This work was supported by intramural funding from the Institute of Life Sciences, the DBT research grant (BT/PR27622/MED/30/1961/2018), and the Wellcome Trust/DBT India Alliance Fellowship [IA/I/18/2/504017] awarded to Amaresh C. Panda. We thank other lab members for proofreading the article.

RNA is sensitive to RNases; therefore, all reagents, instruments, and workspaces should be RNase-free and handled with care.
1. Divergent primer design for circRNA (see Figure 1)
<ol>
	<li>Retrieve the mature sequence from circRNA annotation data using BEDTools or from the UCSC genome browser by joining the exon/intron sequence present between the backsplice junction coordinates18,<sup class="x...

<ol>
	<li>Salzman, J., Gawad, C., Wang, P. L., Lacayo, N., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNAs+are+the+predominant+transcript+isoform+from+hundreds+of+human+genes+in+diverse+cell+types.">Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types.</a> PLoS One. 7 (2), 30733 (2012).</li><li>Chen, L. L., Yang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+circRNA+biogenesis.">Regulation of circRNA biogenesis.</a> RNA Biology. 12 (4), 381-388 (2015).</li><li>Kristensen, L. 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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+droplet+digital+polymerase+chain+reaction+format+for+DNA+copy+number+quantification.">Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification.</a> Analytical Chemistry. 84 (2), 1003-1011 (2012).</li><li>Hayden, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+droplet+digital+PCR+to+real-time+PCR+for+quantitative+detection+of+cytomegalovirus.">Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus.</a> Journal of Clinical Microbiology. 51 (2), 540-546 (2013).</li><li>Taylor, S. C., Laperriere, G., Germain, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+digital+PCR+versus+qPCR+for+gene+expression+analysis+with+low+abundant+targets:+From+variable+nonsense+to+publication+quality+data.">Droplet digital PCR versus qPCR for gene expression analysis with low abundant targets: From variable nonsense to publication quality data.</a> Scientific Reports. 7, 2409 (2017).</li><li>Hindson, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+droplet+digital+PCR+system+for+absolute+quantitation+of+DNA+copy+number.">High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.</a> Analytical Chemistry. 83 (22), 8604-8610 (2011).</li><li>Ishak, A., AlRawashdeh, M. M., Esagian, S. M., Nikas, I. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic,+prognostic,+and+therapeutic+value+of+droplet+digital+PCR+(ddPCR)+in+COVID-19+patients:+A+systematic+review.">Diagnostic, prognostic, and therapeutic value of droplet digital PCR (ddPCR) in COVID-19 patients: A systematic review.</a> Journal of Clinical Medicine. 10 (23), 5712 (2021).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+droplet+digital+PCR+to+detect+the+pathogens+of+infectious+diseases.">Application of droplet digital PCR to detect the pathogens of infectious diseases.</a> Bioscience Reports. 38 (6), (2018).</li><li>Lee, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+UCSC+genome+browser+database:+2022+update.">The UCSC genome browser database: 2022 update.</a> Nucleic Acids Research. 50, 1115-1122 (2022).</li><li>Quinlan, A. R., Hall, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BEDTools:+A+flexible+suite+of+utilities+for+comparing+genomic+features.">BEDTools: A flexible suite of utilities for comparing genomic features.</a> Bioinformatics. 26 (6), 841-842 (2010).</li><li>Untergasser, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer3--new+capabilities+and+interfaces.">Primer3--new capabilities and interfaces.</a> Nucleic Acids Research. 40 (15), 115 (2012).</li><li>Das, A., Das, D., Das, A., Panda, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quick+and+cost-effective+method+for+DNA-free+total+RNA+isolation+using+magnetic+silica+beads.">A quick and cost-effective method for DNA-free total RNA isolation using magnetic silica beads.</a> bioRxiv. , (2020).</li><li>Oberacker, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+Synthesis+of+Functionalized+Paramagnetic+Beads+for+Nucleic+Acid+Purification+and+Manipulation.">Simple Synthesis of Functionalized Paramagnetic Beads for Nucleic Acid Purification and Manipulation.</a> Bio-protocol. 9 (20), 3394 (2019).</li><li>Ra&#269;ki, N., Dreo, T., Gutierrez-Aguirre, I., Blejec, A., Ravnikar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+transcriptase+droplet+digital+PCR+shows+high+resilience+to+PCR+inhibitors+from+plant,+soil+and+water+samples.">Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples.</a> Plant Methods. 10 (1), 42 (2014).</li><li>Taylor, S. C., Carbonneau, J., Shelton, D. N., Boivin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+droplet+digital+PCR+from+RNA+and+DNA+extracts+with+direct+comparison+to+RT-qPCR:+Clinical+implications+for+quantification+of+Oseltamivir-resistant+subpopulations.">Optimization of droplet digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR: Clinical implications for quantification of Oseltamivir-resistant subpopulations.</a> Journal of Virological Methods. 224, 58-66 (2015).</li></ol>

The authors declare no conflicts of interest.

CircRNA research has grown in the last decade with the discovery of high-throughput sequencing technologies. As a result, it has been considered a potential molecule for future RNA therapeutics. In addition, it is known to act as a biomarker in several diseases, including cancer and cardiovascular diseases4,8. However, the identification of circRNA is tricky because of its low abundance and it having only one specific backsplice junction sequence that differentia...

Recent advancements in RNA sequencing technologies and novel computational algorithms have discovered a new member of the growing family of non-coding RNAs, called circular RNA (circRNA)1. As the name suggests, circRNAs are a family of single-stranded RNA molecules with no free ends. They are formed by non-canonical head-to-tail splicing called back-splicing, where the upstream splice acceptor site joins covalently with the downstream splice donor site to form a stable RNA circle1,2. This process could be mediated by several factors, including inverted Alu repetitive elements present in the upstream and downstream of circularized exons, or can be mediated by some splicing factors or RNA binding proteins (RBPs)2,3. Circular RNAs generated exclusively from the exonic or intronic sequence are classified as exonic circRNA and circular intronic RNAs or ci-RNAs, whereas some exonic circRNAs retain the intron and are called exon-intron circRNAs (EIcircRNAs)3,4. The functions of circRNAs are multifaceted, including sponging miRNA and/or RBP, regulating transcription, and regulating cellular function by translating into peptides3,5,6,7. Several reports have highlighted the significance of circRNAs in various diseases and physiological processes8. Furthermore, tissue-specific expression patterns and resistance to exonuclease digestion make it a functional biomarker for disease diagnosis and it can also be used as a suitable therapeutic target8. Considering its importance in regulating health and diseases, the accurate quantification of circRNA expression is the need of the hour.
Several biochemical methods have been developed to quantify circRNAs in biological samples9. One of the most convenient and widely accepted methods for circRNA quantification is reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) using divergent primer pairs10. However, the majority of circRNAs are in low abundance compared to linear mRNAs, which makes it challenging to quantify them11. To overcome this issue, we sought to use digital droplet PCR (dd-PCR) to quantify the number of circRNAs in a given sample accurately. The dd-PCR is an advanced PCR technology that follows the microfluidics principle; it generates multiple aqueous droplets in oil, and the PCR occurs in each droplet as an individual reaction12. The reaction occurs in individual droplets and is analyzed using a droplet reader, which gives the number of positive or negative droplets for the gene of interest12. It is the most sensitive technique to accurately quantify a gene of interest, even if there is only a single copy in a given sample. Decreased sensitivity toward inhibitors, better precision, and omitting the reference gene for quantification make it more advantageous than conventional qPCR13,14,15. It has been widely used as a research and diagnostic tool for the absolute quantification of a gene of interest16,17. Here, we describe the detailed dd-PCR protocol for circRNA quantification in differentiating mouse C2C12 myotubes and proliferating mouse C2C12 myoblasts using divergent primers.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >1.5 ml microcentifuge tube</td>
			<td >Tarson</td>
			<td >500010</td>
			<td ></td>
		</tr>
		<tr >
			<td >0.2 ml tube strips with cap</td>
			<td >Tarson</td>
			<td >610020, 510073</td>
			<td></td>
		</tr>
		<tr >
			<td >Filter Tips</td>
			<td >Tarson</td>
			<td >528104</td>
			<td></td>
		</tr>
		<tr >
			<td >DNase/RNase-Free Distilled Water</td>
			<td >Thermo Fisher Scientific</td>
			<td >10977023</td>
			<td></td>
		</tr>
		<tr >
			<td >Phosphate-buffered saline (PBS)</td>
			<td >Sigma</td>
			<td >P4417</td>
			<td></td>
		</tr>
		<tr >
			<td >Cell scrapper</td>
			<td >HiMedia</td>
			<td >TCP223</td>
			<td></td>
		</tr>
		<tr >
			<td >Chloroform</td>
			<td >SRL</td>
			<td >96764</td>
			<td></td>
		</tr>
		<tr >
			<td >DNA diluent</td>
			<td >HiMedia</td>
			<td >MB228</td>
			<td></td>
		</tr>
		<tr >
			<td >Random primers</td>
			<td >Thermo Fisher Scientific</td>
			<td >48190011</td>
			<td></td>
		</tr>
		<tr >
			<td >dNTP set</td>
			<td >Thermo Fisher Scientific</td>
			<td >R0181</td>
			<td></td>
		</tr>
		<tr >
			<td >Murine RNase inhibitor</td>
			<td >NEB</td>
			<td >M0314S</td>
			<td></td>
		</tr>
		<tr >
			<td >Maxima reverse transcriptase</td>
			<td >Thermo Fisher Scientific</td>
			<td >EP0743</td>
			<td></td>
		</tr>
		<tr >
			<td >QX200 dd-PCR Evagreen Supermix</td>
			<td >Bio-Rad</td>
			<td >1864033</td>
			<td></td>
		</tr>
		<tr >
			<td >Droplet generation oil for Evagreen</td>
			<td >Bio-Rad</td>
			<td >1864006</td>
			<td></td>
		</tr>
		<tr >
			<td >PCR Plate Heat Seal, foil, pierceable</td>
			<td >Bio-Rad</td>
			<td >1814040</td>
			<td></td>
		</tr>
		<tr >
			<td >DG8 Cartridges and Gaskets</td>
			<td >Bio-Rad</td>
			<td >1864007</td>
			<td></td>
		</tr>
		<tr >
			<td >DG8 Cartridge holder</td>
			<td >Bio-Rad</td>
			<td >1863051</td>
			<td></td>
		</tr>
		<tr >
			<td >QX200 Droplet Generator</td>
			<td >Bio-Rad</td>
			<td >1864002</td>
			<td></td>
		</tr>
		<tr >
			<td >ddPCR 96-Well Plates</td>
			<td >Bio-Rad</td>
			<td >12001925</td>
			<td></td>
		</tr>
		<tr >
			<td >PX1 PCR Plate Sealer</td>
			<td >Bio-Rad</td>
			<td >1814000</td>
			<td></td>
		</tr>
		<tr >
			<td >C1000 Touch Thermal Cycler with 96&#8211;Deep Well Reaction Module</td>
			<td >Bio-Rad</td>
			<td >1851197</td>
			<td></td>
		</tr>
		<tr >
			<td >QX200 Droplet Reader</td>
			<td >Bio-Rad</td>
			<td >1864003</td>
			<td></td>
		</tr>
		<tr >
			<td >Quantasoft Software</td>
			<td >Bio-Rad</td>
			<td >1864011</td>
			<td></td>
		</tr>
		<tr >
			<td >Silica column</td>
			<td >Umbrella Life Science</td>
			<td >38220090</td>
			<td></td>
		</tr>
		<tr >
			<td >UCSC Genome Browser</td>
			<td ></td>
			<td >https://genome.ucsc.edu/</td>
			<td></td>
		</tr>
		<tr >
			<td >Primer 3</td>
			<td ></td>
			<td >https://primer3.ut.ee/</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of circular rnas using digital droplet pcr

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 water-in-oil droplets, and the PCR occurs in the individual droplets. The dd-PCR has several advantages over conventional real-time qPCR, including increased accuracy in detecting low-abundance targets, omitting reference genes for quantification, eliminating technical replicates for samples, and showing high resilience to inhibitors in the samples. Recently, dd-PCR has become one of the most popular methods for accurately quantifying target DNA or RNA for gene expression analysis and diagnostics. Circular RNAs (circRNAs) are a large family of recently discovered covalently closed RNA molecules lacking 5' and 3' ends. They have been shown to regulate gene expression by acting as sponges for RNA-binding proteins and microRNAs. Furthermore, circRNAs are secreted into body fluids, and their resistance to exonucleases makes them serve as biomarkers for disease diagnosis. This article aims to show how to perform divergent primer design, RNA extraction, cDNA synthesis, and dd-PCR analysis to accurately quantify specific circular RNA (circRNA) levels in cells. In conclusion, we demonstrate the precise quantification of circRNAs using dd-PCR.

The absolute number of circRNAs in each sample can be derived from the exported dd-PCR data. Real-time quantitative PCR analysis suggested differential expression of circBnc2 in the differentiated C2C12 myotubes (data not shown). Here, we wanted to check the absolute copy number of circBnc2 in proliferating C2C12 myoblasts and myotubes. Since the expression of circBnc2 is compared in two conditions, it is really important to process all samples for RNA isolation, cDNA synthesis, and PCR simulta...

Watch this Scientific Journal Video about Quantification of Circular RNAs Using Digital Droplet PCR at JoVE.com

Quantification of Circular RNAs Using Digital Droplet PCR

This protocol describes the detailed method of digital droplet PCR (dd-PCR) for precise quantification of circular RNA (circRNA) levels in cells using divergent primers.

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 ...

dd-PCR is a highly sensitive and accurate method for absolute quantification of target DNA or RNA. It has been used for disease diagnosis where other conventional methods fail to give accurate results. dd-PCR is the most sensitive technique for detecting very low abundant nucleic acid molecules.Unlike quantitative PCR, dd-PCR doesn't require any housekeeping gene control for quantification. Due to its sensitivity and accuracy, dd-PCR can be used as a tool for molecular diagnostics and research involving low abundant RNA molecules like micro RNAs and circular RNAs. Before using dd-PCR, one should perform RT-PCR or RT-qPCR to confirm that the primers are amplifying their target genes without any non-specific PCR amplification.To begin, prepare a PCR template sequence of 200 nucleotides in length by joining the last 100 nucleotides of the total circular RNA sequence length to the first 100 nucleotides of the circular RNA sequence. Use the template sequence for primer, designed by the Primer 3 web tool. Take one 10 centimeter dish containing about 5 million proliferating C2C12 myoblast cells and one four-day differentiated C2C12 myotube for RNA isolation.Wash the cells with 10 milliliters of 1X PBS three times and discard the PBS using a pipette. Add one milliliter of RNA isolation reagent and lyse the cells by vigorous pipetting. Then, add 200 microliters of chloroform and vortex for 15 seconds.Centrifuge the tube at four degrees Celsius for 10 minutes at 12, 000 G.Take 400 microliters of the upper aqueous layer, load it on a silica column, and centrifuge for one minute at 12, 000 G at room temperature. Take the flow through to a new tube and add 600 microliters of 100%ethanol. Add 20 microliters of magnetic silica beads and place the tube on a thermomixer, set at 25 degrees Celsius and 1, 200 RPM for five minutes.Then, place the tube on a magnetic stand for 30 seconds or until the solution becomes clear. Resuspend the magnetic silica beads in 500 microliters of wash buffer containing 90%ethanol. After placing the tube on the magnetic stand for 30 seconds, let the beads settle toward the magnet and discard the buffer using a pipette.Air dry the tube at 50 degrees Celsius for three minutes on a thermomixer, keeping the lid open. Add 20 microliters of nuclease free water and resuspend the beads. Collect the dissolved RNA in a new tube for quantity and quality assessment using a spectrophotometer.Measure the RNA concentration using a spectrophotometer and take one microgram of RNA for cDNA synthesis. Mix one microgram of total RNA with one microliter of dNTP mix, four microliters of 5X reverse transcriptase buffer, two microliters of 10X reverse transcriptase random primer, 0.25 microliters of reverse transcriptase enzyme, and 0.5 microliters of RNase inhibitor. Make up the volume to 20 microliters using nuclease free water.Place the tube at 25 degrees Celsius for 10 minutes, followed by 50 degrees Celsius for one hour. Then place the tube at 85 degrees Celsius for five minutes to inactivate the enzyme. Set up the PCR reaction of 22 microliters in 0.2 milliliter PCR tubes or strips, along with a negative control tube and non-template control tubes.Add 11 microliters of dd-PCR master mix, 5.5 microliters of circular RNA-specific forward and reverse primer mix of one micromolar concentration, and 5.5 microliters of nuclease free water to set up the NTC reaction tubes. Similarly, to set up the test reaction tubes, add 11 microliters of dd-PCR master mix, 5.5 microliters of circular RNA specific forward and reverse primer mix of one micromolar concentration, and 5.5 microliters of cDNA. For the droplet generation, transfer 20 microliters of the PCR mixture from 0.2 milliliter PCR tubes to the sample wells of the droplet generator cartridge.Add 70 microliters of droplet generation oil to the oil wells of the droplet generator cartridge carefully, using a pipette. Cover the droplet generator cartridge with a rubber gasket and place it in the droplet generator machine to get the sample oil droplet mix, generated in the droplet wells of the cartridge. For PCR amplification, transfer 40 microliters of the sample oil droplet mix from the droplet wells of the droplet generator cartridge to a 96-well PCR plate using a pipette.Seal the 96-well PCR plate with the aluminum foil sealer. Place it in the plate sealer machine block, preheated at 180 degrees Celsius, and click seal for sealing the PCR plate. Then, place the plate in the dd-PCR thermal cycler and set the lid temperature to 105 degrees Celsius and a ramp rate to two degrees Celsius per second.Once PCR is over, place the plate on the dd-PCR droplet counter machine with the plate holder in the correct position for counting the droplets. Open the droplet analysis software and set up the run by giving input of the sample information. Click the analyze button to analyze the data after the completion of the droplet reading.Then, click the 1D Amplitude button to see the positive and negative droplets. To segregate the positive droplets from the negative droplets, put a common threshold line for all samples with the same target. Click the export button to export the circular RNA counts data as a csv file.The exported data show the number of circular RNAs present in each sample. Manually calculate the number of circular RNAs in each sample per nanogram of RNA by considering the total amount of cDNA used in each reaction. The data analysis using QuantaSoft Software is shown here.All samples except MT1 showed more than 12, 000 droplet counts. Since the total droplet count was low in MT1, this sample was not considered for the final data analysis. Interestingly, there was a clear difference in the expression pattern of Circ B and C2 in the four day differentiated myotube condition compared to the myoblast cells.The analysis of Circ B and C2 abundance in terms of copy number, indicated that the three replicates of C2C12 myoblasts had 76.8, 67, and 46 copies per nanogram of RNA, while the two myotube samples had 558 and 610 copies per nanogram of RNA. The data in the bar graph represent the mean plus minus standard deviation of two to three biological replicates. ddPCR is one of the most advanced method to measure the accurate differential expression of target circular RNA.This method will be an essential tool to accelerate circular RNA research and diagnostic industries in the coming year.

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3.2K 조회수.  Institute of Life Sciences. dd-PCR은 표적 DNA 또는 RNA의 절대 정량화를 위한 매우 민감하고 정확한 방법입니다. 다른 기존 방법으로는 정확한 결과를 제공하지 못하는 질병 진단에 사용되었습니다. dd-PCR은 매우 낮은 풍부 핵산 분자를 검출하기 위한 가장 민감한 기술입니다.정량적 PCR과 달리 dd-PCR은 정량화를 위한 하우스키핑 유전자 제어가 필요하지 않습니다. 감도와 정확성으로 인해 dd-PCR은 마이?...