Analysis of Extracellular RNA by Digital PCR

Overview

Journal Front Oncol

Specialty Oncology

Date 2014 Jun 14

PMID 24926435

Citations 23

Authors

Kenji Takahashi

Irene K Yan

Chaeyoung Kim

Jungsu Kim

Tushar Patel

Affiliations

Soon will be listed here.

Abstract

The transfer of extracellular RNA is emerging as an important mechanism for inter-cellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

Citing Articles

Non-Coding RNAs in Cancer: Structure, Function, and Clinical Application.

Marton E, Varga A, Domoszlai D, Buglyo G, Balazs A, Penyige A Cancers (Basel). 2025; 17(4).

PMID: 40002172 PMC: 11853212. DOI: 10.3390/cancers17040579.

Approaches and Challenges in Characterizing the Molecular Content of Extracellular Vesicles for Biomarker Discovery.

Kumari S, Lausted C, Scherler K, Ng A, Lu Y, Lee I Biomolecules. 2025; 14(12.

PMID: 39766306 PMC: 11674167. DOI: 10.3390/biom14121599.

Recent Advances in Molecular Diagnosis of Pulmonary Fibrosis for Precision Medicine.

Jeong M, Han H, Lagares D, Im H ACS Pharmacol Transl Sci. 2022; 5(8):520-538.

PMID: 35983278 PMC: 9379941. DOI: 10.1021/acsptsci.2c00028.

The Aflibercept-Induced MicroRNA Profile in the Vitreous of Proliferative Diabetic Retinopathy Patients Detected by Next-Generation Sequencing.

Guo J, Zhou P, Liu Z, Dai F, Pan M, An G Front Pharmacol. 2021; 12:781276.

PMID: 34938191 PMC: 8685391. DOI: 10.3389/fphar.2021.781276.

Extracellular Vesicles and Their Relationship with the Heart-Kidney Axis, Uremia and Peritoneal Dialysis.

Azevedo C, da Cunha R, Junho C, da Silva J, Moreno-Amaral A, Moraes T Toxins (Basel). 2021; 13(11).

PMID: 34822562 PMC: 8618757. DOI: 10.3390/toxins13110778.

References

Skog J, Wurdinger T, van Rijn S, Meijer D, Gainche L, Sena-Esteves M . Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol. 2008; 10(12):1470-6. PMC: 3423894. DOI: 10.1038/ncb1800. View

Noerholm M, Balaj L, Limperg T, Salehi A, Zhu L, Hochberg F . RNA expression patterns in serum microvesicles from patients with glioblastoma multiforme and controls. BMC Cancer. 2012; 12:22. PMC: 3329625. DOI: 10.1186/1471-2407-12-22. View

Thery C . Exosomes: secreted vesicles and intercellular communications. F1000 Biol Rep. 2011; 3:15. PMC: 3155154. DOI: 10.3410/B3-15. View

Cocucci E, Racchetti G, Meldolesi J . Shedding microvesicles: artefacts no more. Trends Cell Biol. 2009; 19(2):43-51. DOI: 10.1016/j.tcb.2008.11.003. View

Arroyo J, Chevillet J, Kroh E, Ruf I, Pritchard C, Gibson D . Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A. 2011; 108(12):5003-8. PMC: 3064324. DOI: 10.1073/pnas.1019055108. View

Kogure T, Patel T . Isolation of extracellular nanovesicle microRNA from liver cancer cells in culture. Methods Mol Biol. 2013; 1024:11-8. DOI: 10.1007/978-1-62703-453-1_2. View

Takahashi K, Yan I, Haga H, Patel T . Long noncoding RNA in liver diseases. Hepatology. 2014; 60(2):744-53. PMC: 4110118. DOI: 10.1002/hep.27043. View

Huggett J, Foy C, Benes V, Emslie K, Garson J, Haynes R . The digital MIQE guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments. Clin Chem. 2013; 59(6):892-902. DOI: 10.1373/clinchem.2013.206375. View

Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee J, Lotvall J . Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007; 9(6):654-9. DOI: 10.1038/ncb1596. View

10.

Hindson C, Chevillet J, Briggs H, Gallichotte E, Ruf I, Hindson B . Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods. 2013; 10(10):1003-5. PMC: 4118677. DOI: 10.1038/nmeth.2633. View

11.

Vickers K, Remaley A . Lipid-based carriers of microRNAs and intercellular communication. Curr Opin Lipidol. 2012; 23(2):91-7. PMC: 5570485. DOI: 10.1097/MOL.0b013e328350a425. View

12.

Hindson B, Ness K, Masquelier D, Belgrader P, Heredia N, Makarewicz A . High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem. 2011; 83(22):8604-10. PMC: 3216358. DOI: 10.1021/ac202028g. View

13.

Turchinovich A, Weiz L, Langheinz A, Burwinkel B . Characterization of extracellular circulating microRNA. Nucleic Acids Res. 2011; 39(16):7223-33. PMC: 3167594. DOI: 10.1093/nar/gkr254. View

14.

Kogure T, Yan I, Lin W, Patel T . Extracellular Vesicle-Mediated Transfer of a Novel Long Noncoding RNA TUC339: A Mechanism of Intercellular Signaling in Human Hepatocellular Cancer. Genes Cancer. 2013; 4(7-8):261-72. PMC: 3807642. DOI: 10.1177/1947601913499020. View

15.

Braconi C, Kogure T, Valeri N, Huang N, Nuovo G, Costinean S . microRNA-29 can regulate expression of the long non-coding RNA gene MEG3 in hepatocellular cancer. Oncogene. 2011; 30(47):4750-6. PMC: 4292930. DOI: 10.1038/onc.2011.193. View

16.

Kogure T, Lin W, Yan I, Braconi C, Patel T . Intercellular nanovesicle-mediated microRNA transfer: a mechanism of environmental modulation of hepatocellular cancer cell growth. Hepatology. 2011; 54(4):1237-48. PMC: 3310362. DOI: 10.1002/hep.24504. View