CN111763734A - Method for amplifying circular RNA, specific amplification primer and kit - Google Patents
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Abstract
A method, specific amplification primers and a kit for amplifying circular RNA (hsa _ circ _0035443), wherein the method comprises the following steps: step 1, synthesizing first strand cDNA; and 2, preparing an amplification system. A specific amplification primer acquisition step: step 1, designing a circRNA molecular amplification primer; step 2, verify the primers by real-timePCR experiments, Sanger sequencing. The kit comprises a specific amplification primer for amplifying the circular RNA, and hsa _ circ _0035443 in the cervical cancer tissue can be detected by using a real-timePCR method for diagnosing the cervical cancer. The invention discovers that the circular RNA is low expressed in the cervical cancer tissue, namely the content of the circular RNA in the cervical cancer tissue is far lower than that of the RNA in the para-carcinoma tissue, and the circular RNAcirc-0035443 can be used as a molecular marker for diagnosing the cervical cancer.
Description
Technical Field
The invention belongs to the field of tumor molecular biology, and particularly relates to a method for amplifying circular RNA (hsa _ circ _0035443), a specific amplification primer and a kit.
Background
Cervical Cancer (CC) is one of the high-incidence cancers in women worldwide, with high morbidity and mortality. Persistent infection with high risk Human Papillomaviruses (HPV) is a major factor contributing to CC. HPV vaccination and early detection programs can reduce the incidence of CC, but the effectiveness of these protocols is limited worldwide. CC has a high incidence of morbidity and mortality in many countries with deficient medical systems, although its incidence is only ranked 11 th in developed countries, but second in developing regions. Current treatment for early stage CC is by radiation or chemotherapy, however most patients have reached an advanced stage at the time of diagnosis and lost the opportunity for surgery. Therefore, it is crucial to study the pathogenesis of CC and to find effective interventions.
Circular RNA (CircRNA) occurs during transcriptional splicing, and single-stranded RNA molecules form loops through covalent bonds. There is increasing evidence that circrnas can be produced from spacer regions or antisense transcripts between protein-encoding genes, trnas and lncrnas. There are several characteristics of CircRNA: (1) mainly expressed in the cytoplasm; (2) regulating expression of the target gene by the response element through interaction with the miRNA; (3) most circrnas are produced by exons; most circrnas regulate the expression of endogenous ncrnas; (4) circRNA is tissue specific and developmental stage specific; (5) are commonly found in extracellular fluids with high expression levels; (6) evolutionary conservation in various species; (7) have a covalent ring closure that is highly resistant to RNA exonuclease or RNaseR.
Functionally, recent studies have shown that circRNA can be involved in a wide variety of processes in cell life activities, such as cell differentiation, proliferation, apoptosis, chromosome modification and inactivation, through regulation of gene expression and function. Compared with the broad spectrum of the expression of the coding gene and miRNA, the expression of the circRNA is often characterized by cell and tissue specificity, space-time difference of differentiation and development and the like. In recent years, with the development of technologies such as sequencing transcription profiling of the new generation, the role of circRNA in cancer is gradually being explored and emphasized by researchers, and more data show that the abnormal expression and/or mutation of circRNA exists in various tumors and is closely related to the occurrence and development of the tumors.
In general, our current research on circRNAs in cervical cancer is still not comprehensive and deep enough, and in the early exploration, we show that the circular RNA hsa _ circ _0035443 is not only significantly down-regulated in cervical cancer tissues, but also has a downward trend in expression in cervical cancer cells through high-throughput sequencing and bioinformatics analysis, and that the molecule is closely related to cervical cancer progression. The hsa _ circ _0035443 is located in the 58284902-58306479 region of human chromosome 15 and is spliced and circularized from the exon (exon)3-8 region of the ALDH1A2 gene. At present, no research report on the molecule in the tumor process exists. In order to further research the functions and corresponding mechanisms of hsa _ circ _0035443 in tumors represented by cervical cancer, the research is carried out deeply by designing specific primers thereof, and the circRNA hsa _ circ _0035443 is expected to be an ideal biomarker for diagnosis, treatment and prognosis tracking of cervical cancer patients.
Until now, research aiming at cervical cancer has found that expression changes of some circRNAs are related to the development of cervical cancer, such as circSLC26a4, circSMARCA5, circCLK3, circEIF4G2 and the like. However, in general, the research on circRNAs in cervical cancer is still not comprehensive and deep enough, and a large research space exists on whether circRNAs can be used as a cervical cancer tumor diagnosis marker.
In order to solve the defects of circular RNA utilization in the prior art, the invention provides a method for amplifying circular RNA (hsa _ circ _0035443), a specific amplification primer and a kit.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for amplifying circular RNA (hsa _ circ _0035443), a specific amplification primer and a kit.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for amplifying a circular RNA (hsa _ circ _0035443), the method for amplifying the circular RNA (hsa _ circ _0035443) comprising:
and 2, performing real-time fluorescence quantitative polymerase chain reaction (real-timePCR) amplification on the first strand cDNA prepared in the step 1: preparing an amplification system, wherein the amplification system comprises the cDNA prepared in the step 1, an upstream primer, a downstream primer and ddH2O, qPCR Mix (containing SYBGREEN dye) was amplified and the reaction was temperature controlled according to a second program.
A specific amplification primer for amplifying circular RNA comprises the following steps:
(2) the design principle is as follows: firstly, following the design principle of a common primer; designing a primer close to a shearing site (backsplicejunction);
(2) and (3) re-splicing sequences: in order to meet the design requirement of crossing the splicing sites, according to the full-length nucleotide sequence of the circRNA in the embodiment 1, intercepting the sequence with the length of 100-;
(3) designing a primer: designing a primer by a conventional method aiming at a sequence obtained by heavy splicing, and writing according to a 5'→ 3' direction by default according to a general sequence linear storage rule;
(5) primer output and specificity debugging: introducing the Primer sequence obtained in step 3) into an NCBI database (http:// blast.ncbi.nlm.nih.gov/blast.cgi), and performing Primer specificity comparison analysis and optimization by adopting a Primer-Blast tool;
(6) obtaining primer information, wherein the primers comprise an upstream primer and a downstream primer;
(1) dissolution curve: the dissolution curve is unimodal, the Tm is within the normal range;
(2) electrophoresis chart: the electrophoresis strip is single, and the size of the strip is correct;
(3) sanger sequencing: sequencing results are unimodal, with correct cyclization sites.
A kit for cervical cancer diagnosis, comprising specific amplification primers for amplifying circular RNA.
Compared with the prior art, the invention has the advantages that:
advantages (1) the present invention found that the circular RNA exhibits low expression in cervical cancer tissues, i.e., the content of the circular RNA in cervical cancer tissues is much lower than that in paracarcinoma tissues, and therefore, the circular RNA circ-0035443 can be used as a molecular marker for cervical cancer diagnosis.
Advantage (2) RNA of example 1 was amplified using the specific amplification primers provided by the present invention, and the amplification results were analyzed as shown in example 1 to obtain a ROC curve, and the area under the ROC curve was 0.821(P ═ 0.037) as shown in fig. 2. The specific amplification primer for amplifying the circular RNA provided by the invention has high stability, specificity and sensitivity to the circular RNA.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the structure and full-length sequence diagram of the hsa _ circ _0035443 gene of the present invention;
FIG. 2 shows a ROC curve for the detection of hsa _ circ _0035443 in cervical cancer tissue according to the invention;
FIG. 3 is a graph showing the results of the verification of the hsa _ circ _0035443 primer of the present invention;
FIG. 4 shows the differential expression profile of real-time PCR detection of hsa _ circ _0035443 in cervical cancer tissue and paracancerous tissue according to the present invention;
FIG. 5 shows the expression profile of real-time PCR detection of hsa _ circ _0035443 in cervical cancer cells according to the invention;
FIG. 6 shows real-time PCR of the invention detecting the localization of hsa _ circ _0035443 in cervical cancer cells;
FIG. 7 shows the stability of expression of hsa _ circ _0035443 of the invention under RNase R conditions.
Detailed Description
Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings, in order that the present disclosure may be more fully understood and fully conveyed to those skilled in the art. While the exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the invention is not limited to the embodiments set forth herein.
A method for amplifying a circular RNA (hsa _ circ _0035443), the method for amplifying the circular RNA (hsa _ circ _0035443) comprising:
In the step 1, the raw materials are mixed according to the following mixture ratio: mu.L of the circular RNA of example 1, 1. mu.L of random primer, 10. mu.L ddH2O, 2 mu L dNTP mixed solution, 4 mu L reverse transcription buffer solution, 1 mu L RNase inhibitor and 1 mu L reverse transcriptase, wherein the dNTP mixed solution comprises dATP, dGTP, dCTP and dTTP.
And 2, performing real-time fluorescence quantitative polymerase chain reaction (real-timePCR) amplification on the first strand cDNA prepared in the step 1: preparing an amplification system, wherein the amplification system comprises the cDNA prepared in the step 1, an upstream primer, a downstream primer and ddH2O, qPCR Mix (containing SYBGREEN dye) was amplified and the reaction was temperature controlled according to a second program. The second procedure is: pre-denaturation at 95 ℃ for 10min, then denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, and carrying out 38 cycles in total; the ABI7500 fluorescence quantitative PCR instrument selects a melting curve program, and continuously collects sample fluorescence signals in the climbing process to obtain a melting curve. In step 2, the amplification system is prepared according to the following mixture ratio: mu.L of the cDNA prepared in step 1, 0.5. mu.L of the upstream primer of example 3, 0.5. mu.L of the downstream primer of example 3, 7. mu.L of ddH2O, 10. mu.L real-time PCR amplified Mix (SYBGREEN dye).
A specific amplification primer for amplifying circular RNA comprises the following steps:
(3) the design principle is as follows: firstly, following the design principle of a common primer; designing a primer close to a shearing site (backsplicejunction);
(2) and (3) re-splicing sequences: in order to meet the design requirement of crossing the splicing sites, according to the full-length nucleotide sequence of the circRNA in the embodiment 1, intercepting the sequence with the length of 100-;
(3) designing a primer: designing a primer by a conventional method aiming at a sequence obtained by heavy splicing, and writing according to a 5'→ 3' direction by default according to a general sequence linear storage rule;
(5) primer output and specificity debugging: introducing the Primer sequence obtained in step 3) into an NCBI database (http:// blast.ncbi.nlm.nih.gov/blast.cgi), and performing Primer specificity comparison analysis and optimization by adopting a Primer-Blast tool;
(6) obtaining primer information, wherein the primers comprise an upstream primer and a downstream primer;
the nucleotide sequence of the upstream primer is as follows: 5'-TTGCATTCACAGGGTCTACTG-3', respectively;
the nucleotide sequence of the downstream primer is as follows: 5'-GTTCTCCTGTGGCTGGATTATAG-3' are provided.
The GC content of the upstream primer is 47.62%, and the GC content of the downstream primer is 47.83%, wherein the GC content refers to the ratio of guanine and cytosine in 4 bases of DNA;
the TM value of the upstream primer is 57.94 degrees, the TM value of the downstream primer is 58.36 degrees, and the TM value refers to the melting temperature of the upstream primer or the downstream primer.
(1) dissolution curve: the dissolution curve is unimodal, the Tm is within the normal range;
(2) electrophoresis chart: the electrophoresis strip is single, and the size of the strip is correct;
(3) sanger sequencing: sequencing results are unimodal, with correct cyclization sites.
A kit for cervical cancer diagnosis, comprising specific amplification primers for amplifying circular RNA. The kit also comprises reverse transcriptase, buffer solution and ddH2O, DNA at least one of polymerase, fluorescent dye and dNTP mixture; the kit can detect hsa _ circ _0035443 in cervical cancer tissues by using a real-time PCR method and is used for diagnosing cervical cancer.
Example 1
The steps for obtaining the nucleotide sequence of the circular RNA are as follows:
the sequence and structure information of the circular RNA hsa _ circ _0035443 are obtained through a circBase, CSCD and a circbank database, and the circular RNA is positioned in the human chromosome 15 58284902-58306479 region and is spliced and cyclized by the 3-8 region of the ALDH1A2 gene exon (exon).
FIG. 1 is a gene structure diagram of a circular RNA, and the entire nucleotide sequence thereof is 681 nt. Wherein, the first two nucleotides and the last two nucleotides are ring-forming binding sites.
Example 2
The present invention provides example 1 use of circular RNA as a diagnostic marker for cervical cancer:
Therefore, the circular RNA hsa _ circ _0035443 can be used for cervical cancer diagnosis.
Example 3
The invention provides a specific amplification primer for amplifying circular RNA in example 1, which comprises the following steps:
(4) the design principle is as follows: firstly, following the design principle of a common primer; ② the primer is designed near the shearing site (backsplicijunction).
(2) And (3) re-splicing sequences: in order to meet the design requirement of crossing the splicing sites, according to the full-length nucleotide sequence of the circRNA in example 1, the sequence with the length of 100-.
(3) Designing a primer: the primers are designed according to the sequence obtained by the heavy splicing by a conventional method, and are written according to the 5'→ 3' direction by default according to a general sequence linear storage rule.
(5) Primer output and specificity debugging: introducing the Primer sequence obtained in step 3) into an NCBI database (http:// Blast. NCBI. nlm. nih. gov/Blast. cgi), and performing Primer-specific comparison analysis and optimization by using a 'Primer-Blast' tool.
(6) The obtained primer information:
the nucleotide sequence of the upstream primer is as follows: 5'-TTGCATTCACAGGGTCTACTG-3', respectively;
the nucleotide sequence of the downstream primer is as follows: 5'-GTTCTCCTGTGGCTGGATTATAG-3' are provided.
In one realizable form, the GC content of the upstream primer is 47.62% and the GC content of the downstream primer is 47.83%, wherein the GC content refers to the ratio of guanine and cytosine in 4 bases of DNA. The TM value of the upstream primer is 57.94 degrees, the TM value of the downstream primer is 58.36 degrees, and the TM value refers to the melting temperature of the upstream primer or the downstream primer.
FIG. 2 is a schematic of the circular RNA primer design, showing that the amplification product will contain a cleavage site.
(1) dissolution curve: the dissolution curve is unimodal, the Tm is within the normal range;
(2) electrophoresis chart: the electrophoresis strip is single, and the size of the strip is correct;
(3) sanger sequencing: sequencing results are unimodal, with correct cyclization sites.
FIG. 3 shows the verification result of the circular RNA primer, and it can be seen that the dissolution curve shows a single peak, the electrophoresis band of the PCR product is single, and the size of the band is correct (refer to FIG. 7); the PCR product is taken for Sanger sequencing, and the sequencing result shows a single peak and the cyclization site is correct. Thus, the primer verification of the circular RNA was completed.
Example 4
The present invention provides a method for amplifying the circular RNA of example 1 (hsa _ circ _0035443), the method comprising:
and 2, performing real-time fluorescence quantitative polymerase chain reaction (real-timePCR) amplification on the first strand cDNA prepared in the step 1: preparing an amplification system, wherein the amplification system comprises the step 1The cDNA thus prepared, the forward primer shown in example 3, the reverse primer shown in example 3, and ddH2O, qPCR Mix (containing SYBGREEN dye) was amplified and the reaction was temperature controlled according to a second program.
In an achievable mode, in the step 1, the raw materials are mixed according to the following mixture ratio: mu.L of the circular RNA of example 1, 1. mu.L of random primer, 10. mu.L of ddH2O, 2 mu L dNTP mixed solution, 4 mu L reverse transcription buffer solution, 1 mu L RNase inhibitor and 1 mu L reverse transcriptase, wherein the dNTP mixed solution comprises dATP, dGTP, dCTP and dTTP;
(1) the first procedure is: reacting at 42 deg.C for 15min, reacting at 70 deg.C for 5min, and storing cDNA at-80 deg.C for use.
(2) In step 2, the amplification system is prepared according to the following mixture ratio: mu.L of the cDNA prepared in step 1, 0.5. mu.L of the upstream primer of example 3, 0.5. mu.L of the downstream primer of example 3, 7. mu.L of ddH2O, 10. mu.L real-time PCR amplification Mix (SYBGREEN dye); the second procedure is: pre-denaturation at 95 ℃ for 10min, followed by denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 38 cycles. The ABI7500 fluorescence quantitative PCR instrument selects a melting curve program, and continuously collects sample fluorescence signals in the climbing process to obtain a melting curve.
Example 5
The invention also provides application of the specific amplification primer in preparation of a kit for diagnosing cervical cancer.
Example 6
The invention also provides a kit for diagnosing cervical cancer, which comprises the specific amplification primer in the embodiment 3.
In one implementation, the kit further comprises reverse transcriptase, buffer, ddH2O, DNA polymerase, fluorescent dye and dNTP mixture. The kit can detect hsa _ circ _0035443 in cervical cancer tissues by using a real-time PCR method and is used for diagnosing cervical cancer.
In the following examples, reagents and biomaterials used were commercially available unless otherwise specified. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The molecular biological experiments, which are not specifically described in the examples, were carried out according to the methods specified in molecular cloning, A laboratory Manual (third edition) J. SammBruke, or according to the kit and product instructions.
Example 7
Real-time PCR reaction detected the expression of hsa _ circ _0035443 in cervical cancer tissue.
1. Designing a specific amplification primer:
the sequences and related information of specific amplification primers designed by the present inventors are shown in table 1:
TABLE 1 specific amplification primer sequences and related information
2. Total RNA extraction:
(1) extracting the total RNA of the cervical cancer tissue by a Trizol method: taking about 0.2g of cervical cancer tissue, grinding the cervical cancer tissue under the condition of liquid nitrogen until the tissue is in a powder state, then adding 1mL of Trizol, fully grinding the cervical cancer tissue and uniformly blowing the cervical cancer tissue;
(2) cracking the liquid sample prepared in the step (1) at room temperature for 5min, adding chloroform according to the proportion that 0.2mL of chloroform is added into 1mL of Trizol, covering a pipe cover tightly, carrying out vortex oscillation for 15s, standing for 5min until layering occurs, centrifuging at 12000rpm for 15min at 4 ℃, layering the mixed liquid into a lower-layer chloroform phase, a middle-layer protein layer and an upper-layer colorless water phase, and distributing all RNA into the water phase;
(3) transferring the water phase into a new centrifuge tube, adding isopropanol with the same volume, uniformly mixing, standing at-20 ℃ for 30min, and centrifuging at 12000rpm at 4 ℃ for 10min to obtain a precipitate, wherein the RNA is completely present in the precipitate;
(4) removing the supernatant, adding 1mL of 75% ethanol into the system to clean the RNA precipitate, and centrifuging at 7500rpm at 4 ℃ for 5 min;
(5) repeating the step (4);
(6) removing ethanol solution, drying at room temperature for 5-10min until ethanol volatilizes, and removing ddH without RNase2Adding O water into the centrifuge tube, and fully dissolvingResolving to obtain total RNA;
(7) and (3) measuring the concentration and purity of the RNA by using the NanoDrop ND-2000, subpackaging and storing at-80 ℃ after the quality of the RNA reaches the standard.
3. Synthesis of first strand cDNA sequence:
taking a PCR tube to configure a reverse transcription system, wherein the reverse transcription system is as follows: 1 μ L of total RNA (about 500-1000ng), 1 μ L of random primer, 10 μ L of ddH2O, 2. mu.L dNTP mixture (dATP, dGTP, dCTP and dTTP), 4. mu.L reverse transcription buffer (Thermo Co.), 1. mu.L RNase inhibitor, 1. mu.L reverse transcriptase, total volume of 20. mu.L; the reaction conditions are as follows: the reaction was carried out at 42 ℃ for 60min and at 70 ℃ for 5 min. The cDNA obtained by reverse transcription is stored at-80 ℃ for later use.
4. And (3) verifying the cDNA amplification of the Hsa _ circ _0035443 gene:
taking 2 mu L of cDNA obtained by reverse transcription in the step 3, and preparing a system for PCR amplification;
(1) the PCR amplification system is as follows: mu.L of cDNA, 0.5. mu.L of the forward primer shown in Table 1, 0.5. mu.L of the reverse primer shown in Table 1, 7. mu.L of ddH2O, 10 mu LPCR amplification Mix with the total volume of 20 mu L;
(2) the reaction conditions were pre-denaturation at 95 ℃ for 10min, followed by denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 38 cycles in total. Finally, the reaction was carried out at 72 ℃ for 5 min.
(3) mu.L of the reaction product was taken to verify whether the PCR product was a monospecific amplification band under conditions of 2.0g agarose/100 mL 1 XTAE buffer, 120V voltage, 20 min.
5. The Real-time PCR amplification reaction was detected using a cDNA sample obtained by reverse transcription.
(1) The reaction system is as follows: mu.L cDNA, 0.8. mu.L forward primer, 0.8. mu.L reverse primer, 6.4. mu.L ddH2O, 10 μ LqPCR amplification Mix (SYBGREEN dye) in a total volume of 20 μ L;
(2) real-time PCR reaction conditions: pre-denaturation at 95 ℃ for 10min, followed by denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 38 cycles. The ABI7500 fluorescence quantitative PCR instrument selects a melting curve program, and continuously collects sample fluorescence signals in the climbing process to obtain a melting curve.
(3) The amplification reaction was performed on a real-time fluorescent quantitative PCR instrument ABI7500, GAPDH was amplified as an internal control while the target gene was amplified, and the relative expression level of the gene was calculated by the 2(— Δ CT) method.
For example 7, different samples were taken and repeated 6 times. Among them, in the internal reference GAPDH gene and hsa _ circ _0035443 gene real-time PCR dissolution curve chart, it can be seen that the amplification peak is single, no miscellaneous peak, the primer specificity is good, and the amplification experiment is normal.
Example 8
The Real-time PCR reaction detected the expression of hsa _ circ _0035443 in the para-carcinoma tissue of cervical carcinoma.
The procedure of example 7 was repeated except that: in step 2, total RNA is extracted from the tissues adjacent to the cervical cancer.
For example 8, different samples were taken and repeated 6 times.
Example 9
Real-time PCR reaction detected the expression of hsa _ circ _0035443 in cervical cancer cells.
The procedure of example 7 was repeated except that: in step 2, total RNA of human cervical cancer cells is extracted.
For example 9, different samples were taken and repeated 5 times.
Real-time PCR results analysis of examples 7-9.
1. Analysis of gene expression:
the expression result of hsa _ circ _0035443 is shown in FIG. 4 and FIG. 5:
(1) in the 7 pairs of tissues of fig. 4, the expression of the circular RNA hsa _ circ _0035443 was significantly down-regulated in cervical cancer tissues relative to paracervical cancer tissues;
(2) in the 4 cervical cancer cell samples of fig. 5, the expression of circular RNA hsa _ circ _0035443 was significantly down-regulated in the cervical cancer cell samples relative to 293T cells.
The above results indicate that the circular RNA hsa _ circ _0035443 has differential expression in cervical cancer tissues and paracancerous tissues, and also has differential expression in cervical cancer cells and non-cervical cancer cells, specifically, the circular RNA hsa _ circ _0035443 has low expression in both cervical cancer tissues and cells, so the circular RNA hsa _ circ _0035443 can be used for diagnosis of cervical cancer.
Example 10
The positional expression of circular RNA hsa _ circ _0035443 in cells.
The nucleoplasm separation kit is derived from a Biyunshi cell nuclear protein and cytoplasm protein extraction kit, and the steps of the kit are further optimized as follows:
1. preparing a solution: three reagents (a cytoplasmic protein extraction reagent A, a cytoplasmic protein extraction reagent B and a cell nucleus protein extraction reagent) in the kit are thawed at room temperature, immediately placed on ice after being dissolved, and evenly mixed. An appropriate amount of the cytoplasmic protein extraction reagent A was taken for use, and PMSF was added several minutes before use to give a final concentration of 1mM of PMSF. An appropriate amount of the nuclear protein extraction reagent was prepared, and PMSF was added to the reagent several minutes before use to give a final concentration of 1mM PMSF.
2. For adherent cells: the cells were washed once with PBS, scraped with a cell scraper, or treated with EDTA solution so that the cells were no longer adherent and blown down with a pipette. Cells were collected by centrifugation and the supernatant was aspirated with maximum effort, leaving the cell pellet ready for use. The cell is prevented from being digested by pancreatin to prevent the pancreatin from degrading the target protein to be extracted.
3. 200. mu.l of a cell plasma protein extraction reagent A containing PMSF was added to 20. mu.l of the cell pellet.
4. The Vortex was vigorous at maximum speed for 5 seconds to completely suspend and disperse the cell pellet. (if the cell pellet is not completely suspended and dispersed, the vortex time can be extended appropriately.)
5. Ice-bath for 10-15 min.
6. mu.L of the cytoplasmic protein extraction reagent B10 was added. Vortex 5 seconds at maximum speed and ice bath 1 minute.
7. The highest speed Vortex was vigorous for 5 seconds, and centrifuged at 12,000-16,000g for 5 minutes at 4 ℃.
8. The supernatant was aspirated (about 100. mu.l of supernatant was aspirated from the tip to the middle, and the tip was not downward, but was essential) into pre-cooled EP tubes. 500 microliters of Trizol was added directly to the EP tube and mixed until ready for use.
9. And (4) sucking off the supernatant in the rest precipitate, and sucking the supernatant clean again by using a white gun head after the supernatant is taken out by using a yellow gun head.
10. Add cytoplasmic protein extraction reagent A to the sediment for 5 seconds at the highest speed, suspend and disperse the cell sediment completely, ice-wash for 10-15 minutes. (second washing)
Centrifuging at 11.4 ℃ for 5 minutes at 12,000-.
12. For the precipitation, 50. mu.l of a reagent for extracting nuclear protein to which PMSF was added.
13. The highest speed, vigorous Vortex 15-30 seconds, completely suspended and dispersed the cell pellet. Then adding Trizol and mixing evenly for standby.
The procedure of example 7 was repeated except that the cervical cancer cells and the above total RNA of the nucleus and cytoplasm were extracted.
Analysis of gene expression: the expression result of hsa _ circ _0035443 is shown in FIG. 6:
in FIG. 6 it can be seen that the circular RNA hsa _ circ _0035443 is mainly expressed in the cytoplasm and is relatively low expressed in the nucleus.
Example 11
RNase R detects the expression stability of circular RNA hsa _ circ _ 0035443.
RNase R (Ribonucleae R) is a 3 '-5' exonuclease from the RNR superfamily of E.coli that cleaves RNA stepwise from the 3 '-5' direction into di-and tri-nucleotides. RNase R digests almost all linear RNA molecules, but does not readily digest circular RNA, lasso structures or double stranded RNA molecules with less than 7 nucleotides at the 3' overhang. RNase R is commonly used for gene expression and variable splicing studies and can digest linear RNA to enrich for circular RNA (circRNAs) or lasso-structured RNA (lariat RNA).
As can be seen in fig. 7: both circular and linear RNAs were detectable without RNase R addition, whereas linear RNA was not detectable after RNase R addition, while circular was still detectable, indicating that circular RNA hsa _ circ _0035443 is stable, is not easily digested by RNase R, and conforms to the characteristics of circular RNA.
ROC curve analysis.
The experimental data obtained in example 7 were analyzed to obtain an ROC curve, as shown in fig. 2, in which AUC, the area under the curve, was 0.821(P ═ 0.037), indicating that the target of detection can be used as a specific marker for detecting the circular RNA.
Those skilled in the art know that the area under the ROC curve is between 1.0 and 0.5, and that with AUC >0.5, the closer the AUC is to 1, indicating better diagnostic results. The AUC has lower accuracy at 0.5-0.7, certain accuracy at 0.7-0.9, and higher accuracy at more than 0.9, and the value greater than 0.7 indicates that the detection target can be used as a specific marker for the detection.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described above with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
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Claims (10)
1. A method for amplifying a circular RNA (hsa _ circ _0035443), wherein the method for amplifying the circular RNA (hsa _ circ _0035443) comprises:
step 1, first strand cDNA synthesis: mixing raw materials including the circular RNA, the random primer and ddH in example 12O, dNTP mixing solution, reverse transcription buffer solution, RNase inhibitor and reverse transcriptase, and controlling temperature according to a first program;
step 2, performing real-time fluorescent quantitative polymerase chain reaction amplification on the first strand cDNA prepared in the step 1: preparing an amplification system, wherein the amplification system comprises the cDNA prepared in the step 1, an upstream primer, a downstream primer and ddH2O, qPCR amplify Mix, following a second temperature-programmed reaction.
2. The method for amplifying circular RNA (hsa _ circ _0035443) according to claim 1, wherein the raw materials are mixed in the following ratio in step 1: mu.L of the circular RNA of example 1, 1. mu.L of random primer, 10. mu.L of ddH2O, 2 mu L dNTP mixed solution, 4 mu L reverse transcription buffer solution, 1 mu L RNase inhibitor and 1 mu L reverse transcriptase, wherein the dNTP mixed solution comprises dATP, dGTP, dCTP and dTTP.
3. The method for amplifying circular RNA (hsa _ circ _0035443) according to claim 1, wherein the first procedure is: reacting at 42 deg.C for 15min, reacting at 70 deg.C for 5min, and storing cDNA at-80 deg.C for use.
4. The method of claim 1, wherein in step 2, the amplification system is formulated as follows: 2 μ L of the cDNA prepared in step 1, 0.5 μ L of the forward primer, 0.5 μ L of the reverse primer, 7 μ L of ddH2O, 10. mu.L real-time PCR amplified Mix.
5. The method for amplifying circular RNA (hsa _ circ _0035443) according to claim 1, wherein the second procedure is: pre-denaturation at 95 ℃ for 10min, then denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, and carrying out 38 cycles in total; the ABI7500 fluorescence quantitative PCR instrument selects a melting curve program, and continuously collects sample fluorescence signals in the climbing process to obtain a melting curve.
6. A specific amplification primer for amplifying circular RNA is characterized in that the specific amplification primer is obtained by the following steps:
step 1, design of amplification primers of circRNA molecules:
(1) the design principle is as follows: firstly, following the design principle of a common primer; designing a primer close to a shearing site (backsplicejunction);
(2) and (3) re-splicing sequences: in order to meet the design requirement of crossing the splicing sites, according to the full-length nucleotide sequence of the circRNA in the embodiment 1, intercepting the sequence with the length of 100-;
(3) designing a primer: designing a primer by a conventional method aiming at a sequence obtained by heavy splicing, and writing according to a 5'→ 3' direction by default according to a general sequence linear storage rule;
(5) primer output and specificity debugging: introducing the Primer sequence obtained in the step 3) into an NCBI database, and performing Primer specificity comparison analysis and optimization by adopting a Primer-Blast tool;
(6) obtaining primer information, wherein the primers comprise an upstream primer and a downstream primer;
step 2, on the basis of the step 1, further verifying the primers by real-time PCR experiment and Sanger sequencing:
(1) dissolution curve: the dissolution curve is unimodal, the Tm is within the normal range;
(2) electrophoresis chart: the electrophoresis strip is single, and the size of the strip is correct;
(3) sanger sequencing: sequencing results are unimodal, with correct cyclization sites.
7. The specific amplification primer for amplifying a circular RNA according to claim 6,
the nucleotide sequence of the upstream primer is as follows: 5'-TTGCATTCACAGGGTCTACTG-3', respectively;
the nucleotide sequence of the downstream primer is as follows: 5'-GTTCTCCTGTGGCTGGATTATAG-3' are provided.
8. The specific amplification primer for amplifying a circular RNA according to claim 6,
the GC content of the upstream primer is 47.62%, and the GC content of the downstream primer is 47.83%, wherein the GC content refers to the ratio of guanine and cytosine in 4 bases of DNA;
the TM value of the upstream primer is 57.94 degrees, the TM value of the downstream primer is 58.36 degrees, and the TM value refers to the melting temperature of the upstream primer or the downstream primer.
9. A kit for diagnosing cervical cancer, comprising the specific amplification primer for amplifying the circular RNA according to any one of claims 6 to 8.
10. The kit for cervical cancer diagnosis according to claim 9, characterized in that the kit further comprises at least one of reverse transcriptase, buffer, ddH2O, DNA polymerase, fluorescent dye and dNTP mixture; the kit can detect hsa _ circ _0035443 in cervical cancer tissues by using a real-time PCR method and is used for diagnosing cervical cancer.
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| CN113718035A (en) * | 2021-09-30 | 2021-11-30 | 安徽同科生物科技有限公司 | Application of circular RNA hsa _ circ _0003552 and kit for detecting circular RNA hsa _ circ _0003552 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113528662A (en) * | 2021-05-21 | 2021-10-22 | 山西医科大学第二医院 | CircRNA marker, specific primer pair, kit and application for detecting cervical cancer |
| CN113528662B (en) * | 2021-05-21 | 2022-10-28 | 山西医科大学第二医院 | CircRNA marker, specific primer pair, kit and application for detecting cervical cancer |
| CN113718035A (en) * | 2021-09-30 | 2021-11-30 | 安徽同科生物科技有限公司 | Application of circular RNA hsa _ circ _0003552 and kit for detecting circular RNA hsa _ circ _0003552 |
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