CN111471769A

CN111471769A - Method for amplifying circular RNA, specific amplification primer and application

Info

Publication number: CN111471769A
Application number: CN202010324035.6A
Authority: CN
Inventors: 袁奕; 谢依; 张梦娇; 唐林香; 何胜祥
Original assignee: Jiangsu Toneker Medical Technology Co ltd
Current assignee: Jiangsu Toneker Medical Technology Co ltd
Priority date: 2020-04-22
Filing date: 2020-04-22
Publication date: 2020-07-31
Anticipated expiration: 2040-04-22
Also published as: CN111471769B

Abstract

A method, specific amplification primers and use for amplifying circular RNA (hsa _ circ _0005841), the method comprising: step 1, synthesizing first strand cDNA, and controlling temperature according to a first program; and 2, preparing an amplification system, and controlling the temperature according to a second program. The specific amplification primer acquisition steps are as follows: step 1, designing a circ RNA molecular amplification primer; and 2, further verifying the primers through real-time PCR experiments and Sanger sequencing on the basis of the step 1. The kit comprises specific amplification primers for amplifying the circular RNA. Wherein, the circular RNA presents low expression in cervical cancer tissues and cells, namely the content of the circular RNA in the cervical cancer tissues is far lower than that in paracarcinoma tissues, therefore, the circular RNAhsa _ circ _0005841 can be used as a molecular marker for diagnosing cervical cancer.

Description

Method for amplifying circular RNA, specific amplification primer and application

Technical Field

The invention belongs to the field of tumor molecular biology, and particularly relates to a method for amplifying circular RNA (hsa _ circ _0005841), a specific amplification primer and application.

Background

Cervical Cancer (CC) is one of the high-incidence cancers in women worldwide, with high morbidity and mortality. The global cancer statistics in 2018 show that there are about 57 million new cases of cervical cancer each year, 31 million people die of them, of which more than 80% of cervical cancer patients are in developing countries. As one of the most serious countries for cervical cancer burden, China has about 13 ten thousand cases each year, which account for about 20% of the worldwide cervical cancer incidence rate, and some regions have the tendency of increasing incidence rate and the affected population tending to be younger. With the widespread application of cervical cytology diagnosis and the increasing maturity of surgical therapies, the diagnosis rate and the therapeutic machine of cervical cancer are obviously improved, but the diagnosis rate and the therapeutic machine of cervical cancer still have not been satisfactory for middle and late stage patients, and many patients still die from local recurrence and distant metastasis. The control rate of local advanced cervical cancer is low, the invasiveness is high, and tumor cells are transferred and spread to adjacent and distant organs such as pelvic lymph nodes, retroperitoneum and the like through various ways, so that new tumors appear, and the treatment effect is poor. The elucidation of the mechanism of CC relapse and metastasis, and the accurate intervention, efficacy monitoring and prognosis evaluation are the key to improve the survival rate of the CC patients in middle and late stages. The discovery of key regulatory molecules and pathological mechanisms of CC relapse and metastasis and the search and identification of CC specific biomarkers are scientific problems to be solved urgently in the field, and have important significance for improving the current situation that the treatment of patients with late-stage metastatic CC is poor.

Circular RNA (circ RNA) is a special non-coding RNA molecule (a small amount of RNA is proved to be capable of coding protein), and is different from the traditional linear RNA (1 initial RNA, containing 5 'end and 3' end), the circ RNA molecule is in a closed circular structure, is not influenced by RNA exonuclease, is more stable in expression and is not easy to degrade. Functionally, recent studies have shown that circ RNA can be involved in a wide variety of processes in cell life activities, such as cell differentiation, proliferation, apoptosis, chromosome modification and inactivation, through pathways that regulate gene expression and function. Compared with the broad spectrum of the expression of the coding gene and miRNA, the expression of the circ RNA is often characterized by cell and tissue specificity, space-time difference of differentiation and development and the like. The function of circ RNA, which is currently being studied more, is to function as a miRNA sponge (miRNA sponge). miRNA is a single-stranded micro RNA with the length of about 22-23 nucleotides, and can inhibit the translation of a target gene or mediate the degradation process of the target gene by being combined with the mRNA 3' UTR region of the target gene, thereby playing a role in negatively regulating the expression of the target gene. Research suggests that mirnas can regulate about 30% of human genes. One gene may be regulated by multiple mirnas, while one miRNA may regulate the expression of multiple target genes to form a complex miRNA regulation network. In the occurrence and development of cancer, the circ RNA is taken as miRNA sponge to be combined with miRNA, so that functional miRNA is reduced, the function of miRNA for negatively regulating the target gene is inhibited, the abnormal expression of the gene is further caused, and the physiological and pathological processes are finally caused or changed. The above characteristics of the CircRNA make the CircRNA have potential to be used as a tumor diagnosis marker and applied to treatment.

So far, research aiming at cervical cancer discovers that expression changes of some circS RNAs are related to the occurrence and development of cervical cancer, such as circS L C26A4, circSMARCA5, circC L K3, circEIF4G2 and the like.

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 _0005841), a specific amplification primer and application.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a method for amplifying circular RNA (hsa _ circ _0005841), a specific amplification primer and application.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a method for amplifying a circular RNA (hsa _ circ _0005841), the method for amplifying a circular RNA (hsa _ circ _0005841) comprising:

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;

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 comprising the cDNA prepared in step 1, the forward primer shown in example 3, the reverse primer shown in example 3, 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:

step 1, designing a circ RNA molecule amplification primer:

(2) the design principle is that ① follows the common primer design principle, ② primer is designed across 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 circ RNA in the embodiment 1, a 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 a5 '→ 3' direction by default according to a general sequence linear storage rule;

(5) primer output and specificity debugging: the primer sequence obtained in 3) was introduced into NCBI database (http: v/Blast. nih. gov/Blast. cgi) using "Primer-Blast" tool for Primer-specific comparison analysis and optimization;

(6) the obtained primer information: the obtained 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.

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:

advantage (1): the invention provides a circular RNA hsa _ circ _0005841, a specific amplification primer and application thereof, wherein the circular RNA presents low expression in cervical cancer tissues and cells, namely the content of the circular RNA in the cervical cancer tissues is far lower than that of the circular RNA in para-carcinoma tissues, so the circular RNA circ-0044506 can be used as a molecular marker for diagnosing cervical cancer.

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 an ROC curve, and the area under the ROC curve was 0.800(P ═ 0.033) as shown in fig. 3. 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 _0005841 gene of the present invention;

FIG. 2 shows a design of the hsa _ circ _0005841 primer of the present invention;

FIG. 3 is a graph showing the results of the verification of the hsa _ circ _0005841 primer according to the present invention;

FIG. 4 shows the differential expression profile of real-time PCR detection of hsa _ circ _0005841 in cervical cancer tissue and paracancerous tissue according to the present invention;

FIG. 5 shows the real-time PCR detection of hsa _ circ _0005841 expression profile in cervical cancer cells of the invention;

FIG. 6 shows a ROC curve for the detection of hsa _ circ _0005841 in cervical cancer tissue of the invention.

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.

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; 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.

In step 1, the raw materials were mixed in the following ratio, 1. mu. L circular RNA of example 1, 1. mu. L 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 comprising the cDNA prepared in step 1, the forward primer shown in example 3, the reverse primer shown in example 3, 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 was prepared from 2. 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, and 7. mu. L ddH₂O, 10 μ L real-time PCR amplified Mix (SYBGREEN dye).

step 1, designing a circ RNA molecule amplification primer:

(3) the design principle is that ① follows the common primer design principle, ② primer is designed across the shearing site (backsplicijunction);

the nucleotide sequence of the upstream primer is as follows: 5'-ACTCCTAGAAGGTGTTCAAGATAA-3', respectively;

the nucleotide sequence of the downstream primer is as follows: 5'-AACAACTCTGAATGTCGGAATAAG-3' are provided.

Both the upstream primer and the downstream primer have 37.5 percent of GC content, wherein the GC content refers to the ratio of guanine to cytosine in 4 bases of DNA;

the TM value of the upstream primer is 56.82 degrees, the TM value of the downstream primer is 57.67 degrees, and the TM value refers to the melting temperature of the upstream primer or the downstream primer.

Wherein 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 _0005841 in cervical cancer tissues by using a real-time PCR method and is used for diagnosing cervical cancer.

Example 1

A cyclic RNA, the nucleotide sequence of which is obtained by the following steps:

the sequence and structure information of the circular RNA hsa _ circ _0005841 are obtained through a circBase, CSCD and a circbank database, and the circular RNA is positioned in the human chromosome 1 sense strand 213251037-213303232 region and is formed by reversely cutting and looping the 5-9-th exon of the RPS6KC1 gene.

FIG. 1 is a gene structure diagram of a circular RNA having a nucleotide sequence of 694nt in total length. Wherein, the first two nucleotides and the last two nucleotides are ring-forming binding sites.

Example 2

Use of circular RNA as a diagnostic marker for cervical cancer:

step 1, obtaining a cervical cancer circ RNA expression profile by high-throughput sequencing of 5 paired cervical cancer patients and para-carcinoma tissues, wherein the circular RNA hsa _ circ _0005841 is obviously low-expressed in the cervical cancer tissues;

step 2, further, detecting the expression levels of 10 matched cervical cancer patient cancers and the adjacent tissue circular RNA hsa _ circ _0005841 by real-time PCR, and verifying the sequencing result;

step 3, further detecting the expression level of the 4 cervical cancer cell circular RNA hsa _ circ _0005841 by real-time PCR;

step 4, further, through bioinformatics prediction, the cyclic RNA hsa _ circ _0005841 is found to be capable of driving a cerRNA network (hsa _ circ _0005841/miR-22-5p/RASSF4), and a target gene RASSF4(Ras Association domain Family Member 4) of the cyclic RNA is remarkably reduced in the mRNA expression profile of the cancer tissue of the cervical cancer patient and is positively correlated with the survival rate of the cervical cancer patient, and the cyclic RNA can play a role in tumor inhibition by inducing tumor cell apoptosis and inhibiting tumor cell growth.

Therefore, the circular RNA hsa _ circ _0005841 can be used for cervical cancer diagnosis.

Example 3

A specific amplification primer for amplifying the circular RNA in example 1 is obtained by the following steps:

step 1, designing a circ RNA molecule amplification primer:

(4) design rules ① follow the general primer design rules, ② primers were designed across the cleavage site (backsplicituunction).

(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 circ RNA 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: the primer sequence obtained in 3) was introduced into NCBI database (http: v/Blast. nih. gov/Blast. cgi), Primer-Blast tool was used for Primer-specific alignment analysis and optimization.

(6) The obtained primer information:

In a practical mode, the GC contents of the upstream primer and the downstream primer are both 37.5%, wherein the GC contents refer to the ratio of guanine and cytosine in 4 bases of DNA. Further, the TM value of the forward primer was 56.82 degrees, and the TM value of the reverse primer was 57.67 degrees, where the TM value refers to the melting temperature of the forward primer or the reverse primer.

FIG. 2 is a schematic of the circular RNA primer design, showing that the amplification product will contain a cleavage site.

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; 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

A method of amplifying the circular RNA of example 1, the method comprising:

step 1, first strand cDNA synthesis: mixing raw materials, wherein the raw materials comprise the circular RNA, the random primer, ddH2O, dNTP mixed solution, reverse transcription buffer solution, RNase inhibitor and reverse transcriptase in the embodiment 1, and performing temperature control reaction according to a first program; 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.

In step 1, raw materials are mixed according to the following ratio of 1 mu L example 1 circular RNA, 1 mu L 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, the upstream primer shown in the example 3, the downstream primer shown in the example 3, ddH2O and a Mix (containing SYBGREEN dye) amplified by qPCR, and controlling the temperature according to a second program. 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.

In step 2, the amplification system was prepared from cDNA prepared in step 1, 2. mu. L, upstream primer of example 3, 0.5. mu. L, downstream primer of example 3, 0.5. mu. L, 7. mu. L ddH2O, 10. mu. L real-time PCR amplified Mix (SYBGREEN dye).

Example 5

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 6

Real-time PCR reaction detected the expression of hsa _ circ _0005841 in cervical cancer tissue.

1. Designing a specific amplification primer:

the sequences and related information of specific amplification primers designed by the applicant are shown in table 1:

TABLE 1 specific amplification primer sequences and related information

2. Total RNA extraction:

extracting total RNA of a cervical cancer tissue by a Trizol method, namely taking about 0.2g of the cervical cancer tissue, grinding the cervical cancer tissue under the condition of liquid nitrogen until the tissue is in a powder state, then adding 1m L Trizol, fully grinding and uniformly blowing;

step (2), cracking the liquid sample prepared in the step (1) at room temperature for 5min, adding chloroform according to the proportion that 0.2m L of chloroform is added into Trizol of every 1m L, tightly covering a tube cover, 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 after centrifugation, and distributing all RNA into the water phase;

transferring the water phase into a new centrifugal 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;

removing the supernatant, adding 1m L75% ethanol into the system to clean the RNA precipitate, and centrifuging at 7500rpm at 4 ℃ for 5 min;

step (5) repeating step (4);

removing the ethanol solution, drying at room temperature for 5-10min until the ethanol is volatilized, adding the ddH2O water without the RNase into a centrifuge tube, and fully dissolving to obtain total RNA;

and (7) 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.

Step 3, synthesizing a first strand cDNA sequence:

a PCR tube is taken to be configured with a reverse transcription system, the reverse transcription system comprises 1 mu L total RNA (about 500-1000ng), 1 mu L random primer, 10 mu L ddH2O, 2 mu L dNTP mixed solution (dATP, dGTP, dCTP and dTTP), 4 mu L reverse transcription buffer (Thermo company), 1 mu L RNase inhibitor and 1 mu L reverse transcriptase, the total volume is 20 mu L, the reaction conditions are that the reaction is carried out at 42 ℃ for 60min, the reaction is carried out at 70 ℃ for 5min, and the cDNA obtained by reverse transcription is stored at-80 ℃ for standby.

4. And (3) verifying the cDNA amplification of the Hsa _ circ _0005841 gene:

taking cDNA obtained by reverse transcription in the step 3 of 2 mu L, and preparing a system for PCR amplification;

the PCR amplification system in the step (1) is 2 mu L cDNA, 0.5 mu L upstream primer shown in Table 1, 0.5 mu downstream primer shown in Table 1, 7 mu L ddH2O and 10 mu L PCR amplification Mix with the total volume of 20 mu L;

the reaction condition of the step (2) 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 38 cycles are carried out. Finally, the reaction was carried out at 72 ℃ for 5 min.

Step (3) taking 2 mu L reaction products, and verifying whether the PCR products are monospecific amplification bands or not under the conditions of 2.0g agarose/100 m L1 × TAE buffer solution, 120V voltage and 20 min.

5. The Real-time PCR amplification reaction was detected using a cDNA sample obtained by reverse transcription.

The reaction system in the step (1) is 2 mu L cDNA, 0.8 mu L upstream primer, 0.8 mu L downstream primer, 6.4 mu L ddH2O, 10 mu L Mix (SYBGREEN dye) amplified by qPCR, and the total volume is 20 mu L;

step (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.

And (3) carrying out amplification reaction on a real-time fluorescent quantitative PCR instrument ABI7500, amplifying a target gene and GAPDH as an internal reference control, and calculating the relative expression quantity of the gene by a 2 (-Delta CT) method.

For example 6, different samples were taken and repeated 10 times. Among them, in the internal reference GAPDH gene and bsa _ circ _0005841 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 7

The Real-time PCR reaction detected the expression of hsa _ circ _0005841 in the para-carcinoma tissue of cervical carcinoma.

The procedure of example 6 was repeated except that: in step 2, total RNA is extracted from the tissues adjacent to the cervical cancer.

For example 7, different samples were taken and repeated 10 times.

Example 8

Real-time PCR reaction detected the expression of hsa _ circ _0005841 in cervical cancer cells.

The procedure of example 6 was repeated except that: in step 2, total RNA of human cervical cancer cells is extracted.

For example 8, different samples were taken and repeated 5 times.

Analysis of real-time PCR results for examples 6-8,

1. analysis of Gene expression

The expression result of hsa _ circ _0005841 is shown in FIG. 4-FIG. 5:

(1) in the paired tissues of fig. 4, the expression of circular RNA hsa _ circ _0005841 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 _0005841 was significantly down-regulated in the cervical cancer cell samples relative to 293T cells.

The above results indicate that the circular RNA hsa _ circ _0005841 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 _0005841 has low expression in both cervical cancer tissues and cells, so the circular RNA hsa _ circ _0005841 can be used for diagnosis of cervical cancer.

2. ROC curve analysis

The analysis of the test data obtained in example 1 gave an ROC curve, as shown in fig. 6, in which the area under the curve AUC was 0.800(P ═ 0.033), indicating that the target of detection could be 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 the AUC is closer to 1 for AUC > 0.5, 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.

Sequence listing

<110> Jiangsu Cochinological medicine science and technology Co., Ltd

<120> method for amplifying circular RNA, specific amplification primer and application

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>24

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>1

actcctagaa ggtgttcaag ataa 24

<210>2

<211>24

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>2

aacaactctg aatgtcggaa taag 24

<210>3

<211>694

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>3

ataattgtat ggaagagata cagtgatttt aagaaactac acaaagaact atggcaaatt 60

cacaaaaact tattccgaca ttcagagttg tttcctccat ttgctaaagg aatagtgttt 120

gggcgatttg atgaaactgt tatcgaagag agaagacaat gtgctgaaga cctgctacag 180

ttctctgcca atattcctgc tctttacaat agtaaacagc ttgaagactt tttcaagggt 240

ggaataatta atgatagttc tgaattaatt ggtcctgctg aagctcactc agattccctc 300

attgatacct ttcctgagtg tagtacggaa ggcttctcca gtgacagtga tctggtatct 360

cttactgttg atgtggattc tcttgctgag ttagatgatg gaatggcttc caatcaaaat 420

tctcccatta gaacttttgg tctcaatctt tcttcggatt cttcagcact aggggctgtt 480

gcttctgaca gtgaacagag caaaacagaa gaagaacggg aaagtcgtag cctctttcct 540

ggcagtttaa agccgaagct tggcaagaga gattatttgg agaaagcagg agaattaata 600

aagctggctt taaaaaagga agaagaagac gactatgaag ctgcttctga tttttatagg 660

aagggagttg atttactcct agaaggtgtt caag 694

<210>4

<211>694

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>4

ggtatctctt actgttgatg tggattctct tgctgagtta gatgatggaa tggcttccaa 60

tcaaaattct cccattagaa cttttggtct caatctttct tcggattctt cagcactagg 120

ggctgttgct tctgacagtg aacagagcaa aacagaagaa gaacgggaaa gtcgtagcct 180

ctttcctggc agtttaaagc cgaagcttgg caagagagat tatttggaga aagcaggaga 240

attaataaag ctggctttaa aaaaggaaga agaagacgac tatgaagctg cttctgattt 300

ttataggaag ggagttgatt tactcctaga aggtgttcaa gataattgta tggaagagat 360

acagtgattt taagaaacta cacaaagaac tatggcaaat tcacaaaaac ttattccgac 420

attcagagtt gtttcctcca tttgctaaag gaatagtgtt tgggcgattt gatgaaactg 480

ttatcgaaga gagaagacaa tgtgctgaag acctgctaca gttctctgcc aatattcctg 540

ctctttacaa tagtaaacag cttgaagact ttttcaaggg tggaataatt aatgatagtt 600

ctgaattaat tggtcctgct gaagctcact cagattccct cattgatacc tttcctgagt 660

gtagtacgga aggcttctcc agtgacagtg atct 694

Claims

1. A method for amplifying a circular RNA (hsa _ circ _0005841), wherein the method for amplifying the circular RNA (hsa _ circ _0005841) comprises:

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, the upstream primer, the downstream primer and the ddH in the step 1₂O, qPCR amplify Mix, following a second temperature-programmed reaction.

2. The method of claim 1, wherein the circular RNA (hsa _ circ _0005841) is prepared by mixing the starting materials at a ratio of 1 μ L for circular RNA of example 1, 1 μ L random primer, and 10 μ L ddH₂O, 2 mu L dNTP mixed solution, 4 mu L reverse transcription buffer solution and 1 muL RNase inhibitor, 1 μ L reverse transcriptase, wherein the dNTP mixture comprises dATP, dGTP, dCTP and dTTP.

3. The method of claim 1, wherein the amplification system in step 2 is prepared from cDNA prepared in step 1 of 2 μ L, 0.5 μ L upstream primer, 0.5 μ L downstream primer, and 7 μ L ddH₂O, 10 μ L real-time PCR amplified Mix.

4. The method for amplifying circular RNA (hsa _ circ _0005841) 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.

5. The method for amplifying circular RNA (hsa _ circ _0005841) 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, designing a circ RNA molecule amplification primer:

(1) the design principle is that ① follows the common primer design principle, ② primer is designed across the shearing site (backsplicijunction);

(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;

7. The specific amplification primer for amplifying a circular RNA according to claim 6,

8. The specific amplification primer for amplifying the circular RNA as claimed in claim 6, wherein the GC contents of the upstream and downstream primers are both 37.5%, wherein the GC contents refer to the ratio of guanine and cytosine in 4 bases of DNA;

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 thatCharacterized in that 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 _0005841 in cervical cancer tissues by using a real-time PCR method and is used for diagnosing cervical cancer.