Detection and Analysis of Circular RNAs by RT-PCR
采用RT-PCR检测和分析环状RNA
发布: 2018年03月20日第8卷第6期 DOI: 10.21769/BioProtoc.2775 浏览次数: 29187
评审: Gal HaimovichOmar AkilWilliam C. W. Chen
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The authors used this protocol in:
Jul 2017
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Abstract
Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) and the development of innovative bioinformatic pipelines have begun to allow the systematic identification of circRNAs (Szabo and Salzman, 2016; Panda et al., 2017b; Panda et al., 2017c). However, the validation of true circRNAs identified by RNA sequencing requires other molecular biology techniques including reverse transcription (RT) followed by conventional or quantitative (q) polymerase chain reaction (PCR), and Northern blot analysis (Jeck and Sharpless, 2014). RT-qPCR analysis of circular RNAs using divergent primers has been widely used for the detection, validation, and sometimes quantification of circRNAs (Abdelmohsen et al., 2015 and 2017; Panda et al., 2017b). As detailed here, divergent primers designed to span the circRNA backsplice junction sequence can specifically amplify the circRNAs and not the counterpart linear RNA. In sum, RT-PCR analysis using divergent primers allows direct detection and quantification of circRNAs.
Keywords: Circular RNA (环状RNA)
Backsplice junction (反向剪接连接)
Divergent primer (发散引物)
RT-PCR (RT-PCR)
RNase R (RNase R)
Background
CircRNAs are covalently closed, single-stranded RNAs lacking 5’ or 3’ ends. Although their genesis is poorly understood, they can arise from pre-mRNAs by a process called backsplicing (Panda et al., 2017d; Jeck et al., 2013). CircRNAs have been reported to be abundant, ubiquitously expressed, and conserved across species (Jeck et al., 2013). A number of studies have established that circRNAs can regulate gene expression by acting as competitors of pre-mRNA splicing, as decoys for microRNAs, as sponges for RBPs, and possibly also as substrates for translation (Panda et al., 2017d). In recent years, more than one hundred thousand circRNAs have been reported bioinformatically from high-throughput RNA sequencing (RNA-seq) (Glazar et al., 2014). Unfortunately, there is little overlap among different bioinformatic pipelines and there is no ‘gold standard’ method to validate the accuracy of circRNAs identified by different bioinformatic tools (Szabo and Salzman, 2016). However, RT-PCR has been widely used for validation of circRNAs identified by RNA-seq. This protocol describes the design of divergent primers which face away from each other on the linear RNA, so that they can only amplify the circRNAs, and not the linear RNAs with the same sequence. The PCR amplicon for the detection of circRNAs using divergent primers spans the backsplice junction of circRNAs. This method has been successfully used in several studies for the detection and quantification of circRNAs.
Materials and Reagents
- Standard pipette tips with a volume capacity of 10 µl, 20 µl, 200 µl, and 1 ml
- Nuclease-free 1.7-ml microcentrifuge tubes (Denville Scientific, catalog number: C2171 )
- ThermoGridTM rigid strip 0.2-ml PCR tubes [(Denville Scientific, catalog number: C18064 (1000859) ]
- MicroAmp® optical 384-well reaction plate (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4309849 )
- Optical adhesive film (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4311971 )
- Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14040-133 )
- Total RNA isolation-miRNeasy Mini Kit (QIAGEN, catalog number: 217004 )
- (Optional) TRIzol reagent (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 15596018 )
- Nuclease-free water (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9930 )
- RiboLock RNase inhibitor (40 U/µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EO0381 )
- RNase R (Lucigen, Epicentre, catalog number: RNR07250 )
- dNTP mix (10 mM each) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0193 )
- Random primers (150 ng/µl) (Sigma-Aldrich, Roche Diagnostics, catalog number: 11034731001 )
- Maxima reverse transcriptase (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EP0741 )
- KAPA SYBR® FAST ABI prism 2x qPCR master mix (Kapa Biosystems, catalog number: KK4605 ), or SYBR Green from other vendors
- QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704 )
- TBE Buffer, 10x, Molecular Biology Grade (Sigma-Aldrich, catalog number: 574795-1L )
- 1 Kb Plus DNA Ladder (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10787018 )
- UltraPureTM Agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500500 )
- Ethidium bromide solution (Sigma-Aldrich, catalog number: E1510-10ML )
- 2% agarose gel (see Recipes)
Equipment
- Manual Pipettes set of 2 µl, 20 µl, 200 µl and 1,000 µl (Mettler-Toledo, Rainin, catalog number: 17014393 , 17014392 , 17014391 , and 17014382 )
- Cell scraper (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 179707PK )
- Vortex mixer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88880018 )
- UV transilluminator
- Refrigerated centrifuge (Eppendorf, model: 5430 R )
- NanoDropTM One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDrop OneTM , catalog number: ND-ONE-W)
- PCR strip tube rotor, mini centrifuge C1201 [Denville Scientific, catalog number: C1201-S (1000806) ]
- Eppendorf® Thermomixer® C (Eppendorf, model: Thermomixer® C , catalog number: 5382000015)
- Veriti® 96-well thermal cycler (Thermo Fisher Scientific, Applied BiosystemsTM, model: VeritiTM 96-Well, catalog number: 4375786 )
- OwlTM EasyCastTM B1 Mini Gel Electrophoresis Systems (Thermo Fisher Scientific, Thermo ScientificTM, model: OwlTM EasyCastTM B1 , catalog number: B1)
- Gel imaging system (ProteinSimple, catalog number: FluorChem E system )
- MPS 1000 mini plate spinner (Next Day Science, catalog number: C1000 )
- QuantStudio 5 Real-Time PCR System, 384-well (Thermo Fisher Scientific, Applied BiosystemsTM, model: QuantStudioTM 5, catalog number: A28140 )
Procedure
文章信息
版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Panda, A. C. and Gorospe, M. (2018). Detection and Analysis of Circular RNAs by RT-PCR. Bio-protocol 8(6): e2775. DOI: 10.21769/BioProtoc.2775.
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分类
癌症生物学 > 通用技术 > 分子生物学技术
分子生物学 > RNA > RNA 检测
分子生物学 > RNA > qRT-PCR
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