Experiment Summary

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

3. Rigid strip 0.2-ml PCR tubes

4. Optical 384-well reaction plate

5. Optical adhesive film

6. Phosphate-buffered saline (DPBS)

7. Total RNA isolation-miRNeasy Mini Kit

8. (Optional) TRIzol reagent

9. Nuclease-free water

10. RNase inhibitor (40 U/µl)

11. RNase R

12. dNTP mix (10 mM each)

13. Random primers (150 ng/µl)

14. Maxima reverse transcriptase

15. FAST ABI prism 2x qPCR master mix

16. Quick Gel Extraction Kit

17. TBE Buffer, 10x, Molecular Biology Grade

18. 1 Kb Plus DNA Ladder

19. Agarose

20. Ethidium bromide solution

21. 2% agarose gel

Equipment

Manual pipettes (set of 2 µl, 20 µl, 200 µl and 1,000 µl), cell scraper, vortex mixer, UV transilluminator, microvolume UV-Vis spectrophotometer, PCR strip tube rotor, mini centrifuge C1201, 96-well thermal cycler, OwlTM EasyCastTM B1 mini gel electrophoresis systems, gel imaging system, MPS 1000 mini plate spinner, QuantStudio 5 eal-Time PCR system.

Procedure

A. Divergent primer design

1. Get the mature sequence of circular RNA (https://genome.ucsc.edu/) using the genomic coordinates.

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.

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.

5. Design divergent primers using the CircInteractome webtool (Dudekula et al., 2016) (http://circinteractome.irp.nia.nih.gov/Divergent_Primers/divergent_primers.html).

B. 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.

C. 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.

D. 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.

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.

E. 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.

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.

6. The percentage (%) RNA left after RNase R treatment using the delta CT method as described in Table (Figure 3).

Fig. 3 Hypothetical qPCR data showing the resistance of circRNA to RNase R treatment as calculated in Table.

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. 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).