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Detection and Analysis of Circular RNAs by RT-PCR
采用RT-PCR检测和分析环状RNA   

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Nucleic Acids Research
Jul 2017

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

  1. Standard pipette tips with a volume capacity of 10 µl, 20 µl, 200 µl, and 1 ml
  2. Nuclease-free 1.7-ml microcentrifuge tubes (Denville Scientific, catalog number: C2171 )
  3. ThermoGridTM rigid strip 0.2-ml PCR tubes [(Denville Scientific, catalog number: C18064 (1000859) ]
  4. MicroAmp® optical 384-well reaction plate (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4309849 )
  5. Optical adhesive film (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4311971 )
  6. Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14040-133 )
  7. Total RNA isolation-miRNeasy Mini Kit (QIAGEN, catalog number: 217004 )
  8. (Optional) TRIzol reagent (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 15596018 )
  9. Nuclease-free water (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9930 )
  10. RiboLock RNase inhibitor (40 U/µl) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EO0381 )
  11. RNase R (Lucigen, Epicentre, catalog number: RNR07250 )
  12. dNTP mix (10 mM each) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0193 )
  13. Random primers (150 ng/µl) (Sigma-Aldrich, Roche Diagnostics, catalog number: 11034731001 )
  14. Maxima reverse transcriptase (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EP0741 )
  15. KAPA SYBR® FAST ABI prism 2x qPCR master mix (Kapa Biosystems, catalog number: KK4605 ), or SYBR Green from other vendors
  16. QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704 )
  17. TBE Buffer, 10x, Molecular Biology Grade (Sigma-Aldrich, catalog number: 574795-1L )
  18. 1 Kb Plus DNA Ladder (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10787018 )
  19. UltraPureTM Agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500500 )
  20. Ethidium bromide solution (Sigma-Aldrich, catalog number: E1510-10ML )
  21. 2% agarose gel (see Recipes)

Equipment

  1. Manual Pipettes set of 2 µl, 20 µl, 200 µl and 1,000 µl (Mettler-Toledo, Rainin, catalog number: 17014393 , 17014392 , 17014391 , and 17014382 )
  2. Cell scraper (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 179707PK )
  3. Vortex mixer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88880018 )
  4. UV transilluminator
  5. Refrigerated centrifuge (Eppendorf, model: 5430 R )
  6. NanoDropTM One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDrop OneTM , catalog number: ND-ONE-W)
  7. PCR strip tube rotor, mini centrifuge C1201 [Denville Scientific, catalog number: C1201-S (1000806) ]
  8. Eppendorf® Thermomixer® C (Eppendorf, model: Thermomixer® C , catalog number: 5382000015)
  9. Veriti® 96-well thermal cycler (Thermo Fisher Scientific, Applied BiosystemsTM, model: VeritiTM 96-Well, catalog number: 4375786 )
  10. OwlTM EasyCastTM B1 Mini Gel Electrophoresis Systems (Thermo Fisher Scientific, Thermo ScientificTM, model: OwlTM EasyCastTM B1 , catalog number: B1)
  11. Gel imaging system (ProteinSimple, catalog number: FluorChem E system )
  12. MPS 1000 mini plate spinner (Next Day Science, catalog number: C1000 )
  13. QuantStudio 5 Real-Time PCR System, 384-well (Thermo Fisher Scientific, Applied BiosystemsTM, model: QuantStudioTM 5, catalog number: A28140 )

Procedure

  1. Divergent primer design
    1. Get the mature sequence of circular RNA from the UCSC genome browser (https://genome.ucsc.edu/) using the genomic coordinates (Note 4).
    2. As shown in Figure 1, make the PCR amplicon template by joining the 100 nt sequence from the 3’ end to 100 nt sequence at the 5’ end of the circRNA (Note 5).


      Figure 1. Schematic illustration of circRNA biogenesis from backsplicing of pre-mRNA (top) and schematic representation of the design of divergent primers using the circRNA junction as template for PCR amplification (bottom)

    3. Use the above PCR amplicon template sequence to design PCR primers using the Primer 3 webtool (http://bioinfo.ut.ee/primer3/) or NCBI primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/).
    4. Make sure the PCR amplicon is between 120-200 nt long (Note 6).
    5. If you know the CircBase ID of your circRNA (Glazar et al., 2014), you may design divergent primers using the CircInteractome webtool (Dudekula et al., 2016) (http://circinteractome.irp.nia.nih.gov/Divergent_Primers/divergent_primers.html).

  2. Total RNA isolation
    1. Take ~2 million cultured cells and remove the culture media.
    2. Wash the cells three times with cold PBS at 4 °C.
    3. Immediately scrape the cells and transfer them to a 1.7-ml tube using cold DPBS to rinse the plate.
    4. Collect the cell pellet by centrifugation at 500 x g for 5 min at 4 °C.
    5. Immediately add 700 µl of QIAzol Lysis Reagent provided in the miRNeasy Kit and disrupt the cell pellet by pipetting.
    6. Prepare the total RNA using the miRNeasy Kit following the manufacturer’s instructions (Note 1).
    7. The RNA in nuclease-free water can be stored for 6 months at -20 °C or -80 °C, or used immediately for RNase R digestion and cDNA synthesis.

  3. Degradation of linear RNA by digestion with RNase R and cDNA synthesis
    1. Measure RNA concentration with a NanoDrop spectrophotometer.
    2. Prepare an RNase R digestion reaction containing 2 µg of prepared RNA, 1 µl RiboLock, 2 µl 10x RNase R reaction buffer, and 1 µl of RNase R; adjust the volume to 20 µl with nuclease-free water (Note 2).
    3. Prepare a control reaction exactly the same as the RNase R reaction but without RNase R.
    4. Incubate the reactions at 37 °C for 30 min and immediately proceed to RNA isolation.
    5. Prepare the RNA from the RNase R and control treated samples using miRNeasy Kit following the protocol provided by the manufacturer and elute in 40 µl of nuclease-free water.
    6. Prepare the cDNA synthesis reaction containing 12 µl of prepared RNA, 1 µl RiboLock, 1 µl dNTP mix, 1 µl random primers, 4 µl 5x RT buffer, and 1 µl Maxima reverse transcriptase (Note 2).
    7. Prepare No-RT reaction containing everything except the Maxima reverse transcriptase (Note 3).
    8. Mix the reaction gently and centrifuge for 10 sec to settle the reaction at the bottom of the tube.
    9. Incubate the reaction at 25 °C for 10 min followed by 30 min incubation at 50 °C for cDNA synthesis.
    10. Inactivate the reverse transcriptase by incubating the reaction at 85 °C for 5 min.
    11. The prepared cDNA can be stored at -20 °C or used immediately for PCR analysis.

  4. PCR and circRNA sequencing
    1. Prepare the forward and reverse divergent primer mix at a final concentration of 1 µM in nuclease-free water for the circRNA.
    2. Prepare the PCR reactions containing 25 µl of 2x SYBR Green mix, 0.1 µl cDNA, 12.5 µl divergent primer mix, and adjust the volume to 50 µl with nuclease-free water (Notes 2 and 10).
    3. Prepare another reaction same as above with 0.1 µl of no-RT instead of cDNA.
    4. Mix the reaction by tapping the tube with finger and centrifuge for a few seconds to settle the reactions at the bottom of the tube.
    5. Perform the PCR on a thermal cycler with a cycle setup of 3 min at 95 °C and 35 cycles of 5 sec at 95 °C plus 5 sec at 60 °C.
    6. Prepare ethidium bromide-containing 2% agarose gel (see Recipes) in 1x TBE buffer and resolve the whole 50 µl PCR product at 100 V until the loading dye reaches 3/4 of the gel.
    7. Visualize the PCR products on an ultraviolet transilluminator to confirm the size of the PCR product amplified (Note 7).
    8. Purify the PCR product from the agarose gel using the QIAquick Gel Extraction Kit following the manufacturer’s instructions (Figure 2).
    9. Quantify the PCR product concentration in the prepared DNA sample.
    10. Sequence the amplified PCR products with forward or reverse primers to find the backsplice junction sequence.


      Figure 2. Example circRNA PCR product resolved and visualized on ethidium bromide-stained agarose gel. The PCR product is submitted for DNA sequencing after gel purification.

  5. Quantitative PCR (qPCR) analysis of circRNA
    1. Prepare forward and reverse primer mixes for target mRNAs and circRNAs at a final concentration of 1 µM in nuclease-free water.
    2. Prepare the qPCR reactions in a 384-well plate containing 10 µl of 2x SYBR Green mix, 0.1 µl cDNA, and 5 µl primer mix. Adjust the volume to 20 µl with nuclease-free water (Notes 2 and 11).
    3. Vortex the reaction plate for few seconds after sealing the plate with optical adhesive film.
    4. Spin the plate for a few seconds to settle the reactions at the bottom of the wells.
    5. Set up the qPCR reaction cycle for 2 min at 95 °C and 40 cycles of 2 sec at 95 °C and 10 sec at 60 °C on QuantStudio 5 Real-Time PCR System (Note 2).
    6. The percentage (%) RNA left after RNase R treatment using the delta CT method as described in Table 1 (Notes 8 and 9) (Figure 3).

      Table 1. % of RNA left after RNase R treatment relative to control. The example CT values for linear and circRNAs in control and RNase R-treated samples, and calculation of RNA left after RNase R treatment.



      Figure 3. Hypothetical qPCR data showing the resistance of circRNA to RNase R treatment as calculated in Table 1. The qPCR results showing the levels of circRNAs and linear RNAs in RNase R (black) treated sample compared with the control treatment (grey).

Data analysis

To validate the existence of a circRNA, Sanger sequencing is to be performed on the PCR product amplified with the divergent primers (Figure 2). The PCR product sequence should match exactly the expected circRNA junction sequence as predicted from the RNA-seq (Panda et al., 2017a and 2017c). However, this analysis does not inform on whether the backsplice junction sequence is coming from a scrambled exon linear transcript or a real backsplice junction. To study this possibility, RNA is digested with RNase R, a 5’ to 3’ exonuclease known to degrade linear RNAs. As shown in Figure 3, following RNase R treatment, the linear X mRNA and Y mRNA are depleted to a level lower than 10%, while Y circRNA was not degraded (Table 1). The fact that Y circRNA level did not show depletion while the counterpart linear Y mRNA depleted to a minimal level with RNase R treatment supports the notion that RNase R degrades linear RNAs specifically leading to enrichment of circRNA population (Figure 3) (Panda et al., 2017c).

Notes

  1. Total RNA can also be prepared with the TRIzol reagent (Thermo Fisher Scientific) or any other total RNA isolation kit.
  2. To avoid contamination, a PCR workstation may be used to prepare the reaction mixtures for RNase R treatment, cDNA synthesis, RT-PCR, and qPCR.
  3. A ‘No-RT’ cDNA reaction serves as a negative control for cDNA synthesis and only low-level background should be amplified in the RT-PCR using specific primer sets.
  4. The mature sequence of circRNA can be obtained by joining the exon sequence present between the backsplice site coordinates in the genome.
  5. If the circular RNA is shorter than 200 nt, then the mature circRNA sequence can be divided into two halves and the template for primer design can be generated by joining the 3’ half to the 5’ end of 5’ half.
  6. The PCR amplicon of the divergent primers should span the circRNA backsplice junction; care should be taken that no primer overlap with the junction sequence.
  7. The PCR product on agarose gels should show a single product of the expected size and the ‘No-RT’ reaction should not amplify a product.
  8. The dissociation curve analysis for each primer set should show a single peak.
  9. CT values should represent the average of triplicate reactions. No normalization is needed for the qPCR analysis. The actual level of linear RNA depletion in RNase R treatment can be estimated by normalizing it to the level of counterpart circRNA.
  10. Any Taq polymerase in place of SYBR green mix can be used for this PCR reaction.
  11. As pipetting 0.1 µl of cDNA is difficult and error-prone, a master mix of cDNA and water is prepared depending on the final number of reactions required for each cDNA sample. Alternatively, the 20 µl prepared cDNA can be diluted to 1 ml with nuclease-free water and 5 µl of the diluted cDNA can be used in the qPCR reaction.

Recipes

  1. 2% agarose gel
    2 g of agarose in 100 ml of 1x TBE
    Ethidium bromide (EtBr) at a final concentration of approximately 0.2 μg/ml

Acknowledgments

ACP was supported by the Science & Engineering Research Board, a statutory body of the Department of Science &Technology (DST), Government of India (SERB/F/6890/2017-18). ACP and MG were supported by the National Institute on Aging, Intramural Research Program, National Institutes of Health.
This protocol was adapted from the previously published papers (Dudekula et al., 2016 and Panda et al., 2017b). The protocol was tested and optimized by different researchers in the Gorospe laboratory, National Institute on Aging, NIH (Panda et al., 2017c and Abdelmohsen et al., 2017). The authors have no conflicts of interest or competing interests to declare.

References

  1. Abdelmohsen, K., Panda, A. C., De, S., Grammatikakis, I., Kim, J., Ding, J., Noh, J. H., Kim, K. M., Mattison, J. A., de Cabo, R. and Gorospe, M. (2015). Circular RNAs in monkey muscle: age-dependent changes. Aging (Albany NY) 7(11): 903-910.
  2. Abdelmohsen, K., Panda, A. C., Munk, R., Grammatikakis, I., Dudekula, D. B., De, S., Kim, J., Noh, J. H., Kim, K. M., Martindale, J. L. and Gorospe, M. (2017). Identification of HuR target circular RNAs uncovers suppression of PABPN1 translation by CircPABPN1. RNA Biol 14(3): 361-369.
  3. Dudekula, D. B., Panda, A. C., Grammatikakis, I., De, S., Abdelmohsen, K. and Gorospe, M. (2016). CircInteractome: A web tool for exploring circular RNAs and their interacting proteins and microRNAs. RNA Biol 13(1): 34-42.
  4. Glazar, P., Papavasileiou, P. and Rajewsky, N. (2014). circBase: a database for circular RNAs. RNA 20(11): 1666-1670.
  5. Jeck, W. R. and Sharpless, N. E. (2014). Detecting and characterizing circular RNAs. Nat Biotechnol 32(5): 453-461.
  6. Jeck, W. R., Sorrentino, J. A., Wang, K., Slevin, M. K., Burd, C. E., Liu, J., Marzluff, W. F. and Sharpless, N. E. (2013). Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA 19(2): 141-157.
  7. Panda, A. C., Abdelmohsen, K. and Gorospe, M. (2017a). RT-qPCR detection of senescence-associated circular RNAs. Methods Mol Biol 1534: 79-87.
  8. Panda, A. C., De, S., Grammatikakis, I., Munk, R., Yang, X., Piao, Y., Dudekula, D. B., Abdelmohsen, K. and Gorospe, M. (2017b). High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs. Nucleic Acids Res 45(12): e116.
  9. Panda, A. C., Grammatikakis, I., Kim, K. M., De, S., Martindale, J. L., Munk, R., Yang, X., Abdelmohsen, K. and Gorospe, M. (2017c). Identification of senescence-associated circular RNAs (SAC-RNAs) reveals senescence suppressor CircPVT1. Nucleic Acids Res 45(7): 4021-4035.
  10. Panda, A. C., Grammatikakis, I., Munk, R., Gorospe, M. and Abdelmohsen, K. (2017d). Emerging roles and context of circular RNAs. Wiley Interdiscip Rev RNA 8(2).
  11. Szabo, L. and Salzman, J. (2016). Detecting circular RNAs: bioinformatic and experimental challenges. Nat Rev Genet 17(11): 679-692.

简介

真核细胞中的基因表达在转录和转录后水平受到严格调控。 mRNA转录,mRNA转录和mRNA翻译等后转录过程由RNA结合蛋白(RBPs)和非编码(nc)RNAs控制。大量的ncRNA家族包含多种调控RNA,如microRNAs和长的非编码(lnc)RNAs,但也是探索不足的一类环状RNAs。虽然三十多年前电子显微镜首次发现,但只有高通量RNA测序(RNA-seq)的出现和创新生物信息学管道的开发已经开始允许系统鉴定circRNA(Szabo和Salzman,2016;熊猫,2017b;熊猫等,2017c)。然而,通过RNA测序鉴定的真正的circRNA的验证需要其他分子生物学技术,包括常规或定量(q)聚合酶链反应(PCR)和Northern印迹分析(Jeck和Sharpless,2014)的逆转录(RT)。使用不同引物的环状RNA的RT-qPCR分析已被广泛用于检测,验证和有时定量circRNA(Abdelmohsen等人,2015和2017; Panda等人, ,2017b)。如在此详述的,设计为跨越循环RNA后接连接序列的分歧引物可以特异性扩增circRNA而不是对应的线性RNA。总之,使用不同引物的RT-PCR分析允许直接检测和定量circRNA。

【背景】CircRNAs是共价闭合的,缺少5'或3'末端的单链RNA。虽然它们的起源知之甚少,但它们可以通过称为反向剪接的过程从前体mRNA产生(Panda等人,2017d; Jeck等人,2013)。据报道,CircRNA在物种中是丰富的,无处不在表达的,并且是保守的(Jeck et al。 ,2013)。许多研究已经证实,circRNA可以通过作为前mRNA剪接的竞争者,作为微RNA的诱饵,作为RBP的海绵,并且可能还作为翻译的底物,来调节基因表达(Panda等人,2017d)。近年来,高通量RNA测序(RNA-seq)已经报道了超过10万个circRNAs(Glazar等人,2014)。不幸的是,不同生物信息学管道之间几乎没有重叠,并且没有“金标准”方法来验证不同生物信息学工具鉴定的circRNAs的准确性(Szabo和Salzman,2016)。然而,RT-PCR已被广泛用于验证由RNA-seq鉴定的circRNAs。该协议描述了在线性RNA上彼此背离的分歧引物的设计,以便它们只能扩增circRNA,而不是具有相同序列的线性RNA。用于使用分歧引物检测circRNA的PCR扩增子跨越circRNA的后缝连接。该方法已成功用于几项研究中用于检测和定量circRNAs。

关键字:环状RNA, 反向剪接连接, 发散引物, RT-PCR, RNase R

材料和试剂

  1. 标准移液器吸头,容量为10μl,20μl,200μl和1 ml
  2. 无核酸酶的1.7-ml微量离心管(Denville Scientific,目录号:C2171)
  3. ThermoGrid TM刚性带0.2ml PCR管[(Denville Scientific,目录号:C18064(1000859)]
  4. MicroAmp光学384孔反应板(Thermo Fisher Scientific,Applied Biosystems TM,目录号:4309849)
  5. 光学粘合剂膜(Thermo Fisher Scientific,Applied Biosystems TM,目录号:4311971)
  6. Dulbecco's磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,Gibco TM,目录号:14040-133)
  7. 总RNA分离-miRNeasy Mini Kit(QIAGEN,目录号:217004)
  8. (可选)TRIzol试剂(Thermo Fisher Scientific,Thermo Scientific TM,目录号:15596018)
  9. 无核酸酶的水(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9930)
  10. RiboLock RNA酶抑制剂(40U /μl)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EO0381)
  11. RNase R(Lucigen,Epicentre,目录号:RNR07250)
  12. dNTP混合物(每种10mM)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0193)。
  13. 随机引物(150ng /μl)(Sigma-Aldrich,Roche Diagnostics,目录号:11034731001)
  14. Maxima逆转录酶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EP0741)
  15. KAPA SYBR ®FAST ABI prism 2x qPCR master mix(Kapa Biosystems,产品目录号:KK4605)或其他供应商提供的SYBR Green
  16. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
  17. TBE缓冲液,10倍分子生物学级(Sigma-Aldrich,目录号:574795-1L)
  18. 1 Kb Plus DNA Ladder(Thermo Fisher Scientific,Invitrogen TM,目录号:10787018)
  19. UltraPure TM琼脂糖(Thermo Fisher Scientific,Invitrogen TM,产品目录号:16500500)
  20. 溴化乙锭溶液(Sigma-Aldrich,目录号:E1510-10ML)
  21. 2%琼脂糖凝胶(见食谱)

设备

  1. 2μl,20μl,200μl和1,000μl手动移液器组(Mettler-Toledo,Rainin,产品目录号:17014393,17014392,17014391和17014382)
  2. 细胞刮刀(Thermo Fisher Scientific,Thermo Scientific TM,目录号:179707PK)
  3. 涡旋混合器(Thermo Fisher Scientific,Thermo Scientific TM,目录号:88880018)
  4. 紫外透照器
  5. 冷冻离心机(Eppendorf,型号:5430 R)
  6. NanoDrop TM One / OneC微量紫外可见分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop One TM TM,产品目录号:ND -ONE-W)
  7. PCR带管转子,微型离心机C1201 [Denville Scientific,目录号:C1201-S(1000806)]
  8. Eppendorf®Thermomixer®C(Eppendorf,型号:Thermomixer®C,目录号:5382000015)
  9. Veriti 96孔热循环仪(Thermo Fisher Scientific,Applied Biosystems TM,型号:Veriti TM 96-Well,目录号:4375786)
  10. Owl TM EasyCast TM B1 Mini Gel Electrophoresis Systems(Thermo Fisher Scientific,Thermo Scientific TM,型号:Owl TM TM) EasyCast TM B1,产品目录号:B1)
  11. 凝胶成像系统(ProteinSimple,目录号:FluorChem E系统)
  12. MPS 1000微型平板旋转器(Next Day Science,目录号:C1000)
  13. QuantStudio 5实时PCR系统,384孔(Thermo Fisher Scientific,Applied Biosystems TM,型号:QuantStudio TM 5,目录号:A28140)

程序

  1. 分歧的引物设计
    1. 从UCSC基因组浏览器获取成熟的环状RNA序列( https://genome.ucsc.edu/ )使用基因组坐标(注4)。
    2. 如图1所示,通过将circRNA 5'末端的3'端100nt序列连接到100nt序列(注5),制作PCR扩增子模板。


      图1.来自pre-mRNA反向剪接的circRNA生物合成的示意图(顶部em / em)和使用circRNA连接作为PCR扩增模板的不同引物设计的示意图( bottom )

    3. 使用上述PCR扩增子模板序列,使用Primer 3 webtool设计PCR引物( http://bioinfo.ut.ee / primer3 / )或NCBI引物-BLAST( http:// www.ncbi.nlm.nih.gov/tools/primer-blast/ )。
    4. 确保PCR扩增子长度在120-200nt之间(注6)。
    5. 如果您知道circRNA的CircBase ID(Glazar等人,2014年),您可以使用CircInteractome网络工具设计不同的引物(Dudekula等人,2016年)( http://circinteractome.irp.nia.nih.gov/Divergent_Primers/divergent_primers.html )。

  2. 总RNA分离
    1. 取约200万个培养细胞并除去培养基。

    2. 用冷PBS在4°C清洗细胞三次
    3. 立即刮细胞,并使用冷DPBS将其转移至1.7-ml管中冲洗平板。

    4. 收集细胞颗粒,500×g离心5分钟,4℃。
    5. 立即加入700μl的miRNeasy试剂盒中提供的QIAzol裂解试剂,并通过移液破坏细胞沉淀。

    6. 使用miRNeasy Kit按照制造商的说明准备总RNA(注1)。
    7. 无核酸酶水中的RNA可在-20°C或-80°C下保存6个月,或立即用于RNA酶R消化和cDNA合成。

  3. 用RNA酶R消化和cDNA合成降解线性RNA
    1. 用NanoDrop分光光度计测量RNA浓度。
    2. 准备RNA酶R消化反应,其中包含2μg制备的RNA,1μlRiboLock,2μl10x RNase R反应缓冲液和1μlRNase R;
      使用无核酸酶的水将体积调节至20μl(注2)。
    3. 准备与RNase R反应完全相同但没有RNase R的对照反应。

    4. 在37°C孵育反应30分钟,立即进行RNA分离。
    5. 按照制造商提供的方案,使用miRNeasy试剂盒制备RNA酶R中的RNA,并对照处理的样品,并在40μl不含核酸酶的水中洗脱。
    6. 准备cDNA合成反应,包含12μl制备的RNA,1μlRiboLock,1μldNTP混合物,1μl随机引物,4μl5x RT缓冲液和1μlMaxima逆转录酶(注2)。
    7. 准备包含除Maxima逆转录酶(注3)以外的所有物质的非RT反应。
    8. 轻轻混匀反应液,离心10秒,使反应管底部沉降。
    9. 将反应液在25°C孵育10分钟,然后在50°C孵育30分钟进行cDNA合成。

    10. 在85°C孵育反应5分钟,使逆转录酶失活
    11. 制备的cDNA可以保存在-20°C或立即用于PCR分析。

  4. PCR和circRNA测序
    1. 准备正向和反向分歧的引物混合物,在无核酸酶的水中终浓度为1μM,用于circRNA。
    2. 准备包含25μl2x SYBR Green混合液,0.1μlcDNA,12.5μl不同引物混合液的PCR反应液,并用无核酸酶的水将体积调节至50μl(注2和10)。

    3. 用0.1μlno-RT代替cDNA制备与上述相同的另一个反应。
    4. 用手指轻轻敲击试管并离心几秒钟以解决管底反应。
    5. 在热循环仪上进行PCR,循环设置为95℃3分钟,35个循环,95℃5秒,加上60℃5秒。
    6. 在1x TBE缓冲液中制备含溴化乙锭的2%琼脂糖凝胶(参见食谱),并在100 V下分离全部50μlPCR产物,直至上样染料达到凝胶的3/4。

    7. 在紫外透射仪上显示PCR产物以确认扩增的PCR产物的大小(注7)。
    8. 按照制造商的说明书(图2),使用QIAquick凝胶提取试剂盒从琼脂糖凝胶中纯化PCR产物。
    9. 量化制备的DNA样品中的PCR产物浓度。
    10. 使用正向或反向引物对扩增的PCR产物进行序列分析,以找到反向连接序列。


      图2.在溴化乙锭染色的琼脂糖凝胶上解析并显现的circRNA PCR产物的实例在凝胶纯化后提交PCR产物用于DNA测序。

  5. 定量PCR(qPCR)分析circRNA

    1. 在无核酸酶的水中以1μM的终浓度为目标mRNA和circRNA制备正向和反向引物混合物。
    2. 在含有10μl2x SYBR Green混合物,0.1μlcDNA和5μl引物混合物的384孔板中制备qPCR反应。
      用无核酸酶的水将体积调节至20μl(注2和11)。

    3. 。用光学胶膜密封平板后,将反应板旋转几秒钟。
    4. 旋转板几秒钟以解决在井底部的反应。
    5. 使用QuantStudio 5实时荧光定量PCR系统(注2),在95°C设置qPCR反应循环2分钟,95°C 2秒,60°C 10秒,40次循环。
    6. 使用表1(注8和9)中所述的ΔCT方法(图3)进行RNA酶R处理后剩下的RNA百分比(%)。

      表1.相对于对照,RNA酶R处理后留下的RNA百分比。对照和RNA酶R处理的样品中线性和环状RNA的实例CT值,以及RNA酶R处理后留下的RNA的计算。



      图3.假设的qPCR数据显示了circRNA对RNA酶R处理的耐受性,如表1中所计算的。qPCR结果显示RNase R中的circRNA和线性RNA的水平(黑色 >)处理的样品与对照处理(灰色)相比。

数据分析

为了验证circRNA的存在,Sanger测序将在用不同引物扩增的PCR产物上进行(图2)。如从RNA-seq预测的(Panda等人,2017a和2017c),PCR产物序列应该完全匹配预期的circRNA连接序列。然而,这种分析并不能说明后裂缝连接序列是来自杂乱的外显子线性转录本还是真正的后彩膜连接。为了研究这种可能性,用RNA酶R(一种已知降解线性RNA的5'至3'外切核酸酶)消化RNA。如图3所示,接下来的RNA酶R处理后,线性mRNA和mRNA降低到低于10%的水平,而Y em > circRNA未降解(表1)。当RNase R处理减少至最低水平时,对照的线性Y染色体mRNA支持RNA酶R特异性降解线性RNA的观点,即Y染色circRNA水平不显示消耗的事实以富集circRNA群体(图3)(Panda等人,2017c)。

笔记

  1. 总RNA也可以用TRIzol试剂(Thermo Fisher Scientific)或任何其他总RNA分离试剂盒制备。
  2. 为了避免污染,可以使用PCR工作站来制备用于RNA酶R处理,cDNA合成,RT-PCR和qPCR的反应混合物。
  3. '无RT'cDNA反应可作为cDNA合成的阴性对照,只有低水平背景应在RT-PCR中使用特定引物扩增。
  4. circRNA的成熟序列可以通过连接基因组中的背脊位点坐标之间存在的外显子序列来获得。
  5. 如果环形RNA短于200nt,则可以将成熟的circRNA序列分成两半,通过将5'末端的3'末端连接到5'末端可以产生用于引物设计的模板。
  6. 扩散引物的PCR扩增子应跨越circRNA backsplice接点;应注意没有引物与连接序列重叠。
  7. 琼脂糖凝胶上的PCR产物应该显示预期大小的单一产物,而'无RT'反应不应该放大产物。
  8. 每个引物组的解离曲线分析应该显示一个单峰。
  9. CT值应代表三次反应的平均值。 qPCR分析不需要标准化。 RNase R治疗中线性RNA耗竭的实际水平可以通过将其标准化至对应的circRNA水平来估计。
  10. 任何 Taq 聚合酶代替SYBR绿色混合物都可用于此PCR反应。
  11. 由于吸取0.1μlcDNA很困难且容易出错,根据每个cDNA样品所需的最终反应次数制备cDNA和水的主要混合物。或者,20μl制备的cDNA可用无核酸酶的水稀释至1 ml,5μl稀释的cDNA可用于qPCR反应。

食谱

  1. 2%琼脂糖凝胶
    2克琼脂糖在100毫升1×TBE中
    终浓度约为0.2μg/ ml的溴化乙锭(EtBr)

致谢

ACP得到了科学与技术部门的支持。工程研究委员会,印度政府科学和技术部(DST)的法定机构(SERB / F / 6890 / 2017-18)。 ACP和MG得到了美国国立卫生研究院老龄化,校内研究计划的国家研究所的支持。
他的协议是从以前发表的文章(杜德库拉等人,2016和熊猫等人,2017b)改编的。该议定书由Gorospe实验室,美国国立卫生研究院老年研究所(NIH,Panda等人,2017c和Abdelmohsen等人,2017)中的不同研究人员进行了测试和优化。作者没有利益冲突或竞争利益声明。

参考

  1. Abdelmohsen,K.,Panda,AC,De,S.,Grammatikakis,I.,Kim,J.,Ding,J.,Noh,JH,Kim,KM,Mattison,JA,de Cabo,R.和Gorospe,M (2015)。 猴肌肉中的环状RNA:年龄依赖性变化 衰老(Albany NY) 7(11):903-910。
  2. Abdelmohsen,K.,Panda,AC,Munk,R.,Grammatikakis,I.,Dudekula,DB,De,S.,Kim,J.,Noh,JH,Kim,KM,Martindale,JL和Gorospe,M.( 2017年)。 HuR靶标环状RNA的鉴定揭示了CircPABPN1对PABPN1翻译的抑制。 RNA Biol 14(3):361-369。
  3. Dudekula,D.B.,Panda,A.C.,Grammatikakis,I.,De,S.,Abdelmohsen,K.和Gorospe,M.(2016)。 CircInteractome:探索循环RNA及其相互作用蛋白和microRNA的网络工具 < RNA Biol 13(1):34-42。
  4. Glazar,P.,Papavasileiou,P.和Rajewsky,N。(2014年)。 circBase:环状RNA的数据库 RNA 20 (11):1666-1670。
  5. Jeck,W. R.和Sharpless,N. E.(2014年)。 检测和表征环状RNA Nat Biotechnol 32( 5):453-461。
  6. Jeck,W.R.,Sorrentino,J.A.,Wang,K.,Slevin,M.K。,Burd,C.E.,Liu,J.,Marzluff,W.F。和Sharpless,N.E。(2013)。 环状RNA丰富,保守,并与ALU重复相关。 RNA 19(2):141-157。
  7. Panda,A. C.,Abdelmohsen,K.和Gorospe,M.(2017a)。 RT-qPCR检测衰老相关环状RNA 方法Mol Biol 1534:79-87。
  8. Panda,A. C.,De,S.,Grammatikakis,I.,Munk,R.,Yang,X.,Piao,Y.,Dudekula,D. B.,Abdelmohsen,K.和Gorospe,M.(2017b)。 高纯度环状RNA分离方法(RPAD)揭示大量内含子circRNA。核酸研究45(12):e116。
  9. Panda,A. C.,Grammatikakis,I.,Kim,K. M.,De,S.,Martindale,J. L.,Munk,R.,Yang,X.,Abdelmohsen,K.和Gorospe,M.(2017c)。 衰老相关环RNA(SAC-RNAs)的鉴定揭示了衰老抑制剂CircPVT1。 Nucleic Acids Res 45(7):4021-4035。
  10. Panda,A.C.,Grammatikakis,I.,Munk,R.,Gorospe,M.和Abdelmohsen,K.(2017d)。 环状RNA的新兴角色和背景 Wiley Interdiscip Rev RNA < 8(2)。
  11. Szabo,L。和Salzman,J。(2016)。 检测环状RNAs:生物信息学和实验难题 Nat Rev Genet 17(11):679-692。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
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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walaa nassif
faculty of pharmacy helwan university cairo egypt
also can i Ask if i can do RNase R treatment during the protocol of mirneasy extraction kit after separation of aqueos phase from qiazole and chloroform ??to save the extraction kits used again after rnase R treatment ?
5/27/2018 1:58:21 PM Reply
Amaresh Panda
Institute of Life Sciences

Hello,
We have not tried using RNase R in the aqueous phase from Qiazol preparation. The buffer condition in the aqueous phase may not be suitable for RNase R digestion. If you want to save a column, after RNase R treatment you may use phenol chloroform extraction method and precipitate the RNA using co-precipitant like Glycogen or Glycoblue (ThermoFisher SCientific).

5/28/2018 4:03:07 AM


walaa nassif
faculty of pharmacy helwan university cairo egypt
please sir...
can you clarify me the point please
how can we mesure the relative expression of circRNA in PCR quqntification against linear GADPH in the same sample subjected to Rnase R treatment..
and if no how delta delta CT is mesured???
5/27/2018 1:54:33 PM Reply
Amaresh Panda
Institute of Life Sciences

The level of GAPDH or any linear RNA will be very low after RNase R treatment. You can compare the Ct values of same circRNA or mRNA between RNase R and control treated sample to check their resistance to RNase R as described in the protocol. If you want to see the amount of linear RNA (e.g. mRNA X) left after RNase R digestion, you can use the counterpart circRNA (e.g. circRNA X) for normalisation using the delta delta Ct method.

5/28/2018 4:30:02 AM


an gelo lonoce
university f Bari

Working on human Pvt1 cicular RNA I had the same result with untreated samples and samples subjected to Rnase R .

5/28/2018 7:58:33 AM


walaa nassif
faculty of pharmacy helwan university cairo egypt

Thanks Mr.panda for help but forgive me did u use circular GADPH as a control in rnase treated samples or what ..I didn't get the idea

5/28/2018 8:35:16 AM


walaa nassif
faculty of pharmacy helwan university cairo egypt

Mr lonoce...
Can I get your working experience..
Did u select Linear or circ GADPH as the reference control on space.
If the linear one..how did u calculate felt delta ct of it circular...
Then if results didn't differ before or after Rnase treatment then rnase has no significant effect ..no need for its use

5/28/2018 8:42:42 AM


Amaresh Panda
Institute of Life Sciences

As mentioned in the methods, there was no normalization needed. Only Ct values were taken into calculations. I suggested about normalization using circular GAPDH only if you want to the real amount of linear RNA (GAPDH) left after RNase R treatment. RNase R is very efficient in degrading linear RNA. You should see a significant decrease in linear RNA level while the circRNA level should not change much. If both the linear and circRNA levels change in the same direction after RNase R treatment, then either the RNase R may not be working well or the predicted circRNA is not a real circular RNA.

5/28/2018 8:52:56 AM