the stability of the selected candidate internal controls in another independent group of pancreatitis and pancreatic cancer patients.

[0038] (4) The present invention uses another batch of independent research cohort data to verify that the internal reference miR-423-3p suitable for RT-qPCR quantification of plasma small extracellular vesicle miRNA in pancreatic cancer patients has high stability and can be used to correct the deviation in the experimental process between different samples, better standardize the quantitative data, and improve the accuracy and reliability of pancreatic cancer plasma small extracellular vesicle miRNA as a diagnostic or therapeutic biomarker.

[0039] (5) The present invention selects plasma from patients with chronic pancreatitis and pancreatic cancer as research samples, which is more consistent with the incidence of pancreatic disease patients in the real world. Therefore, the internal reference for RT-qPCR quantification of small extracellular vesicle miRNA in the plasma of pancreatic cancer patients screened by the present invention is more convincing and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0041] Figure 1 Transmission electron microscopy results for identification of small extracellular vesicles;

[0042] Figure 2 This is the result of NTA detection of small extracellular vesicles;

[0043] Figure 3 This is a comparison chart of comprehensive gene stability;

[0044] Figure 4 This is a comparison chart of gene stability analyzed using the △Ct method;

[0045] Figure 5 This is a comparison chart of gene stability analyzed using BestKeeper software;

[0046] Figure 6 This is a comparison chart of gene stability analyzed using normFinder software;

[0047] Figure 7This is a comparison chart of gene stability analyzed using Genorm software. DETAILED DESCRIPTION

[0048] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0049] Example 1

[0050] This embodiment provides a method for preparing an internal reference for RT-qPCR quantification, comprising the following steps:

[0051] (1) Plasma sample collection

[0052] The present invention included a total of 75 samples in the training and validation cohorts, including patients with chronic pancreatitis and pancreatic cancer. Blood samples were collected from patients before surgery or drug treatment into EDTA vacuum tubes (REF367863, BD, USA) and shipped at 4°C within one hour. After receiving the blood samples, plasma was separated using a two-step centrifugation method: the first step was centrifugation at 1600g for 10 minutes at 4°C. The color of the upper plasma layer after centrifugation determined the hemolysis level of the blood sample. Samples with hemolysis levels less than 4 were included in the present invention. The supernatant was transferred to a 1.5ml centrifuge tube and centrifuged at 16000g for 15 minutes at 4°C. The supernatant was then aliquoted into 1ml tubes and stored frozen at -80°C.

[0053] (2) Extraction and characterization of plasma small extracellular vesicles

[0054] 1) Extraction of plasma small extracellular vesicles

[0055] The plasma extracellular vesicles were extracted using the L-type exosome precipitation reagent independently developed by SiliDi (L3525, 3DMed, Shanghai, China). The specific steps are as follows: ① Take out the frozen plasma sample and incubate it at 37°C until it is completely thawed, and centrifuge it at 12000g and 4°C for 10 min; ② Transfer the supernatant to a 0.45μm tube filter (Costar, CLS8163-100EA, Corning, USA), centrifuge it at 12000g and 4°C for 5 min, and filter 500μl each time until all the samples are completely filtered; ③ Transfer the filtrate to a 0.22μm tube filter. ④ Transfer the filtrate to a 1.5 ml centrifuge tube and add 1 / 4 volume of L3525; ⑤ Vortex to mix and incubate at 4°C for 30 min; ⑥ Centrifuge at 4700 g for 30 min; ⑦ After centrifugation, discard the supernatant and add 200 μl PBS (phosphate buffer saline) to thoroughly resuspend the small extracellular vesicle pellet.

[0056] 2) Characteristics of plasma small extracellular vesicles

[0057] To characterize plasma small extracellular vesicles (EVs) from pancreatic cancer patients, this patent uses transmission electron microscopy (TEM) to examine EV morphology and nanoparticle tracking analysis (NTA) to characterize EV particle size. Morphological characterization of plasma EVs: EVs were first resuspended in PBS and fixed with 4% paraformaldehyde. The EVs were then transferred to a carbon-coated electron microscope copper grid. The grid was washed twice with PBS, then rinsed with PBS containing 50 mM glycine for 3 minutes, then washed with PBS containing 0.5% BSA for 10 minutes, and finally stained with 2% uranyl acetate. After staining, the morphological characteristics of the EVs were observed using a transmission electron microscope (H-7650, Hitachi High-Technologies, Japan).

[0058] Plasma EV NTA Assay: EVs were first diluted with PBS to 1 x 10^7-1 x 10^9 / ml and mixed thoroughly by pipetting. Subsequently, the diluted sample was injected into the sample chamber of an NTA instrument (NanoSight NS300, Malvern, UK). A 488 nm excitation module was used, and the camera lens parameters were set to a shutter value of 890, a gain of 146, and a detection threshold of 7. At least 200 complete tracks were analyzed and acquired for each video. Finally, the nanoparticle tracking data of EVs was analyzed using NTA Analysis Software (version 2.3).

[0059] (3) Extraction and expression detection of plasma small extracellular vesicle miRNA

[0060] 1) Extraction of plasma small extracellular vesicle miRNA

[0061] Plasma extracellular vesicle miRNAs were extracted using the miRNeasy Serum / Plasma Kit (217184, QIAGEN, Shanghai, China). For detailed procedures, refer to the product manual. MiRNA quantification and fragment distribution were performed using an Agilent 2100 Bioanalyzer chip (5067-1548, Agilent, USA).

[0062] 2) Expression detection of plasma small extracellular vesicle miRNA

[0063] This patent uses the NEBNext, Multiplex Small RNA Library Prep Set for Illumina (E7300L, NEB, USA) kit for library construction and small RNA sequencing to detect the expression level of plasma small extracellular vesicle miRNA. The operation process refers to the product manual. The general process is to load 100ng of sample for each sample library construction, with a volume of no more than 6μl, and then perform 3' adapter ligation, reverse transcription primer hybridization, 5' adapter ligation, reverse transcription and PCR amplification respectively. Finally, the PCR product is purified using the NucleoSpin Gel and PCR Clean-up (740609.50, MACHEREY-NAGEL, Germany) kit, and the library DNA is eluted with 30μl enzyme-free water. The library quality control was performed using a GX Touch™ HT nucleic acid analyzer and its associated chips (CLS138948, PerkinElmer, USA) and reagents (CLS760672, PerkinElmer, USA). 20–25 libraries were mixed in equal molar ratios and sequenced in lanes using the Illumina HiSeq PE150 sequencing platform.

[0064] (4) Sequencing data analysis process

[0065] 1) Sequencing data alignment. After removing the sequencing adapters from the small RNA sequencing data, the sequencing data were aligned to the human reference genome hg19 (genome download link: http: / / hgdownload.soe.ucsc.edu / goldenPath / hg19 / bigZips / ) using BWA software (version: 0.7.12-r1039), and the number of reads aligned to the miRNA was counted.

[0066] 2) miRNA annotation. MiRNAs were annotated using the Gencode v25 and miRBase v21 databases, and those annotated as known mature miRNAs were retained for subsequent analysis.

[0067] 3) miRNA filtering: Mature miRNAs with a length of 30 nt or less and covered by at least 2 reads per sample were retained for subsequent analysis.

[0068] 4) Normalization of miRNA expression levels. The trimmed mean of M-values ​​(TMM) method and the limma-voom method in the limma analysis package in R were used to normalize miRNA expression levels in the training cohort and validation cohort samples, respectively.

[0069] (5) Screening of candidate internal references

[0070] Based on small RNA sequencing technology, the expression levels of miRNAs in the patient's peripheral blood extracellular vesicles were obtained, and candidate internal controls were screened according to the following principles:

[0071] 1) The expression level is high in patients with chronic pancreatitis and pancreatic cancer, and the normalized expression level is required to be greater than 8;

[0072] 2) Statistical analysis was performed based on the coefficient of variation (CV) of miRNA expression (standard deviation / mean). The top 60 miRNAs ranked by CV value from smallest to largest were selected for stability analysis. Gene stability analysis was performed using the comprehensive online analysis and evaluation software RefFinder, which integrates four internal reference stability analysis methods: geNorm (Vandesompele et al., 2002), NormFinder (Andersen et al., 2004), BestKeeper (Pfaffl et al., 2004), and Delta Ct (Silver et al., 2006).

[0073] (6) Stability verification of candidate internal references

[0074] The copy number of the validation cohort samples detected against the candidate internal reference is imported into the online stability evaluation software RefFinder. The software will give a stability value for the copy number of each miRNA molecule in different samples. The smaller the value, the more stable the expression of the miRNA molecule.

[0075] Example 2

[0076] (1) Study cohort and clinical information

[0077] The training cohort consisted of 52 patients, including 22 with chronic pancreatitis and 30 with pancreatic cancer. The validation cohort consisted of 23 patients, including 9 with chronic pancreatitis and 14 with pancreatic cancer. Table 1 shows the clinical information of the cohorts. Analysis results showed no significant difference in the proportion of patients with chronic pancreatitis or pancreatic cancer between the two cohorts.

[0078] Table 1 Clinical information of patients

[0079]

[0080] (2) Extraction and characterization of plasma small extracellular vesicles

[0081] The present invention uses the small extracellular vesicle (exosome) extraction reagent L3525 independently developed by Shanghai Silidi Biomedical Technology Co., Ltd. to extract small extracellular vesicles from the plasma of pancreatic cancer patients. In order to detect the characteristics of small extracellular vesicles in the plasma of pancreatic cancer patients, the present invention uses transmission electron microscopy to detect the morphology of small extracellular vesicles and uses NTA (Nanoparticle Tracking Analysis) technology to detect the particle size characteristics of small extracellular vesicles. The transmission electron microscopy results show that the small extracellular vesicles have a typical "horseshoe-shaped" morphology (see Figure 1 The NTA test results showed that the average particle size of small extracellular vesicles in the representative samples extracted by this patent was 131.7nm, which is consistent with the particle size distribution of small extracellular vesicles (see Figure 2).

[0082] (3) Screening of candidate internal references

[0083] In the next-generation sequencing data, small extracellular vesicle miRNAs with high expression levels, low coefficient of variation, and stable expression in both pancreatitis and pancreatic cancer patients were selected as candidate internal control miRNAs. First, miRNAs with log2RPM values ​​<8 were eliminated. Then, the coefficient of variation (= standard deviation / mean) for each miRNA was calculated and ranked from smallest to largest (Table 3). The expression levels of the top 60 miRNAs were imported into the online stability evaluation software RefFinder for analysis. The analysis results are shown in Table 2. The top 10 miRNAs with the highest comprehensive stability (miR-423-3p, miR-128-3p, miR-30d-5p, miR-339-3p, miR-625-3p, miR-140-3p, let-7g-5p, miR-148b-3p, miR-191-5p, and miR-24-3p) were selected as candidate internal control miRNAs to be verified.

[0084] (4) Stability verification of candidate internal references

[0085] To verify the expression stability of the above 10 candidate EV miRNA reference genes in patients with pancreatitis and pancreatic cancer, another independent sample set was selected as a validation cohort. Small RNA sequencing was also performed on plasma EVs from 9 patients with chronic pancreatitis and 14 patients with pancreatic cancer. The copy numbers obtained for each sample against the 10 candidate reference genes were imported into the online stability evaluation software RefFinder. The results are shown in Figure 3. Figure 3-Figure 7 Delta Ct, Bestkeeper and NormFinder analysis methods all indicated that miR-423-3p was the most stable candidate internal reference molecule ( Figure 3-6 ), which is completely consistent with the analysis results of the second-generation sequencing data of the discovery cohort, indicating that miR-423-3p has a high stability as an internal reference and can be used to correct the deviations in the experimental process between different samples, thereby improving the accuracy and reliability of pancreatic cancer exosomal miRNA as a diagnostic biomarker.

[0086] The nucleotide sequences of the internal references are shown in Table 2 below:

[0087] Table 2 Candidate miRNA internal reference molecule sequences

[0088]

[0089]

[0090] Table 3 Stability analysis results of four algorithms

[0091]

[0092]

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing an internal reference for RT-qPCR quantification, characterized in that: The internal reference for RT-qPCR quantification is used in RT-qPCR quantification of chronic pancreatitis and pancreatic cancer; the method comprises the following steps: S1. Extraction of plasma small extracellular vesicles; wherein the source sample of the plasma small extracellular vesicles is a plasma sample from a patient with chronic pancreatitis or pancreatic cancer; S2. Extraction and expression detection of plasma small extracellular vesicle miRNAs; S3. Sequencing, annotation, filtering, and expression normalization are performed on the extracted miRNAs to obtain the normalized miRNA expression levels; S4: Screening miRNAs according to the screening criteria to select stable miRNAs as candidate internal controls to be verified; wherein the screening criteria are: The expression level was high in patients with chronic pancreatitis and pancreatic cancer, with the normalized expression level greater than 8; 2) Small coefficient of variation and stable expression; S5. Perform stability verification on the candidate internal references to be verified and select the best final internal reference; The candidate internal references to be verified include miR-423-3p, miR-128-3p, miR-30d-5p, miR-339-3p, miR-625-3p, miR-140-3p, let-7g-5p, miR-148b-3p, miR-191-5p and miR-24-3p; The final internal reference is miR-423-3p, the nucleotide sequence of which is shown in SEQ ID NO.1; Wherein, step S4 specifically includes: S41: miRNAs with expression levels less than 8 after normalization were eliminated; S42: Calculate the coefficient of variation of each remaining miRNA and sort the coefficients of variation from small to large; S43: Select the miRNAs ranked in the top N by coefficient of variation, and perform stability analysis on the expression levels of the selected miRNAs; S44: Select the top M miRNAs with the highest comprehensive stability as candidate internal controls to be verified; Wherein, N and M are natural numbers, and N is greater than M.

2. The preparation method according to claim 1, characterized in that The method for extracting plasma small extracellular vesicles in step S1 includes at least one of: reagent extraction method, ultracentrifugation extraction method, density gradient centrifugation extraction method, ultrafiltration centrifugation extraction method, and magnetic bead immunoextraction method.

3. The preparation method according to claim 1, characterized in that The specific method for extracting plasma small extracellular vesicle miRNA in step S2 is: using the miRNeasy Serum / Plasma Kit to extract plasma small extracellular vesicle miRNA.

4. The preparation method according to claim 1, characterized in that The value of N is 60, and the value of M is 10.

5. Use of an internal reference for RT-qPCR quantification in the preparation of a detection reagent for chronic pancreatitis and pancreatic cancer, characterized in that: The internal reference for RT-qPCR quantification was miR-423-3p, whose nucleotide sequence is shown in SEQ ID NO.

1.

6. Use of an internal reference for RT-qPCR quantification in the preparation of a detection kit for chronic pancreatitis and pancreatic cancer, characterized in that: The internal reference for RT-qPCR quantification was miR-423-3p, whose nucleotide sequence is shown in SEQ ID NO.1.

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