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 coordinates18,19.</li>
	<li>Prepare a PCR template sequence of 200 nucleotides in length by joining the last 100 nucleotides of the total circRNA sequence length to the first 100 nucleotides of the circRNA sequence10. 
	NOTE: If the circRNA length is less than 200 nucleotides, then divide it into two equal halves and join the nucleotides from the last half to the beginning of the first half.</li>
	<li>Use the above template sequence for primer design by using the Primer3 web tool and setting a PCR product length ranging from 120-180 nucleotides in length20.</li>
	<li>Use the default settings of Primer3 for other parameters such as Tm and primer length. The primer sequences for circBnc2 are listed in Table 1.</li>
	<li>Order the divergent primer sequences for synthesis from any oligo synthesizing company.</li>
</ol>
2. RNA isolation
NOTE: Isolate total RNA from the mouse C2C12 cells using any commercially available kits or in-house RNA isolation method. The in-house RNA isolation method used here has been described previously21. The magnetic silica beads are prepared in the lab using the previously published protocol22. These magnetic beads can also be procured from various vendors.
<ol>
	<li>Take one 10 cm dish containing about 5 x 106 proliferating C2C12 myoblast cells and one 4-day differentiated C2C12 myotube for RNA isolation.</li>
	<li>Wash the cells with 10 mL of 1x PBS three times and discard the PBS using a pipette.</li>
	<li>Scrape the cells in 1 mL of 1x PBS using a cell scraper, centrifuge them at 4 &#730;C for 5 min at 750 x g, and discard the PBS using a pipette.</li>
	<li>Add 1 mL of RNA isolation reagent (RIR) and lyse the cells by vigorous pipetting21.</li>
	<li>Add 200 &#181;L (1/5 of the volume of RIR) of chloroform and vortex for 15 s. Centrifuge the tube at 4 &#730;C for 10 min at 12,000 x g.</li>
	<li>Take 400 &#181;L of the upper aqueous layer, load it on a silica column, and centrifuge for 1 min at 12,000 x g at room temperature. Take the flow-through to a new tube and add 600 &#181;L of 100% ethanol.</li>
	<li>Add 20 &#181;L of magnetic silica beads and place the tube on a thermomixer set at 25 &#730;C and 1,200 rpm for 5 min. 
	NOTE: The bead volume can be increased or decreased depending on the initial cell numbers taken for lysis. Magnetic silica beads can be procured from commercial vendors.</li>
	<li>Place the tube on a magnetic stand for 30 s or until the solution becomes clear. Discard the supernatant carefully using a pipette.</li>
	<li>Resuspend the magnetic silica beads in a 500 &#181;L of wash buffer containing 90% ethanol. Place the tube on the magnetic stand for 30 s. Rotate the tube twice on the magnetic stand to wash the beads. Let the beads settle toward the magnet and discard the buffer using a pipette.</li>
	<li>Repeat step 2.9 twice. After the wash, briefly spin the tube and place it back on the magnetic stand for 30 s. Discard the remaining wash buffer. Air-dry the tube at 50 &#730;C for 3 min on a thermomixer, keeping the lid open.</li>
	<li>Add 20 &#181;L of nuclease-free water and resuspend the beads. 
	NOTE: The RNA elution volume can be decreased down to 10 &#181;L to scale up the RNA concentration.</li>
	<li>Place the tubes for 2-3 min at room temperature. Place the tube back on the magnetic stand and let it sit for 30 s. Collect the dissolved RNA in a new tube for quantity and quality assessment using a spectrophotometer. 
	NOTE: RNA can be stored at &#8722;20 &#730;C or &#8722;80 &#730;C for long-term storage. To get an optimum result, it is recommended to proceed with the cDNA synthesis step the next day.</li>
</ol>
3. cDNA synthesis
<ol>
	<li>Measure the RNA concentration using a spectrophotometer and take 1 &#181;g of RNA for cDNA synthesis.</li>
	<li>Mix 1 &#181;g of total RNA with 1 &#181;L of dNTP mix (10 mM each of dATP, dTTP, dGTP, and dCTP), 4 &#181;L of 5x reverse transcriptase (RT) buffer, 2 &#181;L of 10x RT random primer, 0.25 &#181;L of reverse transcriptase enzyme (200 U/&#181;L), and 0.5 &#181;L of RNase inhibitor (40 U/&#181;L), and make up the volume up to 20 &#181;L using nuclease-free water. 
	NOTE: The reverse transcriptase enzyme volume can be altered depending on the initial RNA concentration taken for cDNA synthesis.</li>
	<li>Tap the tube to mix the reaction and spin briefly. Place the tube at 25 &#730;C for 10 min followed by at 50 &#730;C for 1 h.</li>
	<li>To inactivate the enzyme, place the tube at 85 &#730;C for 5 min.</li>
	<li>Ice chill the tube for 2 min and add 480 &#181;L of nuclease-free water to the tube to make the cDNA concentration to 2 ng/&#181;L. 
	NOTE: cDNA can be stored at -20 &#730;C or used immediately for dd-PCR analysis.</li>
</ol>
4. Digital droplet PCR (dd-PCR) workflow
NOTE: The workflow of dd-PCR involves multiple steps, starting from sample preparation, followed by droplet generation, PCR amplification, droplet counting, and data analysis. Each step is crucial for accurate data generation as dd-PCR involves the absolute quantification of products and does not need any standard curve. Hence, each of the different steps of dd-PCR has been described below.
<ol>
	<li>Preparation of the dd-PCR reaction.
	<ol>
		<li>Set up the PCR reaction of 22 &#181;L in 0.2 mL PCR tubes or strips along with a negative control tube and NTC (non-template control) tubes. 
		NOTE: Each different divergent primer pair for the detection of different circRNAs must have an NTC reaction along with test samples.</li>
		<li>Add 11 &#181;L of dd-PCR master-mix (e.g., EvaGreen Supermix) and 11 &#181;L of nuclease-free water to set up the negative control reaction tube. 
		NOTE: Thaw the dd-PCR master-mix at room temperature followed by a brief vortex to make a homogeneous mixture. Use the same nuclease-free water to set up the negative control reaction tube and NTC tubes that were used for diluting the cDNA.</li>
		<li>Add 11 &#181;L of dd-PCR master-mix, 5.5 &#181;L of circRNA specific forward and reverse primer mix of 1 &#181;M concentration, and 5.5 &#181;L of nuclease-free water to set up the NTC reaction tubes. 
		NOTE: Dilute the forward and reverse circular RNA-specific divergent primers of 100 &#181;M stock and mix to prepare a 1 &#181;M working concentration of forward and reverse circRNA primer mix; thus, the final primer concentration is 250 nM in the 22 &#181;L reaction.</li>
		<li>Add 11 &#181;L of dd-PCR master-mix, 5.5 &#181;L of circRNA specific forward and reverse primer mix of 1 &#181;M concentration, and 5.5 &#181;L of cDNA (2 ng/&#181;L) to set up the test reaction tubes.</li>
		<li>Vortex thoroughly followed by briefly centrifuging all the reaction tubes to get a homogeneous reaction mixture at the bottom of the tubes.</li>
	</ol>
	</li>
	<li>Droplet generation.
	<ol>
		<li>Transfer the 20 &#181;L of PCR mixture from 0.2 mL PCR tubes to the sample wells of the droplet generator cartridge.</li>
		<li>Add 70 &#181;L of droplet generation oil to the oil wells of the droplet generator cartridge carefully using a multi-channel pipette. 
		&#8203;NOTE: Incubate the droplet generation oil at room temperature for 15 min before use.</li>
		<li>Cover the droplet generator cartridge with the 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.</li>
	</ol>
	</li>
	<li>PCR amplification.
	<ol>
		<li>Transfer 40 &#181;L of the sample-oil droplet mix from the droplet wells of the droplet generator cartridge to a 96-well PCR plate using a multi-channel pipette. 
		NOTE: Carefully transfer the sample-oil droplet mix while making an angle to the wells through the tips of the multi-channel pipette to avoid breaking the droplets while transferring them to the dd-PCR 96-well plate.</li>
		<li>Seal the 96-well PCR plate with the aluminum foil sealer, place it in the plate sealer machine block preheated at 80 &#730;C, and click seal for sealing the PCR plate.</li>
		<li>Now, place the plate in the dd-PCR thermal cycler and set the program as mentioned in Table 2 with the lid temperature set at 105 &#730;C and a ramp rate of 2 &#730;C/s.</li>
	</ol>
	</li>
	<li>Droplet counting.
	<ol>
		<li>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.</li>
		<li>Open the droplet analysis software and set up the run by giving input for the sample information.</li>
		<li>Click on the setup option. Then, click on the template option to open a new template.</li>
		<li>Define each well by putting the details of the experiment, such as sample name (cDNA used or negative control or NTC), target name (circRNA primer name) and type (unknown), and experiment type as ABS (absolute quantification) and Supermix (dd-PCR EvaGreen Supermix) used, etc.</li>
		<li>Finally, save the template and start the run by clicking on the run option and select counting by row or column-wise. Allow the machine to complete the droplet counting, which takes about 1 min/well.</li>
		<li>Click the Analyze button to analyze the data after the completion of the droplet reading.</li>
		<li>Click the 1D Amplitude button to see the positive and negative droplets</li>
		<li>To segregate the positive droplets from the negative droplets, put a common threshold line for all samples with the same target. 
		NOTE: The total droplets in each sample should be above 10,000 to be considered for absolute quantification. The NTC should not show positive droplet counts.&#160;The presence of positive droplets in the NTC indicates contamination of the reagents or amplification of the primer dimers. It is difficult to find out whether the positive droplets have primer dimers or non-specific products in NTC. It is advised to check the primers and avoid any contamination of the reagents as a general practice for any PCR including dd-PCR.</li>
		<li>Click the Export button to export the circRNA counts data as a .csv file. The exported data show the number of circRNAs present in each sample.</li>
		<li>Manually calculate the number of circRNAs in each sample per nanogram of RNA by considering the total amount of cDNA used in each reaction.</li>
	</ol>
	</li>
</ol>

<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. S., 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+biogenesis,+biology+and+characterization+of+circular+RNAs.">The biogenesis, biology and characterization of circular RNAs.</a> Nature Reviews Genetics. 20 (11), 675-691 (2019).</li><li>Chen, X., Zhou, M., Yant, L., Huang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNA+in+disease:+Basic+properties+and+biomedical+relevance.">Circular RNA in disease: Basic properties and biomedical relevance.</a> Wiley Interdisciplinary Reviews. RNA. , (2022).</li><li>Sinha, T., Panigrahi, C., Das, D., Chandra Panda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNA+translation,+a+path+to+hidden+proteome.">Circular RNA translation, a path to hidden proteome.</a> Wiley Interdisciplinary Reviews. RNA. 13 (1), 1685 (2022).</li><li>Panda, A. C., Xiao, J. <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+Act+as+miRNA+Sponges.">Circular RNAs Act as miRNA Sponges.</a> Circular RNAs: Biogenesis and Functions. , 67-79 (2018).</li><li>Das, A., Sinha, T., Shyamal, S., 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=Emerging+role+of+circular+RNA-protein+interactions.">Emerging role of circular RNA-protein interactions.</a> Non-Coding RNA. 7 (3), 48 (2021).</li><li>Verduci, L., Tarcitano, E., Strano, S., Yarden, Y., Blandino, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CircRNAs:+Role+in+human+diseases+and+potential+use+as+biomarkers.">CircRNAs: Role in human diseases and potential use as biomarkers.</a> Cell Death &amp; Disease. 12 (5), 468 (2021).</li><li>Pandey, P. R., 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=Methods+for+analysis+of+circular+RNAs.">Methods for analysis of circular RNAs.</a> Wiley Interdisciplinary Reviews: RNA. 11 (1), 1566 (2020).</li><li>Das, A., Das, D., 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=Validation+of+circular+RNAs+by+PCR.">Validation of circular RNAs by PCR.</a> PCR Primer Design. Methods in Molecular Biology. 2392, 103-114 (2022).</li><li>Jeck, W. R., 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=Circular+RNAs+are+abundant,+conserved,+and+associated+with+ALU+repeats.">Circular RNAs are abundant, conserved, and associated with ALU repeats.</a> RNA. 19 (2), 141-157 (2013).</li><li>Pinheiro, L. 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 ...

quantification-of-circular-rnas-using-digital-droplet-pcr

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3.3K Views.  Institute of Life Sciences. 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. 

Video: Quantification of Circular RNAs Using Digital Droplet PCR

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

Rapid Genotyping of Mouse Tissue Using Sigma&#039;s Extract-N-Amp Tissue PCR Kit

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Rapid PCR Thermocycling using Microscale Thermal Convection

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed1. Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.
Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations2-9. Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument.
One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time10,11. Here we describe results of our efforts to probe the full 3-D velocity and temperature distributions in microscale convective thermocyclers12. Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

The goal of this video is to demonstrate how to perform&#160;automated DNA extraction from formalin-fixed paraffin-embedded (FFPE) reference standard cell lines and digital droplet PCR&#160;(ddPCR) analysis to detect rare mutations in a clinical setting. Detecting mutations in FFPE samples demonstrates the clinical utility of ddPCR in FFPE samples.

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification.
A fixed volume (200 &#181;L) of EDTA-plasma was prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50). Total RNA was extracted using commercially available system. Plasma miRNAs were quantified by probe-based RT-qPCR assays which contains miRNA-specific forward/reverse PCR primer and probe. Standard curves for absolute quantification were generated using commercially available synthetic RNA oligonucleotides. A synthetic cel-miR-238 was used as an external control for normalization and quality assessment. The miRNAs that showed quantification cycle (Cq) values above 35 were pre-amplified prior to the qPCR step.
Among the 8 miRNAs examined, miR-122, miR-133a, and miR-192 were detectable without pre-amplification, whereas miR-1, miR-206, and miR-499a required pre-amplification because of their low expression levels. MiR-208a and miR-208b were not detectable even after pre-amplification. Sample processing efficiency was evaluated by the Cq values of the spiked cel-miR-238. In this assay method, technical variation was estimated to be less than 3-fold and the lower limit of quantification (LLOQ) was 102 copy/&#181;L, for most of the examined miRNAs.
This protocol provides a better estimate of the quantity of plasma miRNAs, and allows quality assessment of corresponding data from different studies. Considering the low number of miRNAs in body fluids, pre-amplification is useful to enhance detection of poorly expressed miRNAs.

This report describes a protocol for measuring the absolute levels of plasma miRNA, using quantitative real-time reverse transcription PCR with or without pre-amplification. This protocol affords better understanding of the quantity of plasma miRNAs and allows qualitative assessment of corresponding data from different studies or laboratories.

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This ...

Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral lipids encased by a monolayer of phospholipids and proteins. Hepatic LD lipids, such as ceramides, and proteins are implicated in several diseases that cause hepatic steatosis. Although previous methods have been established for LD isolation, they require a time-consuming preparation of reagents and are not designed for the isolation of multiple subcellular compartments. We sought to establish a new protocol to enable the isolation of LDs, endoplasmic reticulum (ER), and lysosomes from a single mouse liver.
Further, all reagents used in the protocol presented here are commercially available and require minimal reagent preparation without sacrificing LD purity. Here we present data comparing this new protocol to a standard sucrose gradient protocol, demonstrating comparable purity, morphology, and yield. Additionally, we can isolate ER and lysosomes using the same sample, providing detailed insight into the formation and intracellular flux of lipids and their associated proteins.

This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit.

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...

Quantification of Viral Vectors in Tears Using ddPCR for Bioshedding Studies

A Validatable Droplet Digital Polymerase Chain Reaction Assay for the Detection of Adeno-Associated Viral Vectors in Bioshedding Studies of Tears

The use of viral vectors to treat genetic diseases has increased substantially in recent years, with over 2,000 studies registered to date. Adeno-associated viral (AAV) vectors have found particular success in the treatment of eye related diseases, as exemplified by the approval of voretigene neparvovec-rzyl. To bring new therapies to market, regulatory agencies typically request qualified or validated bioshedding studies to evaluate release of the vector into the environment. However, no official guidelines for the development of molecular based assays to support such shedding studies have been released by the United States Food and Drug Administration, leaving developers to determine best practices for themselves. The purpose of this protocol is to present a validatable protocol for the detection of AAV vectors in human tears by droplet digital polymerase chain reaction (ddPCR) in support of clinical bioshedding studies. This manuscript discusses current industry approaches to molecular assay validation and demonstrates that the method exceeds the target assay acceptance criteria currently proposed in white papers. Finally, steps critical in the performance of any ddPCR assay, regardless of application, are discussed.

Author Spotlight: Addressing Regulatory Gaps in Molecular Studies by Quantifying Viral Vectors in Complex Matrices 

Here, we present a protocol for the development and validation of good laboratory practices in the compliant detection of adeno-associated viral vectors in human tears by droplet digital polymerase chain reaction in support of clinical development of gene therapy vectors.