Experiment Summary

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). 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.

Materials and Reagents

1. Nuclease-free 1.5-ml and 2-ml microcentrifuge tubes
2. 10 µl, 20 µl, 200 µl, and 1 ml tips
3. 96-well PCR plates
4. Nuclease-free water
5. 0.25% Trypsin
6. Phosphate-buffered saline (PBS)
7. 1 M Tris-HCl, pH 7.5, 1 M Tris-HCl, pH 8.0
8. 2 M Potassium chloride
9. 1 M Magnesium chloride
10. 5 M Sodium chloride
11. Nonidet P-40
12. 0.5 M EDTA, pH 8.0
13. Triton X-100
14. Murine RNase inhibitor
15. 20× Protease inhibitor
16. Streptavidin Dynabeads
17. TRIzol
18. Ethanol
19. Chloroform
20. Isopropanol
21. GlycoBlueTM Coprecipitant (15 mg/ml)
22. Maxima reverse transcriptase
23. dNTP set, 100 mM solutions
24. Random primers
25. PowerUp SYBR Green master mix
26. MicroAmp optical adhesive film
27. Divergent DNA oligo primers synthesized by SIGMA for PCR amplification of circHipk2 (circHipk2-F: 5'-TCCAGACAACCGTACCGAGT-3' and circHipk2-R: 5'-GGCACTTGATTGAAGGGTGT-3')
28. 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])
29. 3× SDS loading dye
30. Polysome extraction buffer
31. 2× Tris, EDTA, NaCl, Triton
32. 1× TENT

Equipment

1. Manual pipette set, 2 µl, 20 µl, 200 µl, and 1 ml
2. Vortex mixer
3. Magnetic stand
4. Tube rotator
5. Refrigerated centrifuge
6. Benchtop microfuge
7. Thermomixer
8. PCR machine
9. QuantStudio 3 real-time PCR system

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

A. Oligo design

1. Obtain the mature sequence of the circRNA. Here, we retrieved the sequence of mouse circHipk2 (chr6|38818229|38819313|-) from the mouse mm10 UCSC genome browser.
2. Design primers and synthesize target sequences.

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

B. 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).

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).

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

C. 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.
4. Incubate the reactions on a rotor at 30 rpm for 90 min at 4°C.

D. 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 180°C on the magnetic rack twice to wash the beads.
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.

E. 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.
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.
8. Allow the beads to settle toward the magnet and discard the buffer.
9. Repeat the washing steps (steps 6-8) twice.
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.

F. 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.
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.
9. Note: Excessive drying of the RNA pellet or residual ethanol may inhibit the solubility of the pellet.
10. 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.
11. 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.
12. Note: The cDNA can be prepared using any standard reverse transcription kit and random primers.
13. Mix the reaction gently and incubate for 10 min at room temperature followed by 1 h at 50°C.
14. Incubate the reaction at 85°C for 5 min to inactivate the reverse transcriptase.
15. Dilute the cDNA with 250 µl nuclease-free water. This can be stored at -20°C or used immediately for PCR analysis.

G. 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. Vortex and centrifuge the plate for a few seconds to bring the reactions to the bottom of the wells.
4. 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.
5. Obtain the average Ct value of the technical replicates for each target in the control and circRNA pulldown samples.
6. 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).

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

H. Detection of interacting miRNAs and RBPs

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

I. Data analysis