CN113604536B - Preparation method of quality control product for microRNA detection - Google Patents
Preparation method of quality control product for microRNA detection Download PDFInfo
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- CN113604536B CN113604536B CN202110684843.8A CN202110684843A CN113604536B CN 113604536 B CN113604536 B CN 113604536B CN 202110684843 A CN202110684843 A CN 202110684843A CN 113604536 B CN113604536 B CN 113604536B
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Abstract
The invention discloses a preparation method of a quality control product for microRNA detection, which comprises the following steps of (1) enriching cells; (2) sample simulation matrix dilution; (3) microRNA release; (4) impurity removal. The quality control product can carry out all-round quality control on microRNA detection, has simple whole preparation flow, low cost, easy storage and transportation and good application prospect.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a preparation method of a quality control product for microRNA detection.
Background
micrornas are a class of single-stranded non-coding RNA molecules of about 19-25 nucleotides in length, which are typically targeted to one or more mrnas, regulating gene expression by inhibiting the translation process of the target gene. Existing studies have shown that common cancers have altered expression of micrornas, and that micrornas are normally located in genomic regions associated with cancers, it is speculated that micrornas may play a dual role in both Cancer suppressor and oncogene (esque-Kerscher, a. And slot, f. J (2006) Nat Rev Cancer 6, 259-269; calin, g.a. and Croce, c.m. (2007) J Clin Invest 117, 2059-2066; blenkire, c.and Miska, e.a. (2007) Hum Mol Genet 16, R106-R113). Mature micrornas will constitute miRNA-protein complexes with other proteins, so they are very stable in vitro (Lu, j. Et al, (2005) Nature 435, 834-838; lim, l.p. et al, (2005) Nature 433, 769-773), have a greater advantage as tumor biomarkers than mRNA, and the demonstrated abnormal expression of micrornas in cancer highlights their potential use as diagnostic and prognostic biomarkers. Therefore, accurate, efficient and precise detection of microRNAs is an important measure for current research and subsequent product development.
Real-time fluorescent quantitative RT-PCR has become an important method for detecting microRNA by virtue of the advantages of high sensitivity, high specificity, good repeatability and the like. However, the whole RT-PCR detection process is complicated and is divided into the steps of nucleic acid extraction and purification, reverse transcription product amplification and the like, so that the RT-PCR detection process is easily influenced by various factors including sample extraction and detection artifacts. Therefore, a strict quality control system is very necessary to be constructed in the detection process, and the condition for constructing the quality control system is that quality control products meeting the condition are introduced to evaluate the whole detection process, so that the method has important significance on the accuracy of microRNA detection results.
At present, quality control products for detecting microRNAs generally use artificial synthetic genes, and the artificial synthetic genes are directly used for subsequent reverse transcription and amplification experiments after being diluted and prepared into the quality control products.
The use of synthetic genes has the following problems:
1. because microRNA sequences are shorter and usually consist of 19-25 nucleotides, and the synthesized genes are not miRNA-protein complexes, the protein protection is avoided, the degradation is easy, and the stability is not good.
2. The quality control of the extraction link cannot be ensured without participating in the extraction process. Even if artificial synthetic genes are used to participate in the extraction link, the sample detection flow cannot be accurately simulated due to the difference of structures of sample matrixes and microRNAs.
Disclosure of Invention
The invention aims to provide a preparation method of a quality control product for microRNA detection, which can carry out omnibearing quality control on microRNA detection, has simple whole preparation flow, low cost, easy storage and transportation and good application prospect.
The technical scheme adopted for solving the technical problems is as follows:
a preparation method of a quality control product for microRNA detection comprises the following steps:
(1) Cell enrichment: centrifuging the counted cells, and discarding the supernatant to obtain precipitated cells;
(2) Sample simulation matrix dilution: diluting the precipitated cells from step (1) with a sample-mimicking matrix;
(3) microRNA release: treating the diluted cells in the step (2) by physical means to break the cells and release microRNA in the cells;
(4) Impurity removal: and (3) freezing and thawing the cells crushed in the step (3) or storing at room temperature to degrade unstable microRNA, centrifuging to remove impurities, and taking the supernatant as a quality control product.
In the step (1), the cells are cells containing the detection target microRNA. Preferably, the cells containing the target microRNA are selected from one or more of human kidney epithelial cell lines (293T cells), CHO-K1 cells and NIH-3T3 cells.
In the step (2), the sample simulation matrix is an artificial synthetic or natural matrix which does not contain detection target microRNA. Preferably, the sample simulation matrix is selected from one or more of artificial serum, artificial urine, artificial saliva and fetal bovine serum.
Preferably, in the step (3), the physical means is selected from one or more of freeze thawing, mechanical crushing and ultrasonic crushing.
Preferably, in the step (3), the physical means is freeze thawing, the number of times of freeze thawing is 1-3, and the freeze thawing temperature is-10 ℃ to-85 ℃.
Preferably, in the step (4), the number of times of freeze thawing is 1 to 5, and the freeze thawing temperature is-10 ℃ to-85 ℃.
Preferably, in the step (4), the room temperature storage time is 12-72 hours.
The quality control product for microRNA detection is prepared by adopting the preparation method.
The beneficial effects of the invention are as follows: the quality control product can participate in all links of microRNA detection, and can strictly control the quality of the whole detection process. The substrate of the quality control product is a simulated sample substrate, so that the possible matrix effect can be avoided, microRNAs in the substrate are of miRNA-protein complex structures, the substrate can be stably stored, the substrate is consistent with the existing structure of microRNAs in a sample, and the sample extraction-detection process can be accurately simulated.
Drawings
FIG. 1 is a comparison of stability studies of quality control prepared according to the present invention and quality control formulated with synthetic genes.
FIG. 2 is a linear relationship between the concentration of a quality control target gene (expressed as CT value) and the number of cells.
FIG. 3 shows quality control results of quality control samples obtained by detecting actual samples using quality control products prepared by the present invention and quality control products prepared by artificially synthesized genes.
Detailed Description
The technical scheme of the invention is further specifically described by the following specific examples.
In the present invention, the materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
Example 1:
a preparation method of a quality control product for microRNA detection comprises the following steps:
step 1, cell enrichment: one tube 293T cell (1X 10) 6 1500g for 15min, removing the supernatant, and collecting the precipitated cells;
step 3microRNA release: cooling diluted cells at-80deg.C for 10min, taking out, thawing at normal temperature, and standing for 5min to obtain 1 times of thawing; breaking cells by 2 times of freeze thawing to release microRNA;
Example 2:
a preparation method of a quality control product for microRNA detection comprises the following steps:
step 1, cell enrichment: one tube of CHO-K1 cells (5X 10) 5 1500g for 15min, removing the supernatant, and collecting the precipitated cells;
step 3microRNA release: cooling diluted cells at-80deg.C for 10min, taking out, thawing at normal temperature, and standing for 5min to obtain 1 times of thawing; breaking cells by 3 times of freeze thawing to release microRNA;
Example 3:
a preparation method of a quality control product for microRNA detection comprises the following steps:
step 1 detailsCell enrichment: one tube of NIH-3T3 cells (1 x 10 4 And then centrifuging for 20min at 1200g, discarding the supernatant, and taking the precipitated cells;
step 3microRNA release: placing the diluted cells in an ultrasonic cell grinder, setting parameters according to the using instruction of equipment, so as to crush the cells and release microRNA;
1. Quality control storage stability assessment
The quality control product prepared in example 1 was kept at room temperature (20 ℃ -25 ℃) and the CT values of the six microRNAs, hsa-miR-29c-3p (G1), hsa-miR-103a-3p (G2), hsa-miR-181a-5p (G3), hsa-miR-21-5p (G4), hsa-miR-30e-5p (G5) and hsa-miR-126-3p (G6), were detected by RT-PCR on days 0, 3 and 7, respectively, and their accelerated stability performance was evaluated, in which the nucleic acid extraction or purification reagents (commercially available) were: hangzhou foraging biotechnology limited, zheHangzhou mechanical equipment 20180690, model MY-02, and commodity number ST1002.
The quality control prepared in example 1 was set as an experimental group, the quality control prepared by using artificial synthetic genes was set as a control group, the prepared matrixes were artificial serum (Sigma), the concentration levels of target genes in the experimental group and the control group are consistent, and the detection result is shown in FIG. 1.
As can be seen from fig. 1, after the experimental group is stored for 7 days at normal temperature, the target gene is not obviously degraded, and the CT value is not obviously changed; after the control group is stored for 7 days at normal temperature, the target gene is seriously degraded, and the CT value is obviously increased. The results showed good storage stability in the experimental group.
2. Linear assessment of quality control quantification
The quality control was prepared using different concentrations of cells according to the method of example 1, the CT value of hsa-miR-29c-3p (G1) in the quality control was detected by RT-PCR, and the linearity of the quality control quantification was assessed by linear fitting and linear relationship between the number of cells and the CT value.
Preparation of quality control product using 293T cells at cell concentrations of 5X 10, respectively 1 、5*10 2 、5*10 3 The nucleic acid extraction or purification reagents used are: hangzhou foraging biotechnology limited, zheHangzhou mechanical equipment 20180690, model MY-02, and commodity number ST1002. The amount of the extracted sample in the extraction kit was 200ul, and the total of the cells 10 used for each extraction 4 、10 5 、10 6 And each. CT values of hsa-miR-29c-3p (G1) in the quality control products are detected by an RT-PCR method, and detection results are shown in figure 2.
As can be seen from FIG. 2, CT values are linear with cell number. The result shows that the content of the target in the quality control product can be controlled through the cell concentration when the quality control product is prepared, and the target concentration can be controlled to be similar to the target concentration in a sample to be detected, so that the detection flow can be monitored more accurately.
3. The quality control product has a quality control effect compared with artificially synthesized genes
The quality control product prepared in example 1 was set as an experimental group, the quality control product prepared by using artificial synthetic genes was set as a control group, the experimental group and the control group were simultaneously detected with a real serum sample, and the CT values of six microRNAs, namely hsa-miR-29c-3p (G1), hsa-miR-103a-3p (G2), hsa-miR-181a-5p (G3), hsa-miR-21-5p (G4), hsa-miR-30e-5p (G5) and hsa-miR-126-3p (G6), were detected by an RT-PCR method to evaluate the quality control effect, wherein the nucleic acid extraction or purification reagents (commercially available) are: hangzhou foraging biotechnology limited, zheHangzhou mechanical equipment 20180690, model MY-02, and commodity number ST1002.
In the RT-PCR detection process, detection reagents and personnel in different batches are respectively adopted to detect, so that sample detection deviation is manufactured, and the detection deviation of the sample is compared with the detection deviation of an experiment group and a control group, and the detection result is shown in figure 3. As can be seen from fig. 3, the experimental group was substantially identical to the true sample. The result shows that the quality control product can well monitor the sample detection flow.
The invention has the following advantages:
the preparation method of the quality control product has the advantages of simple overall process, low cost and easy storage and transportation.
The target in the quality control product prepared by the invention is derived from cells, has a miRNA-protein complex structure, is stable in storage, has a structure consistent with that of a sample to be detected, and can monitor the detection flow of the sample to be detected better.
The use of a sample-simulating matrix substantially identical to the matrix of the sample to be tested can avoid the possible "matrix effects" present.
The target concentration in the quality control product can be controlled to be similar to the concentration of the sample to be detected, so that the effect of better simulating the sample is achieved.
The above-described embodiment is only a preferred embodiment of the present invention, and is not limited in any way, and other variations and modifications may be made without departing from the technical aspects set forth in the claims.
Claims (13)
1. The preparation method of the quality control product for microRNA detection is characterized by comprising the following steps of:
(1) Cell enrichment: centrifuging the counted cells, and discarding the supernatant to obtain precipitated cells; the cells are cells containing detection target microRNA;
(2) Sample simulation matrix dilution: diluting the precipitated cells from step (1) with a sample-mimicking matrix; the sample simulation matrix is one or more of artificial serum, artificial urine, artificial saliva and fetal bovine serum which do not contain detection target microRNA;
(3) microRNA release: treating the diluted cells in the step (2) by physical means to break the cells and release microRNA in the cells;
(4) Impurity removal: and (3) freezing and thawing the cells crushed in the step (3) or storing at room temperature to degrade unstable microRNA, centrifuging to remove impurities, and taking the supernatant as a quality control product.
2. The method according to claim 1, wherein the cells containing the target microRNA are selected from one or more of human kidney epithelial cell line, CHO-K1 cells, NIH-3T3 cells.
3. The method according to claim 1, wherein in the step (3), the physical means is one or more selected from the group consisting of freeze thawing, mechanical disruption, and ultrasonication.
4. The process according to claim 3, wherein in the step (3), the physical means is freeze thawing, the number of times of freeze thawing is 1 to 3, and the temperature of freeze thawing is-10℃to-85 ℃.
5. The method according to claim 4, wherein in the step (3), the physical means is freeze thawing, the number of times of freeze thawing is 2 or 3, and the freeze thawing temperature is-80 ℃.
6. The method according to claim 1, wherein in the step (4), the number of freeze thawing is 1 to 5 and the freeze thawing temperature is-10 ℃ to-85 ℃.
7. The method according to claim 6, wherein in the step (4), the number of times of freeze thawing is 2 and the freeze thawing temperature is-80 ℃.
8. The method according to claim 1, wherein in the step (4), the storage time at room temperature is 12 hours to 72 hours.
9. The method according to claim 8, wherein in the step (4), the storage time at room temperature is 12 hours or 24 hours.
10. The method of manufacturing according to claim 1, comprising the steps of:
(1) Cell enrichment: centrifuging the counted cells of the human kidney epithelial cell line, and discarding the supernatant to obtain precipitated cells;
(2) Sample simulation matrix dilution: cell was resuspended using artificial serum without detection target micrornas, and the cell concentration was diluted to 3 x 10 2 Individual/ul;
(3) microRNA release: cooling diluted cells at-80deg.C for 10min, taking out, thawing at normal temperature, and standing for 5min to obtain 1 times of thawing; breaking cells by 2 times of freeze thawing to release microRNA;
(4) Impurity removal: and (3) placing the crushed cells in the step (3) at room temperature for 12 hours at night to degrade the unstable microRNA, centrifuging to remove impurities, and taking the supernatant to obtain the quality control product.
11. The method of manufacturing according to claim 1, comprising the steps of:
(1) Cell enrichment: centrifuging the counted CHO-K1 cells, and discarding the supernatant to obtain precipitated cells;
(2) Sample simulation matrix dilution: cell concentration was diluted to 1.5 x 10 using artificial saliva resuspended cells without detection target micrornas 2 Individual/ul;
(3) microRNA release: cooling diluted cells at-80deg.C for 10min, taking out, thawing at normal temperature, and standing for 5min to obtain 1 times of thawing; breaking cells by 3 times of freeze thawing to release microRNA;
(4) Impurity removal: and (3) standing the crushed cells in the step (3) at room temperature for 24 hours at night to degrade the unstable microRNA, centrifuging to remove impurities, and taking a supernatant to obtain a quality control product.
12. The method of manufacturing according to claim 1, comprising the steps of:
(1) Cell enrichment: centrifuging the counted NIH-3T3 cells, and discarding the supernatant to obtain precipitated cells;
(2) Sample simulation matrix dilution: re-suspending cells with fetal bovine serum without detection target micrornas, diluting the cell concentration to 50/ul;
(3) microRNA release: carrying out ultrasonic disruption on the diluted cells to release microRNA;
(4) Impurity removal: freezing the crushed cells at-80deg.C for 10min, thawing at normal temperature, and standing for 5min for 1 time; degrading unstable microRNA by 2 times of freeze thawing, centrifuging to remove impurities, and taking supernatant to obtain quality control product.
13. A quality control product for microRNA detection, prepared by the preparation method of any one of claims 1 to 12.
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| CN108048545A (en) * | 2017-11-28 | 2018-05-18 | 北京旌准医疗科技有限公司 | A kind of external diagnosis reagent quality-control product and preparation method and application |
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| CN108048545A (en) * | 2017-11-28 | 2018-05-18 | 北京旌准医疗科技有限公司 | A kind of external diagnosis reagent quality-control product and preparation method and application |
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