CN111763734B - Method for amplifying circular RNA, specific amplification primer and kit - Google Patents

Method for amplifying circular RNA, specific amplification primer and kit Download PDF

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CN111763734B
CN111763734B CN202010323112.6A CN202010323112A CN111763734B CN 111763734 B CN111763734 B CN 111763734B CN 202010323112 A CN202010323112 A CN 202010323112A CN 111763734 B CN111763734 B CN 111763734B
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张梦娇
谢依
袁奕
唐林香
何胜祥
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Jiangsu Toneker Medical Technology Co ltd
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Abstract

A method for amplifying circular RNA (hsa_circ_ 0035443), a specific amplification primer and a kit, wherein the method comprises the following steps: step 1, synthesizing first-strand cDNA; and 2, preparing an amplification system. Specific amplification primer acquisition step: step 1, designing a circRNA molecule amplification primer; step 2, primers were verified by real-timePCR experiments and Sanger sequencing. The kit comprises specific amplification primers for amplifying the circular RNA, and can be used for detecting hsa_circ_0035443 in cervical cancer tissues by using a real-timePCR method and diagnosing cervical cancer. The invention discovers that the annular RNA is expressed in cervical cancer tissues in a low mode, namely the content of the annular RNA in the cervical cancer tissues is far lower than that of the annular RNA in the tissues beside the cancer, and the annular RNAcirc-0035443 can be used as a molecular marker for diagnosing the cervical cancer.

Description

Method for amplifying circular RNA, specific amplification primer and kit
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 global high-grade cancers of women, with higher morbidity and mortality. Persistent infection with high-risk Human Papillomaviruses (HPV) is a major factor leading to CC. HPV vaccination and early detection programs may reduce the incidence of CC, but the efficacy of these regimens is limited worldwide. CC has a high morbidity and mortality in many countries with imperfect medical systems, and although its morbidity is ranked only 11 in developed countries, it is ranked second in developing regions. Current treatment of early CC is by radiation or chemotherapy, however most patients have reached late stages at the time of diagnosis, losing the opportunity for surgery. Therefore, it is important to study the pathogenesis of CC and to find effective interventions.
Circular RNAs (circrnas) occur during transcriptional splicing, with single-stranded RNA molecules forming loops through covalent bonds. There is growing evidence that circRNA can be produced from the spacer region or antisense transcript between the protein-encoding gene, tRNA and lncRNA. The CircRNA has several characteristics: (1) expression predominantly in the cytoplasm; (2) Through interaction with miRNA, expression of the target gene is regulated by the response element; (3) most of the circRNA is produced by exons; most circrnas regulate expression of endogenous ncrnas; (4) The circRNA has tissue specificity and developmental stage specificity; (5) Are typically found in extracellular fluids with high expression levels; (6) evolutionary conservation in various species; (7) Has a high resistance to the covalent closure of RNA exonucleases or RNaseRs.
Functionally, recent researches show that the circRNA can widely participate in a plurality of processes of cell life activities, such as cell differentiation, proliferation, apoptosis, chromosome modification, inactivation and the like through regulating and controlling gene expression, functions and the like. Compared with the broad spectrum of the expression of coding genes and miRNAs, the expression of the circRNA has the characteristics of cell and tissue specificity, space-time difference of differentiation and development and the like. With the development of new generation sequencing transcription profiling and other technologies in recent years, the role of the circRNA in cancer is gradually explored and emphasized by researchers, and more data show that the abnormal expression and/or mutation of the 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 early exploration, we through high-throughput sequencing and bioinformatics analysis, the circular RNA hsa_circ_0035443 is significantly down-regulated in cervical cancer tissues, and the expression in cervical cancer cells is also in a down-trend, so that the molecular is proved to be closely related to the cervical cancer process. hsa_circ_0035443 is located in the region 58284902-58306479 of human chromosome 15 and is formed by cleavage and cyclization of the 3-8 region of exon (exo) of the ALDH1A2 gene. There is no report on the research of the molecule in the tumor progress. In order to further explore the function and the corresponding mechanism of hsa_circ_0035443 in tumors represented by cervical cancer, the specific primers are designed for further research, and the circRNA hsa_circ_0035443 is expected to become an ideal biomarker for diagnosis, treatment and prognosis tracking of cervical cancer patients.
Up to now, research on cervical cancer has found that expression changes of some circRNAs are related to cervical cancer occurrence and development, such as circSLC26A4, circSMARCA5, circCLK3, circEIF4G2, and the like. However, in general, the research of the circRNAs in cervical cancer is still not comprehensive and deep enough, and a large research space exists in the aspects of whether the circRNAs can be used as cervical cancer tumor diagnosis markers or not.
In order to solve the defect of the utilization of circular RNA 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 for solving the technical problems is as follows:
a method of amplifying a circular RNA (hsa_circ_ 0035443), the method of amplifying a circular RNA (hsa_circ_ 0035443) comprising:
step 1, first strand cDNA is synthesized: mixing raw materials including circular RNA of example 1, random primer, ddH 2 O, dNTP, reverse transcription buffer, rnase inhibitor, reverse transcriptase, according to a first procedure;
step 2, carrying out real-time fluorescent quantitative polymerase chain reaction (real-time PCR) amplification on the first-strand cDNA prepared in the step 1: preparing an amplification system comprising the cDNA prepared in the step 1, an upstream primer, a downstream primer and ddH 2 O, qPCR amplification Mix (containing SYBGREEN dye) following the second procedureAnd (3) temperature control reaction.
A specific amplification primer for amplifying a circular RNA, the specific amplification primer being obtained by:
step 1, designing a circRNA molecule amplification primer:
(2) Design principle: (1) following the common primer design principle; (2) primers were designed near the cleavage site (backsplice junction);
(2) Sequence re-splicing: in order to meet the design requirement of crossing the cutting sites, according to the full-length nucleotide sequence of the circRNA of the embodiment 1, a 3 '-end 100-300nt length sequence is intercepted and placed in front of a 5' -end 100-300nt length sequence so as to be spliced again to form a new sequence, and the sequence comprises the cutting sites after loop splicing;
(3) Primer design: designing a primer by a conventional method aiming at the sequences obtained by re-splicing, and writing in a 5'- & gt 3' direction by default according to a linear storage rule of the common sequences;
(5) Primer output and specificity debugging: introducing the Primer sequence obtained in the step 3) into NCBI database (http:// Blast. NCBI. Nlm. Nih. Gov/Blast. Cgi), and performing Primer specificity comparison analysis and optimization by using a Primer-Blast tool;
(6) The primer information obtained, wherein the primer comprises an upstream primer and a downstream primer;
step 2, based on step 1, primers were further verified by real-time PCR experiments, sanger sequencing:
(1) Dissolution profile: the dissolution curve is unimodal, and the Tm value is within the normal range;
(2) Electrophoresis pattern: the electrophoresis strip is single, and the size of the strip is correct;
(3) Sanger sequencing: the sequencing result is unimodal and the cyclization site is correct.
A kit for cervical cancer diagnosis, comprising specific amplification primers for amplifying circular RNAs.
Compared with the prior art, the invention has the advantages that:
advantage (1) the present invention has found that the cyclic RNA exhibits low expression in cervical cancer tissues, i.e., the content of the cyclic RNA in cervical cancer tissues is far lower than that in paracancerous tissues, and thus, the cyclic RNA circ-0035443 can be used as a molecular marker for diagnosis of cervical cancer.
Advantage (2) the RNA of example 1 was amplified using the specific amplification primers of the present invention, and the amplification results were analyzed as shown in example 1 to obtain an 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 invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the hsa_circ_0035443 gene structure and full-length sequence diagram of the present invention;
FIG. 2 shows the ROC curve of hsa_circ_0035443 detection results in cervical cancer tissue according to the present invention;
FIG. 3 shows a diagram of the results of the primer verification of hsa_circ_0035443 of the present invention;
FIG. 4 shows a differential expression pattern of hsa_circ_0035443 detected by real-time PCR in cervical cancer tissue and paracancerous tissue;
FIG. 5 shows the expression pattern of hsa_circ_0035443 in cervical cancer cells detected by real-time PCR of the present invention;
FIG. 6 shows the real-time PCR detection of the localization of hsa_circle_ 0035443 in cervical cancer cells according to the present invention;
FIG. 7 shows the expression stability of hsa_circ_0035443 of the present 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 to provide a more thorough understanding of the present disclosure, and to fully convey the scope of the disclosure to those skilled in the art. While the drawings illustrate exemplary embodiments of the present disclosure, it should be understood that the invention is not limited to the embodiments set forth herein.
A method of amplifying a circular RNA (hsa_circ_ 0035443), the method of amplifying a circular RNA (hsa_circ_ 0035443) comprising:
step 1, first strand cDNA is synthesized: mixing raw materials including circular RNA of example 1, random primer, ddH 2 O, dNTP, reverse transcription buffer, rnase inhibitor, reverse transcriptase, according to a first procedure; the first procedure is: reacting at 42 ℃ for 15min, reacting at 70 ℃ for 5min, and storing cDNA at-80 ℃ for standby.
In the step 1, the raw materials are mixed according to the following proportion: 1. Mu.L of the circular RNA of example 1, 1. Mu.L of random primer, 10. Mu.L of ddH 2 O, 2. Mu.L of dNTP mix, 4. Mu.L of reverse transcription buffer, 1. Mu.L of RNase inhibitor, 1. Mu.L of reverse transcriptase, wherein dNTP mix comprises dATP, dGTP, dCTP and dTTP.
Step 2, carrying out real-time fluorescent quantitative polymerase chain reaction (real-time PCR) amplification on the first-strand cDNA prepared in the step 1: preparing an amplification system comprising the cDNA prepared in the step 1, an upstream primer, a downstream primer and ddH 2 O, qPCR Mix (containing SYBGREEN dye) was amplified and temperature-controlled according to the second procedure. The second procedure is: pre-denaturing at 95 ℃ for 10min, then denaturing at 95 ℃ for 15s, annealing at 60 ℃ for 30s, and extending at 72 ℃ for 30s, and performing 38 cycles in total; the ABI7500 fluorescent quantitative PCR instrument selects a melting curve program, and continuously collects sample fluorescent signals during the ramp up process to obtain a melting curve. In the step 2, the amplification system is prepared according to the following proportion: 2. Mu.L of the cDNA prepared in step 1, 0.5. Mu.L of the upstream primer as in example 3, 0.5. Mu.L of the downstream primer as in example 3, and 7. Mu.L of ddH 2 O, 10. Mu.L real-time PCR amplification Mix (SYBGREEN dye).
A specific amplification primer for amplifying a circular RNA, the specific amplification primer being obtained by:
step 1, designing a circRNA molecule amplification primer:
(3) Design principle: (1) following the common primer design principle; (2) primers were designed near the cleavage site (backsplice junction);
(2) Sequence re-splicing: in order to meet the design requirement of crossing the cutting sites, according to the full-length nucleotide sequence of the circRNA of the embodiment 1, a 3 '-end 100-300nt length sequence is intercepted and placed in front of a 5' -end 100-300nt length sequence so as to be spliced again to form a new sequence, and the sequence comprises the cutting sites after loop splicing;
(3) Primer design: designing a primer by a conventional method aiming at the sequences obtained by re-splicing, and writing in a 5'- & gt 3' direction by default according to a linear storage rule of the common sequences;
(5) Primer output and specificity debugging: introducing the Primer sequence obtained in the step 3) into NCBI database (http:// Blast. NCBI. Nlm. Nih. Gov/Blast. Cgi), and performing Primer specificity comparison analysis and optimization by using a Primer-Blast tool;
(6) The primer information obtained, wherein the primer comprises an upstream primer and a downstream primer;
the nucleotide sequence of the upstream primer is: 5'-TTGCATTCACAGGGTCTACTG-3';
the nucleotide sequence of the downstream primer is: 5'-GTTCTCCTGTGGCTGGATTATAG-3'.
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 upstream primer has a TM value of 57.94 degrees, the downstream primer has a TM value of 58.36 degrees, and the TM value refers to the melting temperature of either the upstream primer or the downstream primer.
Step 2, based on step 1, primers were further verified by real-time PCR experiments, sanger sequencing:
(1) Dissolution profile: the dissolution curve is unimodal, and the Tm value is within the normal range;
(2) Electrophoresis pattern: the electrophoresis strip is single, and the size of the strip is correct;
(3) Sanger sequencing: the sequencing result is unimodal and the cyclization site is correct.
A kit for cervical cancer diagnosis, comprising specific amplification primers for amplifying circular RNAs. The kit also comprises reverse transcriptase, buffer solution and ddH 2 O, DNA polymerase, fluorescent dye and dNTP mix; 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 nucleotide sequence of the circular RNA was obtained as follows:
the sequence and structure information of the circular RNA hsa_circ_0035443 are obtained through circBase, CSCD and circbank databases, and the circular RNA is positioned in the 58284902-58306479 region of a human chromosome 15 and is formed by performing cleavage cyclization on the 3-8 region of the exon (exon) of the ALDH1A2 gene.
FIG. 1 is a gene structure diagram of a circular RNA, wherein the full length of the nucleotide sequence is 681nt. Wherein the first two nucleotides and the last two nucleotides are loop-forming binding sites.
Example 2
The present invention provides the use of the circular RNA of example 1 as a diagnostic marker for cervical cancer:
step 1, obtaining a cervical cancer circRNA expression profile by high-throughput sequencing of 5 cases of paired cervical cancer patient cancers and tissues beside the cancers, wherein the circular RNA hsa_circ_0035443 is remarkably expressed in cervical cancer tissues in a low mode;
step 2, detecting the expression level of circular RNA hsa_circ_0035443 of cancers and tissues beside the cancers of 10 paired cervical cancer patients through real-time PCR, and verifying the sequencing result;
step 3, detecting the expression level of 4 cervical cancer cell circular RNAs hsa_circ_0035443 by real-time PCR;
step 4, through bioinformatic prediction, the response element hsa-miR-1228-3P of the circular RNA hsa_circ_0035443 is found, and the gene is an oncogene, acts by promoting cell cycle progression and cell migration capacity and negatively regulates P53 expression. And researches show that the over-expression of the mutant P53 is a common phenomenon of cervical cancer, and can be used as an early warning signal of cervical cancer occurrence, and the evolution condition of tumors can be judged by detecting the level of the P53 protein.
Thus, the circular RNA hsa_circ_0035443 can be used for cervical cancer diagnosis.
Example 3
The present invention provides a specific amplification primer for amplifying the circular RNA of example 1, which is obtained by the following steps:
step 1, designing a circRNA molecule amplification primer:
(4) Design principle: (1) following the common primer design principle; (2) primers were designed close to the cleavage site (backsplice junction).
(2) Sequence re-splicing: to meet the design requirements of the cross-cut sites, according to the full-length nucleotide sequence of the circRNA of the embodiment 1, a 3 '-end 100-300nt length sequence is cut and placed in front of a 5' -end 100-300nt length sequence to be re-spliced to form a new sequence, and the sequence comprises the cut sites after loop splicing.
(3) Primer design: for the sequences obtained by re-splicing, designing primers by a conventional method, and writing in the 5 '. Fwdarw.3' direction by default according to the linear storage rule of the common sequences.
(5) Primer output and specificity debugging: introducing the Primer sequence obtained in 3) into NCBI database (http:// Blast. NCBI. Nlm. Nih. Gov/Blast. Cgi), and performing Primer specificity comparison analysis and optimization by using a Primer-Blast tool.
(6) The primer information obtained:
the nucleotide sequence of the upstream primer is: 5'-TTGCATTCACAGGGTCTACTG-3';
the nucleotide sequence of the downstream primer is: 5'-GTTCTCCTGTGGCTGGATTATAG-3'.
In one possible implementation, the GC content of the upstream primer is 47.62% and the GC content of the downstream primer is 47.83%, where GC content refers to the ratio of guanine to cytosine in the 4 bases of DNA. The upstream primer has a TM value of 57.94 degrees, the downstream primer has a TM value of 58.36 degrees, and the TM value refers to the melting temperature of either the upstream primer or the downstream primer.
FIG. 2 is a schematic representation of a circular RNA primer design, and it can be seen that the amplified product will contain cleavage sites.
Step 2, based on step 1, primers were further verified by real-time PCR experiments, sanger sequencing:
(1) Dissolution profile: the dissolution curve is unimodal, and the Tm value is within the normal range;
(2) Electrophoresis pattern: the electrophoresis strip is single, and the size of the strip is correct;
(3) Sanger sequencing: the sequencing result is unimodal and the cyclization site is correct.
FIG. 3 shows the results of the circular RNA primer verification, and shows that the dissolution curve shows a single peak, the electrophoresis band of the PCR product is single, and the band size is correct (refer to FIG. 7); the PCR product was taken for Sanger sequencing, showing a single peak of sequencing results and the cyclization site was correct. Thus, primer verification of the circular RNA was completed.
Example 4
The present invention provides a method for amplifying the circular RNA (hsa_circ_ 0035443) of example 1, comprising:
step 1, first strand cDNA is synthesized: mixing raw materials including circular RNA of example 1, random primer, ddH 2 O, dNTP, reverse transcription buffer, rnase inhibitor, reverse transcriptase, according to a first procedure;
step 2, carrying out real-time fluorescent quantitative polymerase chain reaction (real-time PCR) amplification on the first-strand cDNA prepared in the step 1: preparing an amplification system comprising the cDNA prepared in step 1, the upstream primer shown in example 3, the downstream primer shown in example 3, and ddH 2 O, qPCR Mix (containing SYBGREEN dye) was amplified and temperature-controlled according to the second procedure.
In one realizable mode, in step 1, the raw materials are mixed according to the following proportion: 1. Mu.L of the circular RNA of example 1, 1. Mu.L of random primer, 10. Mu.L of ddH 2 O, 2. Mu.L of dNTP mixture, 4. Mu.L of reverse transcription buffer, 1. Mu.L of RNase inhibitor, 1. Mu.L of reverse transcriptase, wherein dNTP mixture comprises dATP, dGTP, dCTP and dTTP;
(1) The first procedure is: reacting at 42 ℃ for 15min, reacting at 70 ℃ for 5min, and storing cDNA at-80 ℃ for standby.
(2) In the step 2, the amplification system is prepared according to the following proportion: 2. Mu.L of the cDNA prepared in step 1, 0.5. Mu.L of the upstream primer as in example 3, 0.5. Mu.L of the downstream primer as in example 3, and 7. Mu.L of ddH 2 O, 10. Mu.L real-time PCR amplification Mix (SYBGREEN dye); the second procedure is: the mixture was pre-denatured at 95℃for 10min, then denatured at 95℃for 15s, annealed at 60℃for 30s, and extended at 72℃for 30s, and the total was subjected to 38 cycles. The ABI7500 fluorescent quantitative PCR instrument selects a melting curve program, and continuously collects sample fluorescent signals during the ramp up process to obtain a melting curve.
Example 5
The invention also provides application of the specific amplification primer in preparing a kit for diagnosing cervical cancer.
Example 6
The invention also provides a kit for diagnosing cervical cancer, which comprises the specific amplification primer of the embodiment 3.
In one embodiment, the kit further comprises reverse transcriptase, buffer, ddH 2 O, 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.
The reagents and biological materials used in the examples below were all commercially available unless otherwise specified. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The experimental methods of molecular biology, which are not specifically described in the examples, are all carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j.
Example 7
Real-time PCR reactions detected hsa_circ_0035443 expression in cervical cancer tissues.
1. Designing specific amplification primers:
the sequences of the specific amplification primers designed by the present inventors and the related information are shown in Table 1:
TABLE 1 specific amplification primer sequences and related information
2. Total RNA extraction:
(1) Extracting total RNA of cervical cancer tissues by a Trizol method: taking about 0.2g of cervical cancer tissue, grinding under the condition of liquid nitrogen until the tissue is in a powder state, then adding 1mL of Trizol, fully grinding and uniformly blowing;
(2) Cracking the liquid sample prepared in the step (1) at room temperature for 5min, adding chloroform according to the proportion of adding 0.2mL chloroform into each 1mL Trizol, covering a tube cover tightly, standing for 5min after vortex shaking for 15s, centrifuging at 12000rpm for 15min at 4 ℃, mixing the liquid after centrifugation, layering into a lower chloroform phase, a middle protein layer, an upper colorless water phase, and distributing all RNA into the water phase;
(3) Transferring the water phase into a new centrifuge tube, adding equal volume of isopropanol, mixing uniformly, standing for 30min at-20 ℃, and centrifuging at 12000rpm for 10min at 4 ℃ to obtain precipitate, wherein all RNA exists in the precipitate;
(4) Removing the supernatant, adding 1mL of 75% ethanol into the system to clean RNA precipitate, and centrifuging at 7500rpm for 5min at 4 ℃;
(5) Repeating step (4);
(6) Removing ethanol solution, drying at room temperature for 5-10min, volatilizing ethanol, and removing RNase-free ddH 2 Adding O water into a centrifuge tube, and fully dissolving to obtain total RNA;
(7) The concentration and purity of RNA are measured by NanoDrop ND-2000, and after the quality of RNA is up to standard, split charging is carried out, and the RNA is preserved at-80 ℃.
3. First strand cDNA sequence Synthesis:
preparing a reverse transcription system by using a PCR tube, wherein the reverse transcription system is as follows: 1. Mu.L total RNA (about 500-1000 ng), 1. Mu.L random primer, 10. Mu.L ddH 2 O, 2. Mu.L dNTP mix (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 20. Mu.L; the reaction conditions are as follows: the reaction is carried out at 42 ℃ for 60min and at 70 ℃ for 5min. The cDNA obtained by reverse transcription is stored at-80 ℃ for standby.
4. Amplification verification of Hsa_circ_0035443 gene cDNA:
2 mu L of cDNA obtained by reverse transcription in the step 3 is taken, and a preparation system is subjected to PCR amplification;
(1) The PCR amplification system is as follows: 2. Mu.L of cDNA, 0.5. Mu.L of the upstream primer shown in Table 1, 0.5. Mu.L of the downstream primer shown in Table 1, 7. Mu.L of ddH 2 O,10 μLPCR amplified Mix, total volume 20 μL;
(2) The reaction conditions were 95℃for 10min, followed by 15s denaturation at 95℃and 30s annealing at 60℃and 30s extension at 72℃for a total of 38 cycles. Finally, the reaction is carried out at 72 ℃ for 5min.
(3) mu.L of the reaction product was taken and the PCR product was verified as a single specific amplified band at a voltage of 120V for 20min in 2.0g agarose/100 mL 1 XTAE buffer.
5. The Real-time PCR amplification reaction was detected using cDNA samples obtained by reverse transcription.
(1) The reaction system is as follows: 2. Mu.L cDNA, 0.8. Mu.L upstream primer, 0.8. Mu.L downstream primer, 6.4. Mu.L ddH 2 O, 10. Mu.L qPCR amplification Mix (SYBGREEN dye), total 20. Mu.L;
(2) Real-time PCR reaction conditions: the mixture was pre-denatured at 95℃for 10min, then denatured at 95℃for 15s, annealed at 60℃for 30s, and extended at 72℃for 30s, and the total was subjected to 38 cycles. The ABI7500 fluorescent quantitative PCR instrument selects a melting curve program, and continuously collects sample fluorescent signals during the ramp up process to obtain a melting curve.
(3) The amplification reaction was performed on a real-time fluorescent quantitative PCR apparatus ABI7500, and GAPDH was amplified as an internal control while amplifying the target gene, and the relative expression amount of the gene was calculated by a2 (. DELTA.CT) method.
For example 7, different samples were taken and repeated 6 times. In the internal reference GAPDH gene and hsa_circ_0035443 gene real-time PCR dissolution graph, the amplification peak is single, and no impurity peak is found, so that the primer specificity is good, and the amplification experiment is normal.
Example 8
Real-time PCR reactions detected hsa_circ_0035443 expression in tissues beside cervical cancer.
The procedure of example 7 was repeated, except that: in step 2, total RNA in the tissues beside the cervical cancer is extracted.
For example 8, different samples were taken and repeated 6 times.
Example 9
Real-time PCR reactions detected hsa_circ_0035443 expression 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 tissue relative to cervical cancer-side tissue;
(2) In the 4 cervical cancer cell samples of fig. 5, the expression of the circular RNA hsa_circ_0035443 was significantly down-regulated in the cervical cancer cell samples relative to 293T cells.
The above results show that the circular RNA hsa_circ_0035443 has differential expression in cervical cancer tissues and tissues beside the cancer, 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 cervical cancer tissues and cells, so the circular RNA hsa_circ_0035443 can be used for diagnosing cervical cancer.
Example 10
The localized expression of the circular RNA hsa_circ_0035443 in cells.
The nuclear separation kit is derived from a Biyun sky nuclear protein and cytoplasmic protein extraction kit, and the following steps of the kit are further optimized:
1. preparing a solution: three reagents (a cytoplasmic protein extraction reagent A, a cytoplasmic protein extraction reagent B and a nuclear protein extraction reagent) in the kit are dissolved at room temperature, and are immediately placed on ice after being dissolved and uniformly mixed. The appropriate amount of the cytoplasmic protein extract reagent A was taken for use, and PMSF was added to a final concentration of 1mM in a few minutes prior to use. The appropriate amount of the nuclear protein extraction reagent was taken for use, and PMSF was added within minutes prior to 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 to keep the cells from sticking tightly, and blown down with a pipette. Cells were collected by centrifugation, with the best effort to drain the supernatant, leaving a pellet of cells ready for use. The cells are prevented from being digested by pancreatin as much as possible so as to prevent the pancreatin from degrading the target protein to be extracted.
3. 200. Mu.l of PMSF-added cytoplasmic protein extraction reagent A was added per 20. Mu.l of cell pellet.
4. The highest velocity, vortex 5 seconds, was used to suspend and disperse the cell pellet completely. (the vortex time may be suitably prolonged if the cell pellet is not fully suspended and dispersed.)
5. Ice bath for 10-15 min.
6. 10. Mu.L of the cytoplasmic protein extraction reagent B was added. The highest speed was severe Vortex for 5 seconds and ice bath for 1 minute.
7. The highest speed is at intense Vortex for 5 seconds and centrifuged at 12,000-16,000g for 5 minutes at 4 ℃.
8. The supernatant (about 100 microliters of supernatant from the tip to the middle, no down from the tip, critical) was aspirated into the pre-chilled EP tube. 500. Mu.l Trizol was added directly to the EP tube and mixed well.
9. The supernatant in the sediment is sucked off, and the supernatant can be sucked off again by the white gun head after the yellow gun head is used for taking the supernatant.
10. The cell plasma protein extraction reagent A was added to the pellet at the highest speed for 5 seconds at severe Vortex, and the cell pellet was completely suspended and dispersed in ice for 10-15 minutes. (second washing)
Centrifuging at 11.4deg.C for 5min at 12,000-16,000g, and discarding supernatant (the supernatant must be removed, and the supernatant can be removed with yellow gun head, and then the supernatant is sucked again with white gun head, so that residual supernatant can not be left after complete suction, and the supernatant can not be sucked completely, thereby causing pollution of cytoplasmic protein).
12. For precipitation, 50. Mu.l of PMSF-added nucleoprotein extraction reagent was added.
13. The cell pellet was completely suspended and dispersed at the highest speed at Vortex 15-30 seconds. And adding Trizol, and uniformly mixing for later use.
The procedure of example 7 was repeated except that cervical cancer cells and total RNA of the above-mentioned nuclei 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 relatively low in the nucleus.
Example 11
RNase R detects the expression stability of the circular RNA hsa_circ_ 0035443.
RNase R (ribonucleotidase R) is a 3'-5' riboexonuclease derived from the E.coli RNR superfamily, which cleaves RNA stepwise from the 3'-5' direction into di-and tri-nucleotides. RNase R digests almost all linear RNA molecules, but does not digest circular RNA, lasso structures, or double stranded RNA molecules with 3' overhangs of less than 7 nucleotides. RNase R is commonly used in gene expression and variable cleavage studies, where linear RNA can be digested to enrich circular RNA (circRNAs) or lasso structure RNA (lariat RNA).
As can be seen in fig. 7: both circular and linear RNAs can be detected without RNase R, but after RNase R is added, the linear RNAs can not be detected, and the circular RNAs can still be detected, which indicates that the circular RNAs hsa_circ_0035443 are stable and are not easy to digest by RNase R, and conform to the characteristics of circular RNAs.
ROC curve analysis.
Analysis of the experimental data obtained in example 7 gave ROC curves, as shown in fig. 2, wherein the area under the curve AUC was 0.821 (p=0.037), indicating that the detection target was able to act as a specific marker for the detection of this circular RNA.
Those skilled in the art will appreciate that the area under the ROC curve is between 1.0 and 0.5, 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, has certain accuracy at 0.7-0.9, has higher accuracy at more than 0.9, and the value being more than 0.7 indicates that the detection target can be used as a specific marker for the detection.
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention are clearly and completely described above in conjunction with the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. The components of the 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.
Sequence listing
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ccagccacag gagaacaggt gtgtgaagtt caagaagcag acaaggcaga tatagacaaa 120
gcagtgcagg cagcccgcct ggctttctct cttggttcag tgtggagaag gatggatgct 180
tcagaaaggg gacgtctgtt ggataagctt gcagacttgg tggaacggga cagggcagtt 240
cttgcaacca tggaatccct aaatggtggc aaaccattcc tgcaagcttt ttatgtggat 300
ttgcagggcg tcatcaaaac ctttcgatat tacgcaggct gggctgataa aattcatggg 360
atgaccattc ctgtagatgg agactatttt acctttacaa gacatgaacc cattggagtg 420
tgtggacaga tcatcccatg gaacttcccc ctgctgatgt ttgcctggaa aatagctcca 480
gctttgtgct gtggcaatac agtagttatt aagccagcag agcaaacacc actcagtgca 540
ctctacatgg gagccctcat caaggaggct ggctttcctc ccggggtcat caatattttg 600
ccaggatatg ggccaacggc tggggcagca atagcttctc acattggcat agacaagatt 660
gcattcacag ggtctactga g 681

Claims (1)

1. Use of a specific amplification primer of circular RNA hsa_circ_0035443 in the preparation of a kit for cervical cancer diagnosis, characterized in that the specific amplification primer is obtained by the following steps:
step 1, designing a circRNA molecule amplification primer:
(1) Design principle: (1) following the common primer design principle; (2) designing a primer at a position close to the shearing site;
(2) Sequence re-splicing: in order to meet the design requirement of cross-cut sites, according to the full-length nucleotide sequence of the circular RNA hsa_circ_0035443, intercepting a sequence with the length of 100-300nt at the 3' end and placing the sequence with the length of 100-300nt at the 5' end in front of the sequence with the length of 100-300nt at the 5' end so as to form a new sequence by re-splicing, wherein the sequence comprises cut sites after circular splicing;
(3) Primer design: designing a primer by a conventional method aiming at the sequences obtained by re-splicing, and writing in a 5'- & gt 3' direction by default according to a linear storage rule of the common sequences;
(5) Primer output and specificity debugging: introducing the Primer sequence obtained in the step 3) into NCBI database, and performing Primer specificity comparison analysis and optimization by using a Primer-Blast tool;
(6) The primer information obtained, wherein the primer comprises an upstream primer and a downstream primer;
step 2, based on step 1, primers were further verified by real-time PCR experiments, sanger sequencing:
(1) Melting curve: the melting curve is unimodal, and the Tm value is within the normal range;
(2) Electrophoresis pattern: the electrophoresis strip is single, and the size of the strip is correct;
(3) Sanger sequencing: the sequencing result is unimodal, and the cyclization site is correct;
the primer is as follows:
the nucleotide sequence of the upstream primer is: 5'-TTGCATTCACAGGGTCTACTG-3';
the nucleotide sequence of the downstream primer is: 5'-GTTCTCCTGTGGCTGGATTATAG-3';
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;
the kit also comprises at least one of reverse transcriptase, buffer solution, ddH2O, DNA polymerase, fluorescent dye and dNTP mixed solution; the kit can detect hsa_circle_ 0035443 in cervical cancer tissues by using a real-time PCR method;
the method for amplifying the circular RNA hsa_circ_0035443 comprises the following steps:
step 1, first strand cDNA is synthesized: mixing raw materials, wherein the raw materials comprise circular RNA hsa_circ_0035443, random primer, ddH2O, dNTP mixed solution, reverse transcription buffer solution, RNase inhibitor and reverse transcriptase, and performing temperature control reaction according to a first program;
step 2, carrying out real-time fluorescent quantitative polymerase chain reaction amplification on the first-chain cDNA prepared in the step 1: preparing an amplification system, wherein the amplification system comprises cDNA, an upstream primer, a downstream primer and ddH2O, qPCR amplification Mix prepared in the step 1, and performing temperature control reaction according to a second program;
in the step 1, the following raw materials are mixed according to the following proportion: 1. Mu.L of circular RNA hsa_circ_0035443,1. Mu.L of random primer, 10. Mu.L of ddH2O, 2. Mu.L of dNTP mixture, 4. Mu.L of reverse transcription buffer, 1. Mu.L of RNase inhibitor, 1. Mu.L of reverse transcriptase, wherein dNTP mixture comprises dATP, dGTP, dCTP and dTTP;
the first procedure is: reacting for 15min at 42 ℃ and for 5min at 70 ℃, and storing cDNA at-80 ℃ for standby;
in the step 2, the amplification system is prepared according to the following proportion: 2. Mu.L of the cDNA prepared in step 1, 0.5. Mu.L of the upstream primer, 0.5. Mu.L of the downstream primer, 7. Mu.L of ddH2O, 10. Mu.L of real-time PCR amplified Mix;
the second procedure is: pre-denaturing at 95 ℃ for 10min, then denaturing at 95 ℃ for 15s, annealing at 60 ℃ for 30s, and extending at 72 ℃ for 30s, and performing 38 cycles in total; the ABI7500 fluorescent quantitative PCR instrument selects a melting curve program, and continuously collects sample fluorescent signals during the ramp up process to obtain a melting curve.
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