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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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)

  1. 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,

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
1.5 ml microcentifuge tubeTarson500010
0.2 ml tube strips with capTarson610020, 510073
Filter TipsTarson528104
DNase/RNase-Free Distilled WaterThermo Fisher Scientific10977023
Phosphate-buffered saline (PBS)SigmaP4417
Cell scrapperHiMediaTCP223
ChloroformSRL96764
DNA diluentHiMediaMB228
Random primersThermo Fisher Scientific48190011
dNTP setThermo Fisher ScientificR0181
Murine RNase inhibitorNEBM0314S
Maxima reverse transcriptaseThermo Fisher ScientificEP0743
QX200 dd-PCR Evagreen SupermixBio-Rad1864033
Droplet generation oil for EvagreenBio-Rad1864006
PCR Plate Heat Seal, foil, pierceableBio-Rad1814040
DG8 Cartridges and GasketsBio-Rad1864007
DG8 Cartridge holderBio-Rad1863051
QX200 Droplet GeneratorBio-Rad1864002
ddPCR 96-Well PlatesBio-Rad12001925
PX1 PCR Plate SealerBio-Rad1814000
C1000 Touch Thermal Cycler with 96–Deep Well Reaction ModuleBio-Rad1851197
QX200 Droplet ReaderBio-Rad1864003
Quantasoft SoftwareBio-Rad1864011
Silica columnUmbrella Life Science38220090
UCSC Genome Browserhttps://genome.ucsc.edu/
Primer 3https://primer3.ut.ee/

References

  1. Salzman, J., Gawad, C., Wang, P. L., Lacayo, N., Brown, P. O. Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. PLoS One. 7 (2), 30733 (2012).
  2. Chen, L. L., Yang, L.

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