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Apr 2017

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Antisense Oligo Pulldown of Circular RNA for Downstream Analysis
用于下游分析的环状RNA的反义Oligo Pulldown   

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Abstract

Circular RNAs (circRNAs) are a large family of noncoding RNA molecules that have emerged as novel regulators of gene expression by sequestering microRNAs (miRNAs) and RNA-binding proteins (RBPs). Several computational tools have been developed to predict circRNA interaction with target miRNAs and RBPs with a view to studying their potential effect on downstream target genes and cellular physiology. Biochemical assays, including reporter assays, AGO2 pulldown, ribonucleoprotein pulldown, and biotin-labeled RNA pulldown, are used to capture the association of miRNAs and RBPs with circRNAs. Only a few studies have used circRNA pulldown assays to capture the associated miRNAs and RBPs under physiological conditions. In this detailed protocol, the circRNA of interest (e.g., circHipk2) was captured using a biotin-labeled antisense oligo (ASO) targeting the circHipk2 backsplice junction sequence followed by pulldown with streptavidin-conjugated magnetic beads. The specific enrichment of circRNA was analyzed using reverse transcription quantitative PCR (RT-qPCR). Furthermore, the ASO pulldown assay can be coupled to miRNA RT-qPCR and western blotting analysis to confirm the association of miRNAs and RBPs predicted to interact with the target circRNA. In summary, the specific pulldown of circRNA using this quick and easy method makes it a useful tool for identifying and validating circRNA interaction with specific miRNAs and RBPs.

Keywords: CircRNAs (环状rna), CircRNA pulldown (环状rna pulldown), Antisense oligo (反义寡核苷酸), Biotin pulldown (生物素pulldown), RT-qPCR (RT-qPCR), miRNA (miRNA), RNA-binding proteins (rna结合蛋白)

Background

The RNA family can be broadly classified into coding (mRNAs) and noncoding RNAs. Interestingly, the vast majority of cellular RNAs are noncoding RNAs, including rRNA, lincRNA, miRNA, snRNA, snoRNA, tRNA, piRNA, and poorly characterized circular (circ) RNAs (Palazzo and Lee, 2015). The use of next-generation RNA sequencing and bioinformatics tools has uncovered more than a hundred thousand circRNAs in humans (Vromman et al., 2020). CircRNAs are generated from pre-mRNAs by head-to-tail splicing of specific exons by a process called backsplicing, which is regulated by transcription speed, RBPs, and inverted repeat sequences. Their length can vary from less than 100 nucleotides to a few thousand nucleotides. Depending on the sequence of circRNA origin from the parent gene, circRNAs have been classified into exonic circular RNA (circRNA), lariat-derived circular intronic RNA (ci-RNA), stable intronic sequence RNA (sisRNA), and exonic-intronic circular RNA (EIcircRNA) (Guria et al., 2019). Although a vast number of circRNAs have been identified in different cellular systems and disease models, only a fraction of circRNAs has been functionally characterized (Vromman et al., 2020). Recent evidence suggests that circRNAs play a critical role in regulating cellular events by interacting with miRNAs and RBPs (Guria et al., 2019). The majority of studies use computational tools to predict the association of circRNAs with cellular miRNAs and proteins that could regulate the expression of downstream target genes (Dudekula et al., 2016). Several biochemical techniques are currently used to analyze the circRNA-miRNA interaction, including luciferase reporter assays, AGO2 pulldown, and fluorescence in situ hybridization (FISH). Similarly, ribonucleoprotein pulldown assays using an antibody against the predicted RBP and biotin-labeled RNA pulldown assays to capture the target RBPs are used to validate the interaction of circRNA with specific RBPs. However, some of these methods are indirect assays to conclude whether the circRNA is associated with target miRNAs or RBPs. Our previous studies have successfully used circRNA pulldown assays with biotin-labeled antisense oligos targeting the circRNA backsplice junction sequence. Furthermore, circRNA-pulldown followed by RT-qPCR and western blotting analysis identified the miRNA or RBP associated with the circRNA of interest (Abdelmohsen et al., 2017; Panda et al., 2017; Pandey et al., 2020). Here, we adapted the published method to successfully pull down circHipk2 in βTC6 cells. The detailed protocol for the pulldown of circRNA to analyze the associated miRNA or RBP is described here.


Materials and Reagents

  1. Nuclease-free 1.5-ml microcentrifuge tubes (Tarson, catalog number: 500010)

  2. Nuclease-free 2-ml microcentrifuge tubes (Tarson, catalog number: 500020)

  3. 10 µl, 20 µl, 200 µl, and 1 ml tips (Tarson, catalog numbers: 524053, 528101, 528104, 528106)

  4. 96-well PCR plates (Thermo Fisher Scientific, catalog number: 4483485)

  5. Nuclease-free water (Thermo Fisher Scientific, catalog number: 10977023)

  6. 0.25% Trypsin (Thermo Fisher Scientific, catalog number: 25200056)

  7. Phosphate-buffered saline (PBS) (Sigma, catalog number: P4417-50TAB)

  8. 1 M Tris-HCl, pH 7.5 (Thermo Fisher Scientific, catalog number: 15567027)

  9. 1 M Tris-HCl, pH 8.0 (Thermo Fisher Scientific, catalog number: 15568025)

  10. 2 M Potassium chloride (Thermo Fisher Scientific, catalog number: AM9640G)

  11. 1 M Magnesium chloride (Thermo Fisher Scientific, catalog number: AM9530G)

  12. 5 M Sodium chloride (Thermo Fisher Scientific, catalog number: AM9759)

  13. Nonidet P-40 (Amresco, catalog number: M158-100ML)

  14. 0.5 M EDTA, pH 8.0 (Amresco, catalog number: E177-100ML)

  15. Triton X-100 (SRL, catalog number: 64518)

  16. Murine RNase inhibitor (NEB, catalog number: M0314S, 40 U/µl)

  17. 20× Protease inhibitor (Sigma, catalog number: S8830)

  18. Streptavidin Dynabeads (NEB, catalog number: S1420S)

  19. TRIzol (Thermo Fisher Scientific, catalog number: 15596018)

  20. Ethanol (HiMedia, catalog number: MB228)

  21. Chloroform (SRL, catalog number: 96764)

  22. Isopropanol (SRL, catalog number: 38445)

  23. GlycoBlueTM Coprecipitant (15 mg/ml) (Thermo Fisher Scientific, catalog number: AM9515)

  24. Maxima reverse transcriptase (Thermo Fisher Scientific, catalog number: EP0743)

  25. dNTP set, 100 mM solutions (Thermo Fisher Scientific, catalog number: R0181)

  26. Random primers (Thermo Fisher Scientific, catalog number: 48190011)

  27. PowerUp SYBR Green master mix (Thermo Fisher Scientific, catalog number: A25778)

  28. MicroAmp optical adhesive film (Thermo Fisher Scientific, catalog number: 4311971)

  29. Divergent DNA oligo primers synthesized by SIGMA for PCR amplification of circHipk2 (circHipk2-F: 5’-TCCAGACAACCGTACCGAGT-3’ and circHipk2-R: 5’-GGCACTTGATTGAAGGGTGT-3’)

  30. Custom antisense oligos labeled with biotin-TEG synthesized by SIGMA for control (Ctrl-ASO: 5’-TGCGTAACGAACGACGAATCGTCGCAGATC-3’[BtnTg]) and circHipk2 (circHipk2-ASO: 5’-CATGTGAGGCCATACCGGTAGTATCTGGAT-3’[BtnTg])

  31. 3× SDS loading dye (NEB, catalog number: B7703S)

  32. Polysome extraction buffer (PEB) (see Recipes)

  33. 2× Tris, EDTA, NaCl, Triton (TENT) buffer (see Recipes)

  34. 1× TENT (see Recipes)

Equipment

  1. Manual pipette set, 2 µl, 20 µl, 200 µl, and 1 ml (various manufacturers)

  2. Vortex mixer (Tarson, catalog number: 3001)

  3. Magnetic stand (Tarson, catalog number: S1509S)

  4. Tube rotator (Tarson, catalog number: 3071)

  5. Refrigerated centrifuge (Eppendorf, model: 5810R)

  6. Benchtop microfuge (Tarson, catalog number: 1010)

  7. Thermomixer (Eppendorf, catalog number: 5384000012)

  8. PCR machine (various manufacturers)

  9. QuantStudio 3 real-time PCR system (Thermo Fisher Scientific, catalog number: A28567)

Software

  1. UCSC genome browser (https://genome.ucsc.edu/)

  2. Primer3 webtool (https://bioinfo.ut.ee/primer3/)

  3. GeneRunner (http://www.generunner.net/)

  4. QuantStudio 3 and 5 system software

  5. Circinteractome website (https://circinteractome.nia.nih.gov/)

Procedure

  1. Oligo design

    1. Obtain the mature sequence of the circRNA of interest from the RNA sequencing data or the UCSC genome browser using the genome coordinates of backsplice sites. Here, we retrieved the sequence of mouse circHipk2 (chr6|38818229|38819313|-) from the mouse mm10 UCSC genome browser (Figure 1).

      Note: The mature splice sequence of multiexonic circRNA can be obtained by combining all the exon sequences between the genomic coordinates of the backsplice site.



      Figure 1. Design of the divergent primers and biotin-labeled antisense oligo targeting circHipk2


    2. As shown in Figure 1, join the last 15 nucleotides of the circRNA sequence to the upstream of the first 15 nucleotides to obtain the 30-nucleotide sequence spanning the circRNA backsplice junction.

    3. To design ASO, generate the reverse complement sequence of the 30-nucleotide junction sequence using GeneRunner. Have this sequence synthesized with biotin-TEG added to the 3’ end by the preferred vendor.

    4. Design primers for the target housekeeping gene mRNAs or rRNAs using the Primer 3 web tool.

    5. Design the divergent primer for the target circRNA as described previously (Panda and Gorospe, 2018) or using the circinteractome website for human circRNAs (Dudekula et al., 2016).


  2. Cell lysis

    1. Take one 100-mm dish of ~70% confluent βTC6 cells and discard the culture media.
      Note: A minimum of 5 million cells should be used for the pulldown assay. A higher amount of cells may help to obtain better pulldown of rare or low copy number circRNAs.

    2. Wash the cells three times with 5-10 ml ice-cold 1× PBS.

    3. Harvest the cells by trypsinization or scraping with a cell scraper.

    4. Pellet the cells by centrifuging at 1,000 × g for 2 min at 4°C and discard the supernatant.

    5. Resuspend the cell pellet in 1 ml ice-cold polysome extraction buffer (PEB).
      Note: Lysis with PEB releases the cytoplasmic fraction, not the nuclear fraction.

    6. Immediately add 5 µl RNase inhibitor and 50 µl 20× protease inhibitor.

    7. Mix well by pipetting ten times and keep on ice for 15 min, pipetting or vortexing for a few seconds every 4-5 min until the cells are lysed.

    8. Centrifuge the lysate at 12,000 × g for 10 min at 4°C.

    9. Collect 900 µl supernatant and proceed to the hybridization step (Figure 2).



      Figure 2. Schematic of the pulldown of circRNA using biotinylated-ASO targeting circHipk2


  3. Antisense oligo hybridization

    1. Add an equal volume (900 µl) of ice-cold 2× Tris, EDTA, NaCl, Triton (TENT) buffer to the supernatant collected in the above step in a 2-ml microcentrifuge tube.

    2. Divide the mixture into two tubes (one for the control-ASO pulldown and the other for the circRNA-ASO pulldown).

    3. Add 1 µl 100 µM (100 pmol) ctrl-ASO and circRNA-ASO to the control and circRNA pulldown tube, respectively.
      Note: The manufacturer's datasheet gives the streptavidin bead binding capacity. The ASO amount should be less than the streptavidin bead binding capacity for the pulldown in the next step. A higher amount of ASO may reduce the pulldown efficiency.

    4. Incubate the reactions on a rotor at 30 rpm for 90 min at 4°C.
      Note: The ASO hybridization reaction can be performed at room temperature or at 37°C to facilitate ASO binding to target circRNA; however, this may affect RNA integrity in samples with high levels of endogenous RNase.


  4. Streptavidin bead preparation

    1. Start the streptavidin bead preparation 15 min before the ASO hybridization step is completed.

    2. Mix well and place 100 µl magnetic streptavidin beads (50 µl for each reaction) into a new tube.

    3. Place the magnetic beads on a magnetic stand for 30 s and discard the supernatant.

    4. Resuspend the magnetic beads in 500 µl ice-cold 1× TENT buffer and place the tube on the magnetic stand for 30 s.

    5. Rotate the tubes 180º on the magnetic rack twice to wash the beads (Video 1).


      Video 1. Magnetic beads washing

    6. Discard the supernatant.

    7. Repeat the washing steps (Steps D4-D6) twice.

    8. Resuspend the magnetic streptavidin beads in 100 µl ice-cold 1× TENT buffer.


  5. Circular RNA pulldown

    1. Add 50 µl washed magnetic streptavidin beads into each hybridization reaction tube (mentioned in Step C4) after completing the 90-minute hybridization step.

    2. Add 1 µl RNase inhibitor and 5 µl 20× protease inhibitor to the tubes and mix well by pipetting.

    3. Rotate both the control and circRNA ASO tubes at room temperature for 30 min on a tube rotator at 30 rpm.

    4. Centrifuge the tubes briefly to bring all the liquid samples to the bottom of the tube without pelleting the beads.

      Note: High-speed centrifugation may pellet the beads, which may affect the washing steps.

    5. Place the tubes on a magnetic rack for 30 s and discard the supernatant.

    6. Resuspend the magnetic beads in 500 µl ice-cold 1× TENT buffer and place the tube on the magnetic stand for 30 s.

    7. Rotate the tube twice on the magnetic stand to wash the beads (Video 1).

    8. Allow the beads to settle toward the magnet and discard the buffer.

    9. Repeat the washing steps (steps 6-8) twice.
      Note: The number of washes may be decided depending on the fold enrichment of the target circRNA. We found that two to three washes are suitable to enrich circHipk2 in our pulldown assays.

    10. After the last wash, centrifuge the tube for a few seconds to settle the beads at the bottom.

    11. Place the tube on the magnetic stand for 30 s and discard the remaining supernatant.

    12. Resuspend the beads in 30 µl ice-cold PEB.

    13. Take 15 µl beads to a fresh tube for RNA isolation and RT-qPCR to analyze the pulldown efficiency of circRNA using the ASO and to detect the circRNA-associated miRNAs.

    14. Add 7 µl 3× SDS loading dye to the remaining 15 µl beads and mix by pipetting, then heat at 95°C for 5 min. This sample can be immediately used for western blotting to identify interacting RBPs or stored at -20°C.


  6. RNA and cDNA preparation from the pulldown sample

    1. Add 250 µl TRIzol reagent to the 15 µl beads for RNA isolation and mix well by pipetting.

      Note: Any similar RNA isolation reagent such as TriSure, RNAzol, TRI reagent, or TriPure can be used to prepare RNA. Column-based RNA isolation kits should be avoided since miRNAs are not purified well using the standard RNA isolation kits. Otherwise, use a kit that can isolate both long RNAs and miRNAs together.

    2. Add 50 µl chloroform and vortex for 15 s.

    3. Centrifuge at 12,000 × g for 15 min at 4°C, then collect 100 µl aqueous layer into a new tube.

    4. Add 100 µl isopropanol and 0.5 µl glycoblue as a co-precipitant.

    5. Mix well and keep at room temperature for 10 min, then centrifuge at 12,000 × g for 10 min at 4°C.

    6. Discard the supernatant, add 500 µl 75% ethanol to the RNA pellet, and vortex the tube for a few seconds.

    7. Centrifuge at 12,000 × g for 5 min at room temperature.

    8. Discard the supernatant and air-dry the RNA pellet for 3-5 min with the lid open.

      Note: Excessive drying of the RNA pellet or residual ethanol may inhibit the solubility of the pellet.

    9. Dissolve the pellet in 20 µl nuclease-free water. The RNA can be stored at -20°C for future use or used immediately for cDNA synthesis.

    10. Prepare a 20-μl cDNA synthesis reaction containing 13 μl prepared RNA, 0.5 μl Maxima reverse transcriptase, 0.5 μl RNase inhibitor, 1 μl 10 mM dNTP mix, 1 μl random primers, and 4 μl 5× RT buffer.

      Note: The cDNA can be prepared using any standard reverse transcription kit and random primers.

    11. Mix the reaction gently and incubate for 10 min at room temperature followed by 1 h at 50°C.

    12. Incubate the reaction at 85°C for 5 min to inactivate the reverse transcriptase.

    13. Dilute the cDNA with 250 µl nuclease-free water. This can be stored at -20°C or used immediately for PCR analysis.


  7. circRNA enrichment analysis by RT-qPCR

    1. Mix 10 μl 100 μM forward and reverse primer stock with 980 μl nuclease-free water to obtain a final concentration of 1 μM primer mix.

    2. Prepare 20-μl reactions in a 96-well plate containing 5 μl cDNA, 5 μl primer mix, and 10 μl 2× SYBR Green mix. Three technical replicates should be prepared.

    3. Seal the plate with an optical adhesive cover and vortex for a few seconds.

    4. Centrifuge the plate for a few seconds to bring the reactions to the bottom of the wells.

    5. Perform quantitative PCR on a QuantStudio 3 Real-Time PCR System with the following reaction setup: initial cycle for 2 min at 95°C followed by 40 cycles of 5 s at 95°C and 20 s at 60°C.

    6. Obtain the average Ct value of the technical replicates for each target in the control and circRNA pulldown samples.

    7. Calculate the percentage (%) enrichment of the target circRNA in the pulldown sample as compared with the control pulldown sample using the delta-CT method (Figure 3) (Livak and Schmittgen, 2001).



      Figure 3. Example data showing the percentage enrichment of circHipk2 in the circRNA-ASO pulldown sample relative to the control ASO pulldown sample


  8. Detection of interacting miRNAs and RBPs

    1. After confirming the enrichment of target circRNA in the circRNA ASO pulldown sample, associated miRNAs and RBPs can be analyzed.

    2. The miRNAs associated with the target circRNA can be analyzed by RT-qPCR. The RNA prepared in the above step can be used to produce miRNA cDNA followed by qPCR analysis to study their specific enrichment in the circRNA pulldown samples.

    3. Perform standard western blotting to detect circRNA-associated RBPs. Briefly, subject the input, control ASO, and circRNA ASO pulldown samples mixed with SDS dye to SDS-PAGE, and transfer the proteins to nitrocellulose membrane. Incubate the membrane with primary antibody against the predicted RBP followed by the appropriate secondary antibody and detect the chemiluminescence signals.

Data analysis

This method describes circular RNA pulldown using an antisense oligo specifically targeting the backsplice junction sequence of the target circRNA. However, the success of this pulldown assay depends on the availability of the circRNA junction sequence, which may be inaccessible due to secondary structures or junction-interacting RBPs, preventing the ASO from binding. Moreover, the PEB used for cell lysis is good for releasing the cytoplasmic fraction, while lysis of the nucleus for the pulldown of nuclear-localized circRNAs remains to be standardized. Since most circRNAs are cytoplasmic in nature (Jeck et al., 2013), we used PEB for the cell lysis and pulldown assay. Use the delta-Ct method to analyze the enrichment of target circRNA in the circRNA ASO pulldown sample compared with the control pulldown using a loading control such as 18S rRNA or Gapdh mRNA (Livak and Schmittgen, 2001). As shown in Figure 3, the circHipk2 levels are more than 2-fold higher in the circHipk2 ASO pulldown than in the control ASO pulldown. Alternatively, the efficiency of circRNA pulldown can be measured by comparing the pulldown samples with the flow through or input. After checking the enrichment of circRNA in the pulldown samples, the remaining RNA can be subjected to miRNA RT-qPCR analysis to check for the specific enrichment of miRNAs predicted to interact with the circRNA of interest. Furthermore, the other half of the pulldown sample may be used for western blotting analysis to evaluate the RBPs associated with the target circRNA. This is a promising method to pulldown the circular RNA of interest and study the circRNA-associated miRNAs and RBPs, which are critical factors for circRNA-mediated gene regulation.

Recipes

  1. Polysome extraction buffer (PEB)

    Reagents Stock   Vol required for 250 ml
    20 mM Tris-HCl, pH 7.5 1 M   5 ml
    100 mM KCl 2 M    12.5 ml
    5 mM MgCl2 1 M   1.25 ml
    0.5% Nonidet P-40 10%   12.5 ml
    Adjust the volume to 250 ml with nuclease-free water

  2. 2× Tris, EDTA, NaCl, Triton (TENT) buffer

    Reagents Stock   Vol required for 100 ml
    20 mM Tris-HCl pH 8.0 1 M   2 ml
    2 mM EDTA pH 8.0 0.5 M   0.4 ml
    500 mM NaCl 5 M   10 ml
    1% v/v Triton X-100 20%   5 ml
    Adjust the volume to 100 ml with nuclease-free water

  3. 1× TENT

    Mix equal volumes of PEB/water and 2× TENT

Acknowledgments

This research was supported by intramural funding from the Institute of Life Sciences and the Wellcome Trust/DBT India Alliance Intermediate Fellowship (IA/I/18/2/504017) provided to ACP. DD and AD were supported by Junior Research Fellowships from the University Grant Commission, India. This protocol was adapted from previously published papers (Abdelmohsen et al., 2017 and Panda et al., 2017). The authors thank our colleagues at the Institute of Life Sciences, Bhubaneswar, for proofreading the article.

Competing interests

The authors declare no conflicts of interest.

References

  1. 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.
  2. 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.
  3. Guria, A., Sharma, P., Natesan, S. and Pandi, G. (2019). Circular RNAs-The Road Less Traveled. Front Mol Biosci 6: 146.
  4. 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.
  5. Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods 25(4): 402-408.
  6. Palazzo, A. F. and Lee, E. S. (2015). Non-coding RNA: what is functional and what is junk? Front Genet 6: 2.
  7. Panda, A. C. and Gorospe, M. (2018). Detection and Analysis of Circular RNAs by RT-PCR. Bio-protocol 8(6): e3241.
  8. Panda, A. C., Grammatikakis, I., Kim, K. M., De, S., Martindale, J. L., Munk, R., Yang, X., Abdelmohsen, K. and Gorospe, M. (2017). Identification of senescence-associated circular RNAs (SAC-RNAs) reveals senescence suppressor CircPVT1. Nucleic Acids Res 45(7): 4021-4035.
  9. Pandey, P. R., Yang, J. H., Tsitsipatis, D., Panda, A. C., Noh, J. H., Kim, K. M., Munk, R., Nicholson, T., Hanniford, D., Argibay, D., Yang, X., Martindale, J. L., Chang, M. W., Jones, S. W., Hernando, E., Sen, P., De, S., Abdelmohsen, K. and Gorospe, M. (2020). circSamd4 represses myogenic transcriptional activity of PUR proteins. Nucleic Acids Res 48(7): 3789-3805.
  10. Vromman, M., Vandesompele, J. and Volders, P. J. (2021). Closing the circle: current state and perspectives of circular RNA databases. Brief Bioinform 22(1): 288-297.

简介

[摘要]环状RNA(circRNAs)是一个大家族的非编码该RNA分子的已通过螯合MI成为基因表达的新的调节剂croRNAs(微RNA)和RNA -结合蛋白(限制性商业惯例)。一些计算工具已经发展到预测与目标miRNA和限制性商业惯例circRNA互动,以期研究荷兰国际集团上下游靶基因和细胞生理学的潜在影响。乙化学分析,包括报告基因分析、AGO2 pulldown、核糖核蛋白 pulldown 和生物素标记的 RNA pulldown,用于捕获 miRNA 和 RBP 与 circRNA 的关联。只有少数研究使用 circRNA pulldown 分析在生理条件下捕获相关的 miRNA 和 RBP。在这种详细的协议中,感兴趣的circRNA(例如,circHipk2 )被捕获使用生物素标记的反义寡核苷酸(ASO)靶向的circHipk2 backsplice结序列,随后与链霉抗缀合的磁珠下拉。使用逆转录定量PCR(RT-qPCR)分析circRNA的特异性富集。此外,Ť他ASO拉下试验可耦合到的miRNA RT-qPCR的和western印迹婷分析,以确认miRNA和限制性商业惯例的关联预测为与目标circRNA相互作用。总之玛丽,circRNA的特定下拉使用此快速和容易的方法使得它用于识别和验证与特定miRNA和限制性商业惯例circRNA相互作用的有用工具。

[背景]的RNA家族可大致分为编码(的mRNA)和非编码RNA。有趣的是,绝大多数细胞 RNA 是非编码 RNA,包括 rRNA、lincRNA、miRNA、snRNA、snoRNA、tRNA、piRNA 和特征不佳的环状 (circ) RNA (Palazzo and Lee, 2015) 。采用新一代RNA测序和生物信息学工具也未在人体中覆盖超过十万circRNAs (Vromman等,2020) 。CircRNAs 是通过特定外显子的头对尾剪接从前体 mRNA 产生的,该过程称为反向剪接,该过程受转录速度、RBP 和反向重复序列的调节。他们的长度C的距离小于100个核苷酸变化到几千个核苷酸。根据从亲代基因circRNA起源的序列,circRNAs被分为外显子圆形RNA(circRNA),套索-衍生的圆形内含子RNA(CI-RNA),稳定内含子序列的RNA(sisRNA),和外显子-内含子环状RNA (EIcircRNA) (古里亚等人,2019) 。尽管已经在不同的细胞系统和疾病模型中鉴定了大量 circRNA,但只有一小部分 circRNA已被功能表征(Vromman等,2020)。最近的证据表明,circRNA通过与 miRNA 和RBP相互作用,在调节细胞事件中发挥着关键作用(Guria等,2019)。大多数研究使用的计算工具来预测circRNAs的关联与细胞的miRNA和蛋白质的是能够调节的表达下游靶基因(Dudekula等人,2016) 。目前有几种生化技术用于分析 circRNA-miRNA 相互作用,包括荧光素酶报告基因检测、AGO2 pulldown 和荧光原位杂交 (FISH)。类似地,核糖核蛋白下拉使用测定的针对预测RBP抗体和生物素标记的RNA拉下试验小号捕获限制性商业惯例用于验证circRNA与特定限制性商业惯例的交互的对象。然而,其中一些方法是间接的测定,以断定是否在circRNA是ASSOC iated与靶miRNA小号或RBP小号。我们前期的研究已经成功地使用circRNA拉下试验小号与生物素标记的反义寡核苷酸小号瞄准circRNA backsplice结序列。此外,circRNA-下拉随后通过RT-qPCR和western印迹婷识别的miRNA或RBP分析相关联的与所关注的circRNA (Abdelmohsen等人,2017;熊猫等人,2017 ;潘迪等人,2020 )。在这里,我们调整了已发表的方法以成功拉低βTC6细胞中的circHipk2 。为详细的协议的circRNA的下拉到ANALY泽相关miRNA或RBP这里描述。

关键字:环状rna, 环状rna pulldown, 反义寡核苷酸, 生物素pulldown, RT-qPCR, miRNA, rna结合蛋白



材料和试剂


1.核酸酶-无1.5毫升微量离心管中小号(Tarson,目录号:500010)     
2.核酸酶-无2-ml离心管小号(Tarson,目录号:500020)     
3.加入10μl,20微升,200微升,和1ml的提示(Tarson,目录号小号:524053,528101,528104,528106)     
4. 96 -孔PCR板(赛默飞世尔科技,产品目录号:4483485 )     
5.无核酸酶水(Thermo Fisher Scientific,目录号:10977023)     
6. 0.25% 胰蛋白酶(Thermo Fisher Scientific,目录号:25200056)     
7.磷酸盐缓冲盐水(PBS)(Sigma,目录号:P4417-50TAB)     
8. 1 M Tris-HCl,pH 7.5(Thermo Fisher Scientific,目录号:15567027)     
9. 1 M Tris-HCl ,pH 8.0(Thermo Fisher Scientific,目录号:15568025)     
10. 2 M氯化钾(Thermo Fisher Scientific,目录号:AM9640G) 
11. 1 M氯化镁(Thermo Fisher Scientific,目录号:AM9530G) 
12. 5 M氯化钠(Thermo Fisher Scientific,目录号:AM9759) 
13. Nonidet P-40(Amresco,目录号:M158-100ML) 
14. 0.5 M EDTA ,pH 8.0(Amresco,目录号:E177-100ML) 
15. Triton X-100(SRL,目录号:64518) 
16.鼠RNase抑制剂(NEB,目录号:M0314S,40 U/μl) 
17. 20    ×蛋白酶抑制剂(Sigma,目录号:S8830)

18.链霉亲和素 Dynabeads(NEB,目录号:S1420S) 
19. TRIzol(Thermo Fisher Scientific,目录号:15596018) 
20.乙醇(HiMedia,目录号:MB228) 
21.氯仿(SRL,目录号:96764) 
22.异丙醇(SRL,目录号:38445) 
23. GlycoBlue TM共沉淀剂(15 mg/ ml )(Thermo Fisher Scientific,目录号:AM9515) 
24.最大值逆转录酶(赛默飞世尔科技,产品目录号:EP0743) 
25. dNT    P set ,1 0 0 mM溶液(Thermo Fisher Scientific,目录号:R0181)

26.随机引物(Thermo Fisher Scientific,目录号:48190011) 
27. PowerUp SYBR G reen master mix(Thermo Fisher Scientific,目录号:A25778) 
28. MicroAmp光学胶膜(Thermo Fisher Scientific ,目录号:4311971) 
29. SIGMA合成的用于 circHipk2 PCR 扩增的Divergent DNA oligo 引物(circHipk2-F: 5'- TCCAGACAACCGTACCGAGT-3'和circHipk2-R: 5'- GGCACTTGATTGAAGGGTGT-3') 
30.用SIGMA合成的生物素-TEG标记的定制反义寡核苷酸用于控制 (Ctrl-ASO: 5'-TGCGTAACGAACGACGAATCGTCGCAGATC-3'[BtnTg]) 和 circHipk2 (circHipk2-ASO: 5'-CATGTGAGGCCATACCGGTAGTATCTGGBtnTg') 
31. 3 × SDS 加载染料(NEB,目录号:B7703S) 
32.多聚体提取缓冲液 (PEB)(参见食谱)                                                                                                                             
33. 2 × Tris、EDTA、NaCl、Triton (TENT) 缓冲液(见配方)                                                                                                                       
34. 1 ×帐篷(见食谱)                                                                                                                       

设备


1.手动p ipette组,2微升,20微升,200微升,和1ml(各个厂商)     
2.涡流混合器(Tarson,目录号:3001)     
3.磁性支架(Tarson,目录号:S1509S)     
4.管旋转器(Tarson,目录号:3071)     
5.冷冻离心机(Eppendorf,型号:5810R)     
6.台式微量离心机(Tarson,目录号:1010)     
7. Thermomixer(Eppendorf,目录号:5384000012)     
8. PCR机(各厂家)     
9. QuantStudio 3 实时 PCR 系统(Thermo Fisher Scientific ,目录号:A28567 )     

软件


1. UCSC 基因组浏览器(https://genome.ucsc.edu/)     
2. Primer3 网络工具 ( https://bioinfo.ut.ee/primer3/ )     
3. GeneRunner ( http://www.generunner.net/ )     
4. QuantStudio 3 和 5 系统软件     
5. Circinteractome 网站(https://circinteractome.nia.nih.gov/)     

程序


                                                                                                                                           寡核苷酸设计
1.利用反向剪接位点的基因组坐标,从RNA测序数据或UCSC基因组浏览器中获取感兴趣的circRNA的成熟序列。在这里,我们从小鼠 mm10 UCSC 基因组浏览器中检索了小鼠 circHipk2 (chr6|38818229|38819313|-) 的序列(图 1)。     
注:多外显子circRNA的成熟剪接序列可以通过反向剪接位点的基因组坐标之间的所有外显子序列组合得到。


图标描述已自动生成

图1 。设计的发散引物和生物素标记的反义寡核苷酸靶向circHipk2 。


2.如图1所示,加入circRNA序列的最后15个核苷酸至第15个核苷酸,以获得30的上游-跨越circRNA backsplice结核苷酸序列。     
3.为了设计ASO,生成30的反向互补序列-核苷酸连接序列使用GeneRunner。与合成该序列b iotin-TEG添加到3'末端通过优选的供应商。     
4.设计引物的使用目标看家基因的mRNA或rRNA的的底漆3网络工具。     
5.如前所述(Panda 和 Gorospe,2018)或使用人类 circRNA 的 circinteractome 网站(Dudekula等,2016 )设计目标 circRNA 的不同引物。     

                                                                                                                                           细胞裂解
              取一个 100 - mm 约 70% 融合的 βTC6 细胞的培养皿并丢弃培养基。
注意:最少应使用500 万个细胞进行下拉测定。细胞的较高量可能有助于以获得稀有或低拷贝数circRNAs更好的下拉。
              用 5-10 ml 冰冷的 1 × PBS清洗细胞3 次。
              通过胰蛋白酶消化或用细胞刮刀刮取细胞。
通过在 4°C 下以 1,000 × g离心2 分钟来沉淀细胞并丢弃上清液。
              在 1 ml 冰冷的多聚体提取缓冲液 (PEB) 中重悬细胞沉淀。
注意:用 PEB 裂解释放细胞质部分,而不是核部分。
              立即加入 5 µl RNase 抑制剂和 50 µl 20 ×蛋白酶抑制剂。
              吹打十倍拌匀,并保持在冰上15分钟,移液或涡旋为几秒钟,每4 - 5分钟,直到细胞被裂解。
              将裂解物在4°C 下以 12,000 × g离心10 分钟。
              收集 900 µl 上清液并进行杂交步骤(图 2)。

图标描述已自动生成

图 2. 使用生物素化-ASO靶向circHipk2的 circRNA 下拉示意图。


                                                                                                                                           反义寡核苷酸杂交
1.添加一个冰冷2米的等体积(900微升)×的Tris,EDTA,氯化钠,曲拉通(TENT)缓冲液把在2以上步骤收集上清液- ml离心管。     
2.鸿沟将混合物分成两管(一个用于所述控制ASO下拉和另一个用于所述circRNA-ASO下拉)。     
3.分别向对照管和 circRNA pulldown 管中加入 1 µl 100 µM (100 pmol) ctrl-ASO 和 circRNA-ASO。注意:制造商的数据表给出了链霉亲和素珠结合能力。ASO 量应小于下一步下拉的链霉亲和素珠结合能力。较高的 ASO 量可能会降低下拉效率。     
4.在转子上以30 rpm 的速度在 4°C 下孵育反应物90 分钟。注:ASO杂交反应可以在室温下或进行在37℃下,以促进ASO双向Ñ丁靶向circRNA ; ħ H但是,这个米AY影响在具有高水平的内源性核糖核酸酶的RNA样品的完整性。     

                                                                                                                                           链霉亲和素珠制备
1.在 ASO 杂交步骤完成前 15 分钟开始制备链霉亲和素珠。     
2.混合均匀,将100 µl磁性链霉亲和素珠(每个反应 50 µl )放入新管中。     
3. P花边上磁性支架30秒磁珠并弃去上清液。     
4. 将磁珠重悬在 500 µl 冰冷的 1 × TENT 缓冲液中,并将管子放在磁力架上 30 秒。     
5.在磁力架上将试管旋转 180º两次以清洗珠子(视频 1)。     



视频 1. 磁珠洗涤


6.弃去上清液。     
7.重复洗涤步骤(小号TEPS d 4- d 6)的两倍。     
8.在 100 µl 冰冷的 1 × TENT 缓冲液中重悬磁性链霉亲和素珠。     

                                                                                                                                            循环RNA p ulldown
1.加入50μl洗涤链亲和素磁性小珠到每个杂交反应管(在提及的小号TEP完成90分钟后C4)UTE杂交步骤。         
2. 向管中加入 1 µl RNase 抑制剂和 5 µl 20 ×蛋白酶抑制剂,并通过移液充分混合。         
3.在试管旋转器上以 30 rpm 的速度在室温下旋转对照管和 circRNA ASO 管 30 分钟。         
4.短暂离心管子,将所有液体样品带到管子底部,而不会使珠粒形成颗粒。         
注意:高速离心可能会使珠粒沉淀,这可能会影响洗涤步骤。

5.将试管置于磁力架上 30 秒,弃去上清液。         
6. 将磁珠重悬在 500 µl 冰冷的 1 × TENT 缓冲液中,并将管子放在磁力架上 30 秒。         
7.在磁力架上旋转试管两次以清洗磁珠(视频 1)。         
8.允许珠以朝向磁铁沉降,弃去缓冲液。         
9.重复洗涤步骤(步骤 6-8)两次。注意:洗涤次数可能取决于目标 circRNA 的富集倍数。我们发现在我们的下拉分析中,两到三次洗涤适合富集 circHipk2。           
10.最后一次洗涤后,离心管几秒钟,使珠子沉淀在底部。       
11.将试管置于磁力架上 30 s,弃去剩余的上清液。       
12.在 30 µl 冰冷的 PEB 中重悬珠子。       
13.取 15 µl 磁珠到新管中进行 RNA 分离和 RT-qPCR,使用 ASO 分析 circRNA 的 pulldown 效率并检测 circRNA 相关的 miRNA。       
14.将 7 µl 3 × SDS 上样染料添加到剩余的 15 µl 珠子中并通过移液混合,然后在 95°C 下加热 5 分钟。该样品可立即用于蛋白质印迹以识别相互作用的 RBP 或储存在 -20°C。       

                                                                                                                                            从RNA和cDNA制备的下拉样
1.将 250 µl TRIzol 试剂加入 15 µl 磁珠中以进行 RNA 分离,并通过移液充分混合。         
不Ë :任何相似的RNA分离试剂如TriSure,RNAzol,TRI试剂,或TriPure可用于制备RNA。应避免使用基于柱的 RNA 分离试剂盒,因为使用标准 RNA 分离试剂盒不能很好地纯化 miRNA 。否则,请使用可以同时分离长RNA和 miRNA的试剂盒。

2.加入 50 µl 氯仿,涡旋 15 秒。         
3.在 4°C 下以 12,000 × g离心15 分钟,然后收集 100 µl 水层到一个新管中。         
4.添加100μl的异丙醇和0.5微升glycoblue作为一个共-沉淀剂。         
5.混合均匀,并保持在室温下10分钟,然后离心机È以12,000 ×克于4℃10分钟。         
6.弃去上清液,向 RNA 沉淀中加入 500 µl 75% 乙醇,涡旋离心管几秒钟。         
7.在室温下以 12,000 × g离心5 分钟。         
8.弃上清,空气干燥RNA沉淀3-5分钟用的盖子打开。         
注:过度干燥的RNA沉淀或残余的乙醇可能抑制颗粒的溶解度。

9.将沉淀溶解在 20 µl 无核酸酶水中。的RNA可以储存在-20℃以备将来使用或用于立即进行cDNA合成。         
10.准备一个20 -微升含13微升cDNA合成反应制备的RNA,0.5μl的最大值逆转录酶,0.5μl的RNase抑制剂,1微升10毫摩尔dNTP混合物,1μl的随机引物,和4微升5 × RT缓冲液。       
注意:可以使用任何标准逆转录试剂盒和随机引物制备 cDNA 。

11.轻轻混合反应液并在室温下孵育 10 分钟,然后在 50°C 下孵育 1 小时。       
12.将反应在 85°C 下孵育 5 分钟以灭活逆转录酶。       
13.用 250 µl 无核酸酶的水稀释 cDNA 。这可以被储存在-20℃或用于立即用于PCR分析。       

                                                                                                                                           RT-qPCR的circRNA富集分析
1.将 10 μl 100 μM 正向和反向引物储备液与 980 μl 无核酸酶水混合,以获得 1 μM 引物混合物的终浓度。         
2.准备20 -在微升反应的96-孔板含有5微升的cDNA,5μl的引物混合,并加入10μl2 × SYBR绿色混合。应准备三个技术重复。         
3.用光学胶盖密封板并涡旋几秒钟。         
4.将板离心几秒钟,使反应物到达孔底。         
5.在QuantStudio 3实时PCR系统中进行定量PCR以在下列反应设置:2分钟,在95初始循环℃,然后4个0 5秒周期在95℃在60℃和20秒℃。         
6.获取的技术重复的平均值Ct值在每个目标的控制和circRNA下拉样品。         
7.计算下拉样品中靶circRNA的百分比(%)浓缩为相比与使用Δ-CT方法(图3),控制下拉样品(Livak和Schmittgen,2001) 。         

图表,条形图描述已自动生成

图3.实施例一MPL Ë数据表示的百分比富集circHipk2在所述circRNA-ASO相对于下拉样品的控制ASO p ulldown样品。


                                                                                                                                           检测相互作用的 miRNA 和 RBP
1.确认circRNA ASO pulldown样本中目标circRNA富集后,可以分析相关的miRNA和RBP 。                                                                                                                                 
2.可以通过RT-qPCR分析与目标circRNA相关的miRNA。上述步骤制备的 RNA 可用于产生miRNA cDNA,然后进行 qPCR 分析,以研究它们在 circRNA pulldown 样本中的特异性富集。                                                                                                                                 
3.执行标准的蛋白质印迹以检测 circRNA 相关的 RBP。简言之,题目输入,控制ASO ,和circRNA ASO用SDS染料混合下拉样到SDS-PAGE,和蛋白质转移到硝酸纤维素膜上。用针对预测的 RBP 的一抗孵育膜,然后用适当的二抗孵育,并检测化学发光信号。                                                                                                                                 

数据分析


该方法描述下拉使用环状RNA的反义寡核苷酸特异性靶向的backsplice接合序列的目标circRNA。然而,日的这个电子成功拉下试验d epends上circRNA接合序列的可用性,其中m AY是不可访问的,由于二级结构或结-相互作用限制性商业惯例,防止从ASO结合。此外,用于细胞裂解的 PEB 有利于释放细胞质部分,而用于下拉核定位 circRNA 的细胞核裂解仍有待标准化。由于大多数 circRNA 本质上是细胞质的(Jeck等人,2013),我们使用 PEB 进行细胞裂解和下拉测定。使用 delta-Ct 方法分析 circRNA ASO pulldown 样本中目标 circRNA 的富集,与使用加载控制(如18S rRNA 或Gapdh mRNA)的对照 pulldown相比(Livak 和 Schmittgen,2001)。如图3所示,circHipk2水平超过2 -在circHipk2 ASO下拉倍比控制ASO下拉。或者,可以通过比较 pulldown 样本与流过或输入来测量 circRNA pulldown 的效率。检查circRNA的下拉样品中富集后,剩余的RNA可进行miRNA的RT-qPCR分析,以检查对于miRNA的特异性富集预测为与感兴趣的circRNA交互。此外,其他的可以使用下拉样品的一半用于免疫印迹婷分析以evalu一个TE的限制性商业惯例associat ED与目标circRNA。这是一种下拉感兴趣的环状 RNA 并研究环状 RNA 相关 miRNA 和 RBP 的有前途的方法,它们是环状 RNA 介导的基因调控的关键因素。


配方小号


1.多聚体提取缓冲液 (PEB)                                                                                                                                 
试剂

库存

250 毫升所需的体积

20 mM Tris-HCl,pH 7.5

1 米

5毫升

100 毫米氯化钾

2米

12.5 毫升

5 毫米氯化镁2

1 米

1.25 毫升

0.5% Nonidet P-40

10%

12.5 毫升

用无核酸酶水将体积调节至 250 ml

2. 2 × Tris、EDTA、NaCl、Triton (TENT) 缓冲液                                                                                                                                 
试剂

库存

100毫升所需的体积

20 mM Tris-HCl pH 8.0

1 米

2毫升

2 mM EDTA pH 8.0

0.5M

0.4 毫升

500 毫米氯化钠

5米

10毫升

1% v/v 海卫 X-100

20%

5毫升

用无核酸酶水将体积调节至 100 ml

3. 1 ×帐篷                                                                                                                                 
混合等体积的 PEB/水和2 × TENT


致谢


这项研究得到了生命科学研究所和惠康信托基金/DBT 印度联盟中级奖学金 (IA/I/18/2/504017) 提供给 ACP 的校内资金支持。DD和AD是由青年研究奖学金支持小号从该大学拨款委员会,印度。该协议改编自先前发表的论文(Abdelmohsen等人,201 7和 Panda等人,2017 年)。作者感谢我们在布巴内斯瓦尔生命科学研究所的同事对文章进行了校对。


利益争夺


作者宣称没有冲突小号的兴趣。


参考


Abdelmohsen, K., Panda, AC, Munk, R., Grammatikakis, I., Dudekula, DB, De, S., Kim, J., Noh, JH, Kim, KM, Martindale, JL 和Gorospe, M. ( 2017)。HuR 目标环状 RNA 的鉴定揭示了 CircPABPN1 对 PABPN1 翻译的抑制。RNA 生物学14(3):361-369。
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Jeck, WR, Sorrentino, JA, Wang, K., Slevin, MK, Burd, CE, Liu, J., Marzluff, WF 和 Sharpless, NE (2013)。环状 RNA 丰富、保守且与 ALU 重复相关。RNA 19(2):141-157。
Livak, KJ 和 Schmittgen, TD (2001)。使用实时定量 PCR 和 2 -ΔΔCT方法分析相关基因表达数据。方法25(4):402-408。              
Palazzo, AF 和 Lee, ES (2015)。非编码 RNA:什么是功能性的,什么是垃圾?前基因6:2。
Panda, AC 和 Gorospe, M.(2018 年)。通过 RT-PCR 检测和分析环状 RNA。生物协议8(6)。
Panda, AC, Grammatikakis, I., Kim, KM, De, S., Martindale, JL, Munk, R., Yang, X., Abdelmohsen, K. 和 Gorospe, M. (2017)。衰老相关环状 RNA(SAC-RNA)的鉴定揭示了衰老抑制因子 CircPVT1。核酸研究45(7):4021-4035。
Pandey, PR, Yang, JH, Tsitsipatis, D., Panda, AC, Noh, JH, Kim, KM, Munk, R., Nicholson, T., Hanniford, D., Argibay, D., Yang, X., Martindale, JL, Chang, MW, Jones, SW, Hernando, E., Sen, P., De, S., Abdelmohsen, K. 和 Gorospe, M. (2020)。circSamd4 抑制 PUR 蛋白的生肌转录活性。核酸研究48(7):3789-3805。              
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引用:Das, D., Das, A. and Panda, A. C. (2021). Antisense Oligo Pulldown of Circular RNA for Downstream Analysis. Bio-protocol 11(14): e4088. DOI: 10.21769/BioProtoc.4088.
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