WO2024032321A1 - 一种检测环状RNA表达量的Touchdown PCR检测方法 - Google Patents

一种检测环状RNA表达量的Touchdown PCR检测方法 Download PDF

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WO2024032321A1
WO2024032321A1 PCT/CN2023/107804 CN2023107804W WO2024032321A1 WO 2024032321 A1 WO2024032321 A1 WO 2024032321A1 CN 2023107804 W CN2023107804 W CN 2023107804W WO 2024032321 A1 WO2024032321 A1 WO 2024032321A1
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circular rna
touchdown pcr
seq
rna
pcr amplification
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孟春燕
刘越
龙奕妃
李雪莹
刘洋
冯福民
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华北理工大学
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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  • the invention belongs to the field of molecular biology, and specifically relates to a Touchdown PCR detection method for detecting circular RNA expression.
  • Circular RNA is a covalently closed circular RNA, which is different from ordinary linear RNA and has the advantage of not being affected by exonucleases.
  • Circular RNA is a covalently closed circular RNA, which is different from ordinary linear RNA and has the advantage of not being affected by exonucleases.
  • circRNA in regulating the occurrence and development of diseases, treatment and diagnosis, etc. It is a hot topic in the field of epigenetics.
  • Circular RNA has many special aspects in research methods. For example, circular RNA does not have a polyA tail, and Oligo dT cannot be used as a reverse transcription primer; circular RNA is a circular structure, and nodes must be designed when designing PCR primers. primer pairs to ensure product specificity.
  • this primer design method limits the selection range of primer pairs and increases the difficulty of design. For this kind of primer, it is difficult to find a suitable annealing temperature when using conventional PCR procedures for amplification. The product is likely to contain dimers, and amplification conditions containing dimers cannot be used for real-time fluorescence quantification.
  • qRT-PCR generally has higher requirements on primer design during the operation process to avoid non-specific products and improve the specificity of the product through higher annealing temperature.
  • the abundance of the target gene in the sample is low, it is difficult to detect it even using real-time fluorescence quantitative PCR technology, and it is even more difficult to achieve relative quantification.
  • the purpose of the present invention is to provide a Touchdown PCR (falling PCR) detection method for detecting the expression level of circular RNA, so as to solve the problems existing in the above-mentioned prior art.
  • the Touchdown PCR detection method can effectively amplify low-abundance circular RNA, is superior to ordinary PCR in terms of sensitivity and specificity, and has an amplification efficiency of up to 97.90%, which can improve the accuracy of quantitative analysis of differential expression. sex.
  • the present invention provides the following solutions:
  • the present invention provides a method for detecting the expression level of circular RNA, which includes the step of detecting the expression level of the circular RNA using a Touchdown PCR amplification method.
  • nucleotide sequence of the circular RNA is shown in SEQ ID NO: 5.
  • primer pairs used to amplify the circular RNA are as shown in SEQ ID NO: 1 ⁇ SEQ ID NO: 2.
  • step S3 the reaction system of the Touchdown PCR amplification is Mix 10 ⁇ L, cDNA 2 ⁇ L, forward primer 0.5 ⁇ L, reverse primer 0.5 ⁇ L, and RNase Free ddH 2 O is supplemented to 20 ⁇ L.
  • step S3 the reaction conditions of the Touchdown PCR amplification are 95°C, 2min; 95°C for 30s, 61-52°C for 20s, 4 cycles, with each cycle decreasing by 3°C; 95°C for 30s, 60°C 30s, 35 cycles; 72°C, 5min.
  • the present invention also provides a primer pair for detecting the expression level of circular RNA.
  • the nucleotide sequence of the primer pair is as shown in SEQ ID NO: 1-SEQ ID NO: 2; the nucleoside of the circular RNA
  • the acid sequence is shown in SEQ ID NO: 5.
  • the present invention also provides the application of the primer pair in detecting the expression level of the circular RNA.
  • the detection is to detect the expression amount of the circular RNA using a Touchdown PCR amplification method; the nucleotide sequence of the circular RNA As shown in SEQ ID NO: 5.
  • the present invention uses Touchdown PCR technology to amplify circular RNA circHDAC2 (circBase ID: hsa_circ_0077692), and applies gradient dilution of cDNA template to conduct real-time fluorescence quantitative detection. It is found that the Touchdown PCR program can accurately distinguish the starting template amount, Ct value and template The linear correlation R 2 of the -log value of the amount reaches 0.9962, and the amplification efficiency reaches 97.90%, which can achieve relative quantification of circular RNA.
  • the present invention compares the amplification effect of the Touchdown PCR program and the ordinary PCR program as well as the amplification effect of the quantitative detection of the Touchdown PCR program. At the same time, the changes in the relative expression of circHDAC2 before and after drug treatment are detected, indicating that the Touchdown PCR method can amplify circRNA. It has good specificity and sensitivity and can be used for qualitative and quantitative detection in laboratories.
  • the method established in the present invention reduces the difficulty of primer design and can efficiently detect the expression level of circular RNA, laying a foundation for in-depth research on circular RNA in the future.
  • Figure 1 shows the amplification products of the same circular RNA produced by two PCR reaction procedures, where M: DNAmarker; S: sample; R: RNA shearing enzyme, repeated 3 times;
  • Figure 2 shows the results of the Touchdown PCR amplification program, where A: amplification curve; B: dissolution curve; C: amplification product sequencing results;
  • Figure 3 shows the standard curve of Touchdown PCR amplification program
  • Figure 4 shows the product gel electrophoresis of Touchdown PCR after 5-fold gradient dilution of cDNA concentration.
  • M DNAmarker
  • 1-5 original cDNA concentration, 51-fold diluted cDNA, 52-fold diluted cDNA, 53-fold diluted cDNA, 54 dilute cDNA;
  • Figure 5 shows the relative expression changes of circular RNA detected by Touchdown PCR program.
  • the main purpose of the present invention is to improve a PCR method for amplifying circular RNA, in which the temperature selected for the annealing step in Touchdown PCR is initially set to be 5°C to 10°C higher than the calculated Tm of the primer. Annealing under high-intensity conditions facilitates perfect primer-template hybridization. In subsequent cycles, the annealing temperature is gradually reduced slightly, so that at the end of PCR, the annealing temperature is 2°C to 5°C lower than the calculated Tm of the primer. Because the efficiency of primer binding to the target fragment is much higher than that of the wrong fragment, when the target sequence undergoes several geometric amplification cycles, it becomes the main product of PCR.
  • PrimeScript TM RT reagent Kit (Shanghai Sangon Bioengineering Co., Ltd.); SYBRgreen with anit-Tap PCR mix (Beijing Jumei Biotechnology Co., Ltd.); isoniazid, rifampicin, pyrazinamide (Beijing Solebao) Technology Co., Ltd).
  • Primer design Search the circHDAC2 (circBase ID: hsa_circ_0077692) sequence from the circBase library, copy the last 20 nucleotide sequences of the sequence and splice it in front of the original sequence, and use Primer Premier 5 software to design a primer pair. It is required that the upstream primer must span the junction position of the circular RNA, and the downstream primer can be selected according to the general principles of primer design.
  • the primers used to amplify the circHDAC2 and internal reference GAPDH gene sequences are shown in Table 1.
  • the nucleotide sequence of the circular RNA circHDAC2 is as shown in SEQ ID NO: 5 (AGGCCCCATAAAGCCACTGCCGAAGAAATGACAAAATATCACAGTGATGAGTATATCAAATTTCTACGGTCAATAAGACCAGATAACATGTCTGAGTATAGTAAGCAGATGCAGAGATTTAATGTTGGAGAAGATTGTCCAGTGTTTGATGGACTCTTTGAGTTTTGTCAGCT CTCAACTGGCGGTTCAGTTGCTGGAGCTGTGAAGTTAAACCGACACAGACTGATATGGCTGTTAATTGGGCTGGAGGATTACATCATGCTAAGAAATCAGAAGCATCAGGATTCTGTTACGTTAATGATATTGTGCTTGCCATCCTTGAATTACTAAAGTATCATCAGAGAGTCTTATATATTGATATAGATATTCATCATCATGGTGATGGTGTTGAAGAAGCTTTATACAACAGATCGTGT
  • HepG2 cells were seeded in a 6-well plate and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 until the cell density reached 80% before drug intervention.
  • three drugs isoniazid + rifampicin + pyrazinamide, were dissolved in DMSO, and the cells were jointly exposed to the poison.
  • the final concentration of the drugs was: 150 ⁇ g/mL + 300 ⁇ g/mL + 800 ⁇ g/mL, while controlling The final DMSO concentration is less than 1%.
  • the experiment set up a blank control group and a solvent control group.
  • Total cellular RNA was extracted strictly according to the instructions of the kit. Add 1mL Trizol to each well, scrape the cells gently, transfer them fully to an RNase-free EP tube, and sonicate on ice for 30 seconds. Add 0.2 mL of chloroform per 1 mL of Trizol, shake and mix, let stand on ice for 2 min, and then centrifuge at 4°C and 12000 rpm for 15 min. Aspirate the water sample layer and place it in a 1.5 mL enzyme-free EP tube, add 0.5 mL isopropanol, mix by inverting up and down (do not shake), place on ice for 10 min, centrifuge at 4°C, 12000 rpm for 10 min, and remove the supernatant.
  • RNA 500 ng was extracted from cells using the Trizol method.
  • reverse transcription was performed using PrimeScript TM RT reagent Kit: PrimeScript RT Master Mix 4 ⁇ L, Total RNA 500 ng, and Nuclease-Free Water was added to make up to 20 ⁇ L.
  • the conditions are 37°C, 15min; 85°C, 6s; 4°CForever.
  • the reverse-transcribed cDNA was detected by RT-PCR using SYBRgreen with anit-Tap kit: Mix 10 ⁇ L, cDNA 2 ⁇ L, Forward primer (10 ⁇ M) 0.5 ⁇ L, reverse primer (10 ⁇ M) 0.5 ⁇ L, RNase Free ddH 2 O supplemented to 20 ⁇ L .
  • RT-PCR program is 95°C, 2min, (95°C, 3s; 60°C, 30s; 72°C, 15s) 40 cycles, 72°C, 5min.
  • Touchdown PCR program 95°C, 2min; 4 cycles of (95°C30s, 61-52°C20s), each cycle decreased by 3°C; (95°C30s, 60°C30s) 35 cycles; 72°C, 5min.
  • the reaction system for both PCR programs is 20 ⁇ L.
  • GAPDH was used as the internal reference for normalization, and the relative expression level of circular RNA was calculated using the 2 - ⁇ Ct method, where the calculation formula of ⁇ Ct is as shown in Formula I;
  • 3 ⁇ L of the PCR amplification product was electrophoresed on a 1% agarose gel at 100V for 45 min, and then observed and imaged using a UVP gel imaging system.
  • Amplification efficiency (10 - (1/k) -1) ⁇ 100% Formula II.
  • RNA shearing enzyme treatment (RNase R enzyme, Epicentre Company) will be performed in this example and will not be treated.
  • the same sample was processed for reverse transcription.
  • the enzyme treatment method is: digest the total RNA at 37°C for 30 minutes according to the ratio of 4U/ ⁇ g RNA, and after the digestion is completed, the enzyme is inactivated at 85°C for 5 minutes. Then calculate the RNA concentration by detecting A260 and A280 absorbance, and use 500ngRNA for reverse transcription.
  • the present invention uses a cDNA template to perform a 5-fold gradient concentration dilution and perform a Touchdown PCR procedure.
  • the gel electrophoresis results show that there is only one target gene and no other non-specific bands, and the bands are clear. The degree is directly related to the amount of starting template, indicating that the Touchdown PCR program has good sensitivity and relative quantitative capabilities (Figure 4).

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Abstract

一种检测环状RNA表达量的Touchdown PCR检测方法，属于分子生物学领域。所述方法通过提取人肝癌细胞系HepG2细胞中总RNA，逆转录成cDNA，以Touchdown PCR程序对circHDAC2片段进行实时荧光定量检测。与普通PCR扩增相比，所述方法对环状RNA的扩增具有更好的特异性和敏感性。所述方法减少了引物设计的难度，并且可以对环状RNA的表达量进行高效的检测，为今后环状RNA的深入研究奠定了基础。

Description

一种检测环状RNA表达量的Touchdown PCR检测方法
本申请要求于2023年06月08日提交中国专利局、申请号为202310676217.3、发明名称为“一种检测环状RNA表达量的Touchdown PCR检测方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于分子生物学领域，具体涉及一种检测环状RNA表达量的Touchdown PCR检测方法。
背景技术
环状RNA是一种共价闭合环状的RNA，不同于普通的线性RNA，具有不受核酸外切酶作用的优点。目前环状RNA在调控疾病发生发展、治疗诊断等方面都有深入的研究，是表观遗传学领域研究的一大热点，也有将环状RNA应用于疫苗开发的研究。
环状RNA在研究方法上具有很多特殊方面，比如环状RNA不具有polyA尾巴，不能采用Oligo dT来作为反转录引物；环状RNA是环形结构，在进行PCR引物设计时必须设计过结点的引物对，以保证产物的特异性。但这种引物设计方式就限制了引物对的选择范围，增大设计难度。而对于这种引物，利用常规PCR程序进行扩增时，很难找到合适的退火温度，产物很可能包含二聚体，而包含二聚体的扩增条件是不能用作实时荧光定量的。另外，qRT-PCR在操作过程中一般对引物设计的要求较高，以避免非特异性的产物，并通过较高的退火温度来提高产物的特异性。但当样品中目的基因丰度较低时，即使利用实时荧光定量PCR技术，也很难检测到，更难做到相对定量。
发明内容
本发明的目的是提供一种检测环状RNA表达量的Touchdown PCR(降落PCR)检测方法，以解决上述现有技术存在的问题。所述Touchdown PCR检测方法能够对低丰度的环状RNA进行有效扩增，在灵敏度、特异度上均优于普通PCR，且扩增效率可达97.90％，能够提高差异性表达定量分析的准确性。
为实现上述目的，本发明提供了如下方案：
本发明提供一种检测环状RNA表达量的方法，包括采用Touchdown PCR扩增方法检测所述环状RNA表达量的步骤。
进一步地，所述环状RNA的核苷酸序列如SEQ ID NO：5所示。
进一步地，用于扩增所述环状RNA的引物对如SEQ ID NO：1～SEQ ID NO：2所示。
进一步地，包括以下步骤：
S1.提取待测样品总RNA；
S2.以所述样品总RNA为模板，逆转录获得cDNA；
S3.以所述cDNA为模板，利用所述引物对进行Touchdown PCR扩增；
S4.根据Ct值计算所述环状RNA的表达量。
进一步地，在步骤S3中，所述Touchdown PCR扩增的反应体系为Mix10μL，cDNA2μL，正向引物0.5μL，反向引物0.5μL，RNase Free ddH2O补充到20μL。
进一步地，在步骤S3中，所述Touchdown PCR扩增的反应条件为95℃，2min；95℃30s，61-52℃20s，4个循环，每个循环降3℃；95℃30s，60℃30s，35个循环；72℃，5min。
本发明还提供一种用于检测环状RNA表达量的引物对，所述引物对的核苷酸序列如SEQ ID NO：1-SEQ ID NO：2所示；所述环状RNA的核苷酸序列如SEQ ID NO：5所示。
本发明还提供所述的引物对在检测环状RNA表达量中的应用，所述检测为采用Touchdown PCR扩增方法检测所述环状RNA的表达量；所述环状RNA的核苷酸序列如SEQ ID NO：5所示。
本发明公开了以下技术效果：
本发明利用Touchdown PCR技术扩增了环状RNA circHDAC2(circBase ID:hsa_circ_0077692)，应用梯度稀释的cDNA模板，进行实时荧光定量检测，发现Touchdown PCR程序可以准确的区分起始模板量，Ct值与模板量的-log值线性相关R2达0.9962，同时扩增效率达97.90％，能够实现环状RNA的相对定量。
本发明比较了Touchdown PCR程序与普通PCR程序扩增的效果以及Touchdown PCR程序定量检测的扩增效果，同时检测了circHDAC2在药物处理前后相对表达量的变化，表明Touchdown PCR方法对环状RNA的扩增具有良好的特异性和敏感性，能够用于实验室的定性和定量检测。
本发明建立的方法减少了引物设计的难度，并且可以对环状RNA的表达量进行高效的检测，为今后环状RNA的深入研究奠定了基础。
附图说明
图1为两种PCR反应程序对同一个环状RNA的扩增产物，其中，M：DNAmarker；S：样本；R：RNA剪切酶，3次重复；
图2为Touchdown PCR扩增程序结果图，其中，A：扩增曲线；B：溶解曲线；C：扩增产物测序结果；
图3为Touchdown PCR扩增程序标准曲线；
图4为5倍梯度稀释cDNA浓度后进行Touchdown PCR的产物凝胶电泳图，其中，M：DNAmarker；1-5：原始cDNA浓度，51倍稀释cDNA，52倍稀释cDNA，53倍稀释cDNA，54倍稀释cDNA；
图5为Touchdown PCR程序检测环状RNA的相对表达量变化。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
本发明的主要目的是改良一种用于扩增环状RNA的PCR方法，其中Touchdown PCR中，选择用于退火步骤的温度最初设定为比引物的计算Tm高5℃～10℃。在高强度条件下退火有利于形成完美的引物-模板杂交。在后续的循环中，退火温度逐渐少量降低，使PCR结束时，退火温度比引物的计算Tm低2℃～5℃。因为引物与目标片段结合的效率远高于错误片段，当目标序列经历几次几何扩增周期后，成为PCR的主要产物。
实施例1
1.材料与方法
1.1材料
主要试剂和材料：人肝癌细胞系HepG2(博士德生物技术股份有限公司)；
PrimeScriptTMRT reagent Kit试剂盒(上海生工生物工程有限公司)；SYBRgreen with anit-Tap PCR mix(北京聚合美生物科技有限公司)；异烟肼、利福平、吡嗪酰胺(北京索莱宝科技有限公司)。
主要仪器：荧光定量PCR仪(ABI StepOne plus，赛默飞世尔科技公司)。
1.2引物序列
引物的设计：从circBase库中查找circHDAC2(circBase ID:hsa_circ_0077692)的序列，将该序列最后20个核苷酸序列复制并拼接在原序列前面，利用Primer Premier 5软件设计引物对。要求其中上游引物必须跨过环状RNA的接点位置，下游引物可以按引物设计的一般原则进行选择。
用于扩增circHDAC2与内参GAPDH基因序列的引物如表1所示，环状RNA circHDAC2的核苷酸序列如SEQ ID NO：5(AGGCCCCATAAAGCCACTGCCGAAGAAATGACAAAATATCACAGTGATGAGTATATCAAATTTCTACGGTCAATAAGACCAGATAACATGTCTGAGTATAGTAAGCAGATGCAGAGATTTAATGTTGGAGAAGATTGTCCAGTGTTTGATGGACTCTTTGAGTTTTGTCAGCTCTCAACTGGCGGTTCAGTTGCTGGAGCTGTGAAGTTAAACCGACAACAGACTGATATGGCTGTTAATTGGGCTGGAGGATTACATCATGCTAAGAAATCAGAAGCATCAGGATTCTGTTACGTTAATGATATTGTGCTTGCCATCCTTGAATTACTAAAGTATCATCAGAGAGTCTTATATATTGATATAGATATTCATCATGGTGATGGTGTTGAAGAAGCTTTTTATACAACAGATCGTGTAATGACGGTATCATTCCATAAATATGGGGAATACTTTCCTGGCACAGGAGACTTGAGG)所示。
表1
1.3实验方法
1.3.1细胞培养及药物性肝损伤细胞模型建立
HepG2细胞接种于6孔板中，使用含10％胎牛血清的DMEM培养基，37℃，5％CO2培养，待细胞密度达到80％，进行药物干预。实验组，将异烟肼+利福平+吡嗪酰胺三种药物溶解在DMSO中，对细胞联合染毒处理，药物作用终浓度为：150μg/mL+300μg/mL+800μg/mL，同时控制DMSO终浓度低于1％。三种药物联合作用24h后，弃去培养基，使用PBS清洗细胞三次，加入1mL Trizol试剂提取细胞总RNA。实验设置空白对照组及溶剂对照组。
1.3.2 RNA的提取和检测
细胞总RNA提取严格按照试剂盒说明书。每孔加入1mL Trizol，轻轻刮取细胞，并充分转移至无RNA酶的EP管中，冰上超声破碎30s。每1mL Trizol加入0.2mL氯仿，震荡混匀，冰上静置2min，后于4℃，12000rpm离心15min。吸取水样层置于1.5mL无酶EP管中，加入0.5mL异丙醇，上下颠倒混匀(不可震荡)，冰上放置10min，于4℃，12000rpm离心10min，移除上清液。加入1mL预冷的75％乙醇(DEPC水配置)，旋涡振荡以充分混合样品，并于4℃，7500rpm离心5min。移去上清液，充分晾干后，吸取10μL DEPC水溶解RNA，然后立刻置于冰上进行RNA浓度测定。
1.3.3 RT-PCR实验
Trizol法提取细胞中总RNA后，使用PrimeScriptTMRT reagent Kit试剂盒进行逆转录：PrimeScript RT Master Mix 4μL，Total RNA 500ng，加Nuclease-Free Water补齐到20μL。条件为37℃，15min；85℃，6s；4℃Forever。
逆转录后的cDNA使用SYBRgreen with anit-Tap试剂盒进行RT-PCR检测：Mix 10μL，cDNA 2μL，Forward primer(10μM)0.5μL，反向引物(10μM)0.5μL，RNase Free ddH2O补充到20μL。
普通RT-PCR程序95℃，2min，(95℃，3s；60℃，30s；72℃，15s)40个循环，72℃，5min。
Touchdown PCR程序：95℃，2min；(95℃30s，61-52℃20s)4个循环，每个循环降3℃；(95℃30s，60℃30s)35个循环；72℃，5min。两种PCR程序反应体系均为20μL。以GAPDH为内参做均一化处理，以2-ΔΔCt法计算环状RNA的相对表达水平，其中ΔCt的计算公式如公式I所示；
ΔCt＝Ct目的-Ct内参，ΔΔCt＝ΔCt染毒组-ΔCt对照组  公式I。
1.3.4电泳
取3μLPCR扩增产物于1％琼脂糖凝胶100V电泳45min，用UVP凝胶成像系统观察并成像分析。
1.3.5扩增效率的计算
取反转录后的cDNA，以5倍浓度进行梯度稀释，分别获得5个不同稀释倍数cDNA模板。利用Touchdown PCR程序对5个浓度的模板进行扩增。以Ct值为纵坐标，以模板量-log值做横坐标，计算扩增标准曲线。曲线的斜率记作k。根据公式II计算扩增效率。
扩增效率＝(10-(1/k)-1)×100％  式II。
1.3.6统计学分析
用SPSS16.0进行统计学分析，使用两独立样本t检验比较两组之间的差异，检测水准α＝0.05。
2.结果
2.1 Touchdown PCR和普通PCR扩增目的基因片段
由于RNA剪切酶能够将线性RNA分解，而环状RNA由于特殊的共价闭合环状结构而被保留，本实施例中将进行RNA剪切酶处理(Rnase R酶，Epicentre公司)和未进行处理的同一样本进行逆转录。其中，酶处理方法为：按照4U/μg RNA的比例对总RNA进行消化处理37℃30min，消化结束后85℃5min令酶失活。然后通过检测A260与A280吸光度计算RNA浓度，并应用500ngRNA进行反转录。
然后利用普通RT-PCR和Touchdown PCR两种方法对每种样本进行扩增，在其他条件相同的情况下，结果显示：1)经过RNA剪切酶处理后RNA丰度下降；2)利用Touchdown PCR程序的特异性明显高于普通PCR程序，并且在不同的样本中Touchdown PCR程序的特异性都优于普通PCR程序(图1)。图中上部为普通RT-PCR程序扩增效果，下图为相同样本相同条件下利用Touchdown PCR程序进行扩增的效果展示。
2.2 Touchdown PCR程序的扩增产物分析
对cDNA模板进行梯度稀释后，利用Touchdown PCR程序对环状RNA以及GAPDH内参进行扩增，发现扩增产物单一(图2的A和图2的B)。随后将环状RNA的产物进行测序，将其与circBase库中的环状RNA序列进行比对，二者序列相吻合，证明扩增产物正确。部分测序结果如图2的C所示。
2.3 Touchdown PCR程序的扩增效率
将模板以梯度稀释的方法对Touchdown PCR程序的扩增效率进行计算。以cDNA模板进行5倍梯度浓度稀释，共获得5个浓度。设定初始模板量为55，稀释后依次为54，53，52，51，以其log值为横坐标，相应的Ct值为纵坐标做散点图，如图3所示。斜率k＝-3.3732，R2为0.9962(图3)，提示Touchdown PCR程序可以有效区分起始模板量的变化，且Ct值与模板量的log值呈线性相关。根据公式III计算扩增效率为97.9％；
扩增效率＝(10(-1/-3.3732)-1)×100％＝97.90％  公式III。
2.4 Touchdown PCR敏感性分析
因样本中环状RNA的丰度较低，本发明以cDNA模板进行5倍梯度浓度稀释，进行Touchdown PCR程序，凝胶电泳结果显示只有一条目的基因，无其他非特异性条带，且条带清晰程度与起始模板量直接相关，说明Touchdown PCR程序具有良好的敏感性以及相对定量能力(图4)。
2.5 Touchdown PCR程序的环状RNA相对定量
细胞经抗结核药物三药联合处理24h后，提取总RNA，利用Touchdown PCR程序对环状RNA进行qRT-PCR相对定量检测，药物处理后(模型组)环状RNA circHDAC2的表达量显著降低(图5)。表明药物处理影响了环状RNA的表达丰度，应用Touchdown PCR程序可以有效检测出环状RNA的表达量变化。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (16)

  1. 一种检测环状RNA表达量的方法，其特征在于，包括采用Touchdown PCR扩增方法检测所述环状RNA表达量的步骤。
  2. 根据权利要求1所述的方法，其特征在于，所述环状RNA的核苷酸序列如SEQ ID NO:5所示。
  3. 根据权利要求2所述的方法，其特征在于，用于扩增所述环状RNA的引物对的核苷酸序列如SEQ ID NO:1-SEQ ID NO:2所示。
  4. 根据权利要求3所述的方法，其特征在于，包括以下步骤：
    S1.提取待测样品总RNA；
    S2.以所述样品总RNA为模板，逆转录获得cDNA；
    S3.以所述cDNA为模板，利用所述引物对进行Touchdown PCR扩增；
    S4.根据Ct值计算所述环状RNA的表达量。
  5. 根据权利要求4所述的方法，其特征在于，在步骤S3中，所述Touchdown PCR扩增的反应体系为Mix 10μL，cDNA 2μL，正向引物0.5μL，反向引物0.5μL，RNase Free ddH2O补充到20μL。
  6. 根据权利要求4或5所述的方法，其特征在于，在步骤S3中，所述Touchdown PCR扩增的反应条件为95℃，2min；95℃ 30s，61～52℃ 20s，4个循环，每个循环降3℃；95℃ 30s，60℃ 30s，35个循环；72℃，5min。
  7. 一种用于检测环状RNA表达量的引物对，其特征在于，所述引物对的核苷酸序列如SEQ ID NO:1-SEQ ID NO:2所示；所述环状RNA的核苷酸序列如SEQ ID NO:5所示。
  8. 权利要求7所述的引物对在检测环状RNA表达量中的应用，其特征在于，所述检测为采用Touchdown PCR扩增方法检测所述环状RNA的表达量；所述环状RNA的核苷酸序列如SEQ ID NO:5所示。
  9. 根据权利要求8所述应用，其特征在于，所述Touchdown PCR扩增的反应体系为Mix 10μL，cDNA 2μL，正向引物0.5μL，反向引物0.5μL，RNase Free ddH2O补充到20μL。
  10. 根据权利要求9所述应用，其特征在于，所述cDNA为逆转录后得到的cDNA进行5倍梯度浓度稀释。
  11. 根据权利要求8或9所述应用，其特征在于，所述Touchdown PCR扩增时，内参基因为GAPDH基因；
    所述GAPDH基因的扩增引物的核苷酸序列为SEQ ID NO:3和SEQ ID NO:4所示。
  12. 根据权利要求8所述应用，其特征在于，所述Touchdown PCR扩增的反应条件为95℃，2min；95℃ 30s，61～52℃ 20s，4个循环，每个循环降3℃；95℃ 30s，60℃ 30s，35个循环；72℃，5min。
  13. 权利要求7所述的引物对在检测肝损伤中的应用，其特征在于，所述检测为采用Touchdown PCR扩增方法检测环状RNA的表达量；所述环状RNA的核苷酸序列如SEQ ID NO:5所示。
  14. 根据权利要求13所述应用，其特征在于，所述肝损伤为抗结核药物性肝损伤。
  15. 根据权利要求14所述应用，其特征在于，所述抗结核药物性肝损伤为异烟肼、利福平和吡嗪酰胺三种药物联合诱导的肝损伤。
  16. 根据权利要求15所述应用，其特征在于，所述异烟肼的药物浓度为150μg/mL；
    所述利福平的药物浓度为300μg/mL；
    所述吡嗪酰胺的药物浓度为800μg/mL。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105018495A (zh) * 2015-07-30 2015-11-04 广州吉赛生物科技有限公司 肝癌中环状rna-met基因及其表达方法和荧光定量pcr检测方法
CN109609656A (zh) * 2018-12-07 2019-04-12 湖北省农业科学院畜牧兽医研究所 一种山羊环状rna及其鉴定方法和功能应用
WO2022183011A1 (en) * 2021-02-26 2022-09-01 Mellios Nikolaos Circular rnas for diagnosis of depression and prediction of response to antidepressant treatment

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105018495A (zh) * 2015-07-30 2015-11-04 广州吉赛生物科技有限公司 肝癌中环状rna-met基因及其表达方法和荧光定量pcr检测方法
CN109609656A (zh) * 2018-12-07 2019-04-12 湖北省农业科学院畜牧兽医研究所 一种山羊环状rna及其鉴定方法和功能应用
WO2022183011A1 (en) * 2021-02-26 2022-09-01 Mellios Nikolaos Circular rnas for diagnosis of depression and prediction of response to antidepressant treatment

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"Master's Thesis", 1 June 2020, NORTH CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY, CN, article XU, JIE: "Competitive Expression of Circ_0077692 and HDAC2 Affects Anti-tuberculosis Drug-induced Liver Injury through Histone Deacetylation", pages: 1 - 71, XP009552551, DOI: 10.27108/d.cnki.ghelu.2020.000034 *
DATABASE Nucleotide 31 May 2023 (2023-05-31), ANONYMOUS : "Homo sapiens histone deacetylase 2 (HDAC2), transcript variant 2, non-coding RNA", XP093137457, retrieved from NCBI Database accession no. NR_033441.2 *
DRULA RARES, BRAICU CORNELIA, CHIRA SERGIU, BERINDAN-NEAGOE IOANA: "Investigating Circular RNAs Using qRT-PCR; Roundup of Optimization and Processing Steps", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, MOLECULAR DIVERSITY PRESERVATION INTERNATIONAL (MDPI), BASEL, CH, vol. 24, no. 6, 16 March 2023 (2023-03-16), Basel, CH , pages 5721, XP093137452, ISSN: 1422-0067, DOI: 10.3390/ijms24065721 *
LIU, CHEN: "Optimization and Application of Rice Circular RNA Experiment Identification Method", CHINESE MASTER’S THESES FULL-TEXT DATABASE, AGRICULTURE SCIENCE AND TECHNOLOGY, 15 January 2019 (2019-01-15) *
ZHANG PEI, FAN YUAN, SUN XIAOPENG, CHEN LU, TERZAGHI WILLIAM, BUCHER ETIENNE, LI LIN, DAI MINGQIU: "profiling reveals universal molecular mechanisms responsive to drought stress in maize and Arabidopsis", THE PLANT JOURNAL, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD., GB, vol. 98, no. 4, 1 May 2019 (2019-05-01), GB , pages 697 - 713, XP093137443, ISSN: 0960-7412, DOI: 10.1111/tpj.14267 *

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