Analysis of Extracellular RNA by Digital PCR
Open accessAbstract
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.
AI snapshot
Written from the paper’s full text.
TL;DR
This methods article describes protocols for isolating extracellular vesicles and RNA and for detecting extracellular RNA by droplet digital PCR (ddPCR) and RainDrop dPCR. It demonstrates sensitive absolute quantification of synthetic and endogenous microRNAs and lncRNAs, including detection below qPCR limits.
Key findings
- dPCR detected synthetic cel-miR-39 input RNA amounts as low as 0.01 pg.
- In HepG2 cells and their EVs, miR-29 concentration in EV was 0.6 copies/μl and was decreased compared with donor cells; based on 12 copies of miR-29 per 1 ng cDNA, the authors estimate a minimum of 1.4 copies of miR-29 per 10^6 vesicles on average.
- TUC339 was not detected in EV derived from human serum by TaqMan RT-qPCR but could be detected by ddPCR with input RNA amounts as low as 0.5 ng/μl (data not shown).
- dPCR provides absolute quantification and is more tolerant to PCR inhibitors because it uses presence/absence endpoint detection.
Methods
- EV were isolated from cell culture supernatants or human serum by sequential centrifugation/ultracentrifugation; RNA was extracted using TRIzol or ExoQuick/SeraMir, then reverse transcribed.
- Two dPCR platforms were used: Bio-Rad QX100 ddPCR and RainDance RainDrop; samples were partitioned into droplets, endpoint PCR was performed, fluorescence was measured, and data were analyzed with QuantaSoft or RainDrop Analyst.
- The Poisson equation was used for absolute quantification: copies per droplet = −ln(1 − p), where p = fraction of positive droplets. Samples included synthetic cel-miR-39, HepG2 cells and their EVs, and human serum EV RNA analyzed for lncRNA 21A, NDM29, and TUC339.
Limitations
- The paper states that limited sample size and low abundance of extracellular RNA necessitate a sensitive method such as dPCR, and that further development and standardization of dPCR-based assays are required; digital MIQE guidelines are cited as a first step.
- The paper states that EV preparations vary with the isolation approach and may contain non-vesicular RNA or restricted EV populations, and that isolation of pure exosomes or other vesicle populations requires additional procedures such as density gradient centrifugation.
- Evident from the text: no formal limitations section is included; some TUC339 detection data are “not shown,” and several analyses use small replicate numbers (e.g., n = 4 replicates in Figure 4). The paper also notes that dPCR may underestimate true concentration because a positive droplet cannot differentiate the number of molecules present, which is addressed by Poisson correction.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
10 of 13 cited sources checked · 7 supported · 0 flagged · accuracy not yet measured
- Premisecitation
Recent studies comparing qPCR and dPCR reported improved precision and reproducibility with dPCR.
“Recent studies have compared the two technologies and reported improved precision and reproducibility with dPCR (11).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [11] Hindson, 2013, Absolute quantification by droplet digital PCR versus analog real-time PCR, Nat Methods source
- Premisecitation
Extracellular RNA is susceptible to degradation by circulating RNAses.
“Extracellular RNA is susceptible to degradation by circulating RNAses (2).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [2] Turchinovich, 2011, Characterization of extracellular circulating microRNA, Nucleic Acids Res source
The Results section (‘Extracellular versus intracellular miRNA expression pattern’; page unavailable) reports that synthetic RNA added to conditioned medium was immediately degraded, supporting susceptibility of unprotected extracellular RNA. But it also reports that endogenous extracellular miRNAs resisted added RNase A; the paper does not establish that extracellular RNA generally is degraded by circulating RNases. (LLM, conf=0.85)
Experimental — accuracy not yet measured
- Premisecitation
Digital PCR provides an alternative approach for detecting gene expression when the amount of target RNA is low and approaching the limits of sensitivity of qPCR.
“Digital polymerase chain reaction (dPCR) provides an alternative approach for the detection of gene expression in the setting where the amount of target RNA is low and approaching the limits of sensitivity of qPCR (10).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [10] Hindson, 2011, High-throughput droplet digital PCR system for absolute quantitation of DNA copy number, Anal Chem source
The Abstract says the droplet digital PCR system provides greater sensitivity than real-time PCR, and Results and Discussion reports detection of rare mutant DNA below the real-time PCR detection limit. The paper's examples concern DNA, however; these passages do not establish its use for detecting gene expression when target RNA is scarce. (LLM, conf=0.86)
Experimental — accuracy not yet measured
- Findingown data
Based on the measured 12 copies of miR-29 per 1 ng of cDNA, there is a minimum of 1.4 copies of miR-29 per 106 vesicles on average.
“Based on the measured 12 copies of miR-29 per 1 ng of cDNA, we estimate a minimum of 1.4 copies of miR-29 per 106 vesicles on average.”
Quote in the paperSubstantial claim - Findingown data
dPCR can detect input RNA amounts as low as 0.01 pg.
“These studies demonstrate the ability of dPCR to detect input RNA amounts as low as 0.01 pg.”
Quote in the paperSubstantial claim
- Findingown data
795.3 ng of RNA was isolated from 6985 × 106 particles as quantitated by nanoparticle tracking analysis using Nanosight.
“795.3 ng of RNA was isolated from 6985 × 106 particles as quantitated by nanoparticle tracking analysis using Nanosight.”
Quote in the paperNot a substantial claim - Premisenone given
The use of qPCR is limited by its sensitivity in detecting the small amounts of RNA obtained from conventional preparations of extracellular RNA.
“Although well-established, and robust, the use of qPCR is limited by the sensitivity in detecting the small amounts of RNA that are obtained from conventional preparations of extracellular RNA.”
Quote in the paperSubstantial claim - Premisereasoning
The true concentration in dPCR may be underestimated because a positive droplet cannot differentiate between the number of molecules present in each partition.
“The true concentration may be underestimated because a positive droplet cannot differentiate between the number of molecules present in each partition.”
Quote in the paperNot a substantial claim - Interpretationown data
ddPCR may be useful as an end-point assay to detect very low expression genes that cannot be detected by qPCR.
“Thus ddPCR may be useful as an end-point assay to detect the presence of very low expression genes that may have biological or translational potential but cannot be detected by qPCR.”
Quote in the paperSubstantial claim
- May combine two claims. 12
- Classified as probably not a substantial claim. 21
May combine two claims.
PremisecitationExtracellular vesicles can transfer RNAs such as non-coding RNAs from one cell to another and thereby contribute to inter-cellular communication.
“Recent studies have confirmed that EV can transfer RNAs such as ncRNAs from one cell to another and thereby contribute to important roles in inter-cellular communication (3–9).”
- Also stated in: § Abstract · plain
“The transfer of extracellular RNA is emerging as an important mechanism for inter-cellular communication.”
- Also stated in: § Introduction · plain
“An emerging mechanism for cell-to-cell communication involves the transfer of biologically active RNA molecules released into the extracellular space and that can be taken up by recipient cells.”
Quote in the paperSubstantial claim5 of 7 sources checked- cites [3] Vickers, 2012, Lipid-based carriers of microRNAs and intercellular communication, Curr Opin Lipidol source
- cites [4] Valadi, 2007, Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells, Nat Cell Biol source
- cites [5] Skog, 2008, Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers, Nat Cell Biol source
- cites [6] Thery, 2011, Exosomes: secreted vesicles and intercellular communications, F1000 Biol Rep source
The Introduction reports that exosomes contain mRNA and microRNA and that, in vitro, some exosomal mRNA was translated in target cells, suggesting transfer of genetic information. The RNA and miRNA in secreted membrane vesicles section confirms miRNA in vesicle preparations but does not establish its transfer to recipient cells; the claim is therefore stronger than this source supports for non-coding RNA. (LLM, conf=0.84)
Experimental — accuracy not yet measured - cites [7] Cocucci, 2009, Shedding microvesicles: artefacts no more, Trends Cell Biol source
- cites [8] Kogure, 2011, Intercellular nanovesicle-mediated microRNA transfer: a mechanism of environmental modulation of hepatocellular cancer cell growth, Hepatology source
- cites [9] Takahashi, 2014, Long non-coding RNA in liver diseases source
- Also stated in: § Abstract · plain
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationGuidelines for digital MIQE have been proposed.
“As a first step toward this, guidelines for digital MIQE have been proposed (16).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [16] Huggett, 2013, The digital MIQE guidelines: minimum information for publication of quantitative digital PCR experiments, Clin Chem source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationmiR-29 can regulate expression of MEG3, a tumor suppressive lncRNA, and miR-29 expression is down regulated in human HCC cells.
“miR-29 can regulate expression of MEG3, a tumor suppressive lncRNA, and miR-29 expression was down regulated in human HCC cells (14).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [14] Braconi, 2011, microRNA-29 can regulate expression of the long non-coding RNA gene MEG3 in hepatocellular cancer, Oncogene source
Probably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataRNA yield from 1 ml of human serum was 183 ng.
“RNA yield was determined using Nanodrop and a total of 183 ng RNA was obtained.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataTUC339 was not detected in EV derived from human serum by TaqMan RT-qPCR, but could be detected by ddPCR with input RNA amounts as low as 0.5 ng/μl.
“TUC339 was not detected in EV derived from human serum by TaqMan RT-qPCR, but could be detected by ddPCR with input RNA amounts as low as 0.5 ng/μl (data not shown).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataAlthough expression of miR-29b was very low in EV, its detection was possible with ddPCR.
“Although the expression of miR-29b expression was very low in EV, their detection was possible with ddPCR.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe concentration of miR-29 in EV was 0.6 copies/μl, and was decreased compared with that in donor cells.
“The concentration of miR-29 in EV was 0.6 copies/μl, and was decreased compared with that in donor cells (Figure 3).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S4 (based).
InterpretationreasoningRNA enclosed within extracellular vesicles may be protected from degradation by circulating RNAses.
“However, RNA enclosed within EV may be protected from such degradation.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
PremisereasoningDetection using dPCR is more tolerant to PCR inhibitors because it is based on detection of presence or absence of a reaction end-point.
“In addition, detection using dPCR is more tolerant to PCR inhibitors because it is based on the detection of presence or absence of a reaction end-point.”
Quote in the paperSubstantial claimMay combine two claims.
PremisereasoningdPCR offers absolute quantification of gene expression, thereby avoiding reliance on invariant genes.
“dPCR offers the ability to obtain absolute quantification of gene expression, thereby avoiding reliance on the need for invariant genes (which may not be possible in the analysis of extracellular RNA).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationTUC339 is a long non-coding RNA that is selectively released in EV from hepatocellular cancer cells and is implicated in modulating tumor cell behavior.
“We also analyzed expression of TUC339, a long non-coding RNA that is selectively released in EV from hepatocellular cancer cells and is foundationally implicated in modulating tumor cell behavior (15).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [15] Kogure, 2013, Extracellular vesicle-mediated transfer of a novel long noncoding RNA TUC339: a mechanism of intercellular signaling in human hepatocellular cancer, Genes Cancer source
Probably not a substantial claim: may not pass S2 (checkable).
InterpretationreasoningDue to limited sample size and low abundance of extracellular RNA, a sensitive method of measuring gene expression such as dPCR is necessary.
“Due to limited sample size and low abundance of extracellular RNA, a sensitive method of measuring gene expression such as dPCR is necessary.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductnone givenThe sequential centrifugation method provides intact EV that can be further analyzed using nanoparticle tracking analysis or electron microscopy.
“This method provides intact EV that can be further analyzed using nanoparticle tracking analysis (NanoSight LM10 instrument, Amesbury, UK, or similar) or examined with electron microscopy.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S2 (checkable). May combine two claims.
Interpretationnone givenFor human cancers, tumor-specific changes in EV RNA content could be potentially used, but such applications require sensitive and accurate determination of their RNA content.
“For human cancers, tumor-specific changes in EV RNA content could be potentially used, but such applications require sensitive and accurate determination of their RNA content.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Premisenone givenIncreasing the number of partitions offers the potential to increase sensitivity for detecting very small amounts of targets.
“Increasing the number of partitions offers the potential to increase sensitivity for the detection of very small amounts of targets, and can be accomplished through microfluidic based PCR platforms such as the BioMark (Fluidigm) and Open Array (Life Technologies).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S4 (based).
Premisenone givenThe ability to detect circulating tumor cell derived extracellular RNA is necessary to enable their use as biomarkers of disease.
“The ability to detect circulating tumor cell derived extracellular RNA is necessary to enable their use as biomarkers of disease.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S2 (checkable), S4 (based).
Premisenone givenThe ability to analyze extracellular RNA is essential for the potential application of extracellular RNA as biomarkers for early diagnosis or as prognostic markers of disease.
“The ability to analyze extracellular RNA is essential for the potential application of extracellular RNA as biomarkers for early diagnosis, or as prognostic markers of disease.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S2 (checkable).
InterpretationreasoningThe use of dPCR could be a very useful tool for the detection and quantitation of extracellular RNA.
“Therefore, the use of dPCR could be a very useful tool for the detection and quantitation of extracellular RNA.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductreasoningEV preparations obtained vary with the isolation approach used and may contain non-vesicular RNA or restricted EV populations.
“It should be noted that the EV preparations obtained vary with the approach used for isolation and depending on the isolation approach used, they may contain non-vesicular RNA or restricted EV populations.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenGating in the RainDrop assay was performed on a negative control sample and applied for all samples from the same experiment.
“Gating is performed on negative control sample and applied for all samples from the same experiment.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S4 (based).
Premisenone givendPCR provides absolute quantification.
“In addition, dPCR provides absolute quantification.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S2 (checkable).
InterpretationreasoningThe use of dPCR could be a very useful tool for the detection and quantitation of RNA biomarkers in the clinic.
“The use of dPCR could be a very useful tool for the detection and quantitation of RNA biomarkers in the clinic.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim. May combine two claims.
Interpretationnone givenThe approaches described may provide a dPCR based method that could be used for detecting the presence and for quantitating circulating RNA, or RNA within EV released from cells in culture.
“The approaches described herein may provide a dPCR based method that could be used for detecting the presence and for quantitating circulating RNA, or RNA within EV released from cells in culture.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenThresholds for detection in ddPCR were set manually based on results from negative control wells containing water instead of RNA.
“The data is analyzed using the QuantaSoft software (Bio-Rad) with the thresholds for detection set manually based on results from negative control wells containing water instead of RNA.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductreasoningFor most isolations, EV were collected from supernatants obtained from at least 16 10-cm culture dishes in order to obtain a sufficient yield for downstream studies.
“For most isolations, we have collected EV from supernatants obtained from at least 16 10-cm culture dishes in order to obtain a sufficient yield for downstream studies.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
InterpretationreasoningdPCR has an advantage for studies where the target of interest is limited or present in low quantities that cannot reliably be detected using qPCR.
“dPCR has an advantage for studies where the target of interest is limited or present in low quantities that cannot reliably be detected using qPCR.”
Quote in the paperSubstantial claimMay combine two claims.
InterpretationreasoningThe use of dPCR offers high sensitivity and enables absolute quantitation of low abundance transcripts that may be present within EV.
“The use of dPCR offers high sensitivity and enables absolute quantitation of low abundance transcripts that may be present within EV.”
Quote in the paperSubstantial claim
The paper relies on this without support.
Extracellular RNA can be released from cells as native RNAs, associated with proteins, or enclosed within extracellular vesicles such as exosomes.
“Extracellular RNA can be released from cells as native RNAs, associated with proteins, or enclosed within extracellular vesicles (EV) such as exosomes.”
The paper relies on this without support.
Extracellular RNA can be detected within extracellular vesicles in the circulation.
“Extracellular RNA can be detected within EV in the circulation.”
The paper relies on this without support.
dPCR has been shown to provide precise estimates of DNA copy number with high-throughput capabilities.
“In other applications, dPCR has been shown to be able to provide precise estimates of DNA copy number with high-throughput capabilities.”
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
HepG2 EV preparation methods differ between results and figure caption
MajorThe results say the HepG2 EV were isolated by differential ultracentrifugation and their RNA isolated as described above, whereas the Figure 3 caption identifies ExoQuick and SeraMir for the EV RNA preparation.
“Supernatant was collected, and EV isolated using differential ultracentrifugation, and RNA isolated from EV preparations as described above.”
“RNA was extracted from HepG2 cells using TRIzol and from extracellular vesicles derived from these cells using ExoQuick and SeraMir (System Biosciences).”
Serum RNA used for reverse transcription exceeds the reported yield
MajorThe results report a total yield of 183 ng RNA from the serum sample, but the Figure 4 caption says 200 ng RNA was used for reverse transcription of that sample.
“RNA yield was determined using Nanodrop and a total of 183 ng RNA was obtained.”
“A total of 200 ng RNA was used for RT and template cDNA was diluted to 2, 1, 0.5, and 0.25 ng for PCR.”
Cites
Valadi, H., Ekström, K., Bossios, A., Sjöstrand, M., Lee, J. J., & Lötvall, J. O. (2007) · Nature Cell Biology · doi:10.1038/ncb1596 · Cited by 12,834 · cited 1× · see citing works
“They are released from a wide variety of normal or diseased cells (4, 5).”
BackgroundSkog, J., Würdinger, T., van Rijn, S., Meijer, D. H., Gainche, L., Curry, W. T., Carter, B. S., Krichevsky, A. M., & Breakefield, X. O. (2008) · Nature Cell Biology · doi:10.1038/ncb1800 · Cited by 5,001 · see citing works
Arroyo, J. D., Chevillet, J. R., Kroh, E. M., Ruf, I. K., Pritchard, C. C., Gibson, D. F., Mitchell, P. S., Bennett, C. F., Pogosova-Agadjanyan, E. L., Stirewalt, D. L., et al. (2011) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1019055108 · Cited by 3,294 · cited 1× · see citing works
“This approach is based on previous reports from our laboratory and others (1, 5, 12, 13) and has been adapted for isolation of EV from cells in culture or from serum.”
MethodHigh-throughput droplet digital PCR system for absolute quantitation of DNA copy number
Hindson, B. J., Ness, K. D., Masquelier, D. A., Belgrader, P., Heredia, N. J., Makarewicz, A. J., Bright, I. J., Lucero, M. Y., Hiddessen, A. L., Legler, T. C., et al. (2011) · Analytical Chemistry · doi:10.1021/ac202028g · Cited by 2,792 · cited 1× · see citing works
“Digital polymerase chain reaction (dPCR) provides an alternative approach for the detection of gene expression in the setting where the amount of target RNA is low and approaching the limits of sensitivity of qPCR (10).”
BackgroundCharacterization of extracellular circulating microRNA
Turchinovich, A., Weiz, L., Langheinz, A., & Burwinkel, B. (2011) · Nucleic Acids Research · doi:10.1093/nar/gkr254 · Cited by 1,941 · cited 1× · see citing works
“Extracellular RNA is susceptible to degradation by circulating RNAses (2).”
BackgroundShedding microvesicles: artefacts no more
Cocucci, E., Racchetti, G., & Meldolesi, J. (2009) · Trends in Cell Biology · doi:10.1016/j.tcb.2008.11.003 · Cited by 1,742 · see citing works
Absolute quantification by droplet digital PCR versus analog real-time PCR
Hindson, C. M., Chevillet, J. R., Briggs, H. A., Gallichotte, E. N., Ruf, I. K., Hindson, B. J., Vessella, R. L., & Tewari, M. (2013) · Nature Methods · doi:10.1038/nmeth.2633 · Cited by 1,488 · cited 1× · see citing works
“Recent studies have compared the two technologies and reported improved precision and reproducibility with dPCR (11).”
BackgroundExosomes: secreted vesicles and intercellular communications
Théry, C. (2011) · F1000 Biology Reports · doi:10.3410/b3-15 · Cited by 989 · see citing works
Huggett, J. F., Foy, C. A., Benes, V., Emslie, K., Garson, J. A., Haynes, R., Hellemans, J., Kubista, M., Mueller, R. D., Nolan, T., et al. (2013) · Clinical Chemistry · doi:10.1373/clinchem.2013.206375 · Cited by 859 · see citing works
microRNA-29 can regulate expression of the long non-coding RNA gene MEG3 in hepatocellular cancer
Braconi, C., Kogure, T., Valeri, N., Huang, N., Nuovo, G., Costinean, S., Negrini, M., Miotto, E., Croce, C. M., & Patel, T. (2011) · Oncogene · doi:10.1038/onc.2011.193 · Cited by 642 · cited 1× · see citing works
“miR-29 can regulate expression of MEG3, a tumor suppressive lncRNA, and miR-29 expression was down regulated in human HCC cells (14).”
BackgroundSelf-citationKogure, T., Lin, W.-L., Yan, I. K., Braconi, C., & Patel, T. (2011) · Hepatology · doi:10.1002/hep.24504 · Cited by 551 · see citing works
Self-citationLipid-based carriers of microRNAs and intercellular communication
Vickers, K. C., & Remaley, A. T. (2012) · Current Opinion in Lipidology · doi:10.1097/mol.0b013e328350a425 · Cited by 314 · see citing works
Kogure, T., Yan, I. K., Lin, W.-L., & Patel, T. (2013) · Genes & Cancer · doi:10.1177/1947601913499020 · Cited by 303 · see citing works
Self-citationNoerholm, M., Balaj, L., Limperg, T., Salehi, A., Zhu, L. D., Hochberg, F. H., Breakefield, X. O., Carter, B. S., & Skog, J. (2012) · BMC Cancer · doi:10.1186/1471-2407-12-22 · Cited by 190 · cited 1× · see citing works
“This approach is based on previous reports from our laboratory and others (1, 5, 12, 13) and has been adapted for isolation of EV from cells in culture or from serum.”
MethodLong noncoding RNA in liver diseases
Takahashi, K., Yan, I., Haga, H., & Patel, T. (2014) · Hepatology · doi:10.1002/hep.27043 · Cited by 184 · see citing works
Self-citationIsolation of Extracellular Nanovesicle MicroRNA from Liver Cancer Cells in Culture
Kogure, T., & Patel, T. C. (2013) · Methods in molecular biology · doi:10.1007/978-1-62703-453-1_2 · Cited by 22 · cited 1× · see citing works
“This approach is based on previous reports from our laboratory and others (1, 5, 12, 13) and has been adapted for isolation of EV from cells in culture or from serum.”
MethodSelf-citation
Flagged first, then by citations
Cited by
Sorted by citations
Extracellular Vesicles: Composition, Biological Relevance, and Methods of Study
Zaborowski, M. P., Balaj, L., Breakefield, X. O., & Lai, C. P. (2015) · BioScience · doi:10.1093/biosci/biv084 · Cited by 1,272 · see citing works
Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles
Bordanaba-Florit, G., Royo, F., Kruglik, S. G., & Falcón‐Pérez, J. M. (2021) · Nature Protocols · doi:10.1038/s41596-021-00551-z · Cited by 328 · see citing works
Buschmann, D., Kirchner, B., Hermann, S., Märte, M., Wurmser, C., Brandes, F., Kotschote, S., von Bonin, M., Steinlein, O. K., Pfaffl, M. W., et al. (2018) · Journal of Extracellular Vesicles · doi:10.1080/20013078.2018.1481321 · Cited by 261 · see citing works
Emerging methods in biomarker identification for extracellular vesicle‐based liquid biopsy
Liang, Y., Lehrich, B. M., Zheng, S., & Lu, M. (2021) · Journal of Extracellular Vesicles · doi:10.1002/jev2.12090 · Cited by 198 · see citing works
“…and optimized the protocol for miRNA and lncRNA dPCR analysis for EVs derived from cell culture medium and serum (Ferracin & Negrini, 2018; Takahashi et al., 2014), while other groups have carefully compared the performance between dPCR and qPCR for EVmiRNA detection in the setting of…”
MethodDigital PCR analysis of circulating nucleic acids
Hudecova, I. (2015) · Clinical Biochemistry · doi:10.1016/j.clinbiochem.2015.03.015 · Cited by 178 · see citing works
“Similarly, encouraging results were observed in a study focused on the measurement of circulating lncRNA expression in human serum [53].”
ResultExtracellular vesicle long noncoding RNA as potential biomarkers of liver cancer
Mohankumar, S., & Patel, T. C. (2015) · Briefings in Functional Genomics · doi:10.1093/bfgp/elv058 · Cited by 98 · see citing works
Self-citationFrom laboratory to clinic: Translation of extracellular vesicle based cancer biomarkers
Yekula, A., Muralidharan, K., Kang, K. M., Wang, L., Balaj, L., & Carter, B. S. (2020) · Methods · doi:10.1016/j.ymeth.2020.02.003 · Cited by 94 · see citing works
Extracellular Vesicles in Oncology: Progress and Pitfalls in the Methods of Isolation and Analysis
Lucchetti, D., Fattorossi, A., & Sgambato, A. (2018) · Biotechnology Journal · doi:10.1002/biot.201700716 · Cited by 89 · see citing works
Huber, H. J., & Holvoet, P. (2015) · Current Opinion in Lipidology · doi:10.1097/mol.0000000000000214 · Cited by 82 · see citing works
Illuminating RNA trafficking and functional delivery by extracellular vesicles
de Voogt, W. S., Tanenbaum, M. E., & Vader, P. (2021) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2021.04.017 · Cited by 64 · see citing works
Navigating the Landscape of Tumor Extracellular Vesicle Heterogeneity
Roy, S., Lin, H.-Y., Chou, C.-Y., Huang, C.-H., Small, J. L., Sadik, N., Ayinon, C., Lansbury, E., Cruz, L., Yekula, A., et al. (2019) · International Journal of Molecular Sciences · doi:10.3390/ijms20061349 · Cited by 38 · see citing works
Balakrishnan, A., Roy, S., Fleming, T., Leong, H. S., & Schuurmans, C. (2020) · Cancers · doi:10.3390/cancers12071964 · Cited by 36 · see citing works
Detection of tumor-derived extracellular vesicles in plasma from patients with solid cancer
Vitale, S. R., Helmijr, J., Gerritsen, M., et al. (2021) · BMC Cancer · doi:10.1186/s12885-021-08007-z · Cited by 32 · see citing works
Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification
Guan, W., Chen, L., Rane, T. D., & Wang, T. (2015) · Scientific Reports · doi:10.1038/srep13795 · Cited by 31 · see citing works
Advances of exosome isolation techniques in lung cancer
Mahgoub, E. O., Razmara, E., Bitaraf, A., Norouzi, F.-S., Montazeri, M., Andouhjerdi, R. B., Falahati, M., Cheng, K., Haik, Y., Hasan, A., et al. (2020) · Molecular Biology Reports · doi:10.1007/s11033-020-05715-w · Cited by 32 · see citing works
Laurenzana, I., Trino, S., Lamorte, D., Girasole, M., Dinarelli, S., Stradis, A. D., Grieco, V., Maietti, M., Traficante, A., Statuto, T., et al. (2021) · International Journal of Nanomedicine · doi:10.2147/ijn.s303391 · Cited by 31 · see citing works
Testa, A., Venturelli, E., & Brizzi, M. F. (2021) · Cancers · doi:10.3390/cancers13112792 · Cited by 28 · see citing works
Non-Coding RNAs in Cancer: Structure, Function, and Clinical Application
Márton, É., Varga, A. I., Domoszlai, D., Buglyó, G., Balázs, A., Penyige, A., Balogh, I., Nagy, B., & Szilágyi, M. (2025) · Cancers · doi:10.3390/cancers17040579 · Cited by 25 · see citing works
“Fluorescence-emitting droplets are quantified, resulting in thousands of data points that enhance the power of statistical analysis [42].”
Zhu, P., Fu, W., Wang, C., Du, Z., Huang, K., Zhu, S., & Xu, W. (2016) · Analytica Chimica Acta · doi:10.1016/j.aca.2016.02.020 · Cited by 22 · see citing works
Kumari, S., Lausted, C. G., Scherler, K., Ng, A. H. C., Lü, Y., Lee, I., Hood, L., & Wang, K. (2024) · Biomolecules · doi:10.3390/biom14121599 · Cited by 25 · see citing works
“As ddPCR is based on the presence or absence of an amplification signal among individual droplets, the process is more tolerant to PCR inhibitors, which are common in various body fluids [125].”
Azevedo, C. A. B., da Cunha, R. S., Junho, C. V. C., da Silva, J. V., Moreno-Amaral, A. N., de Moraes, T. P., Carneiro‐Ramos, M. S., & Stinghen, A. E. M. (2021) · Toxins · doi:10.3390/toxins13110778 · Cited by 20 · see citing works
Circulating Extracellular RNA Markers of Liver Regeneration
Yan, I. K., Wang, X., Asmann, Y. W., Haga, H., & Patel, T. (2016) · PLoS ONE · doi:10.1371/journal.pone.0155888 · Cited by 13 · see citing works
Self-citationCostanzi, E., Simioni, C., Varano, G., Brenna, C., Conti, I., & Neri, L. M. (2021) · Genes · doi:10.3390/genes12060902 · Cited by 8 · see citing works
“The hypersensitivity of dPCR allows detecting RNA molecule targets of low abundance below the qPCR’s sensitivity limit [63,64].”
BackgroundGuo, J., Zhou, P., Liu, Z., et al. (2021) · Frontiers in Pharmacology · doi:10.3389/fphar.2021.781276 · Cited by 8 · see citing works
“NGS is a recently developed sequencing technology that can identify more than 100 million reads per sample (Nakato et al., 2013) and show its advantage at lower detectable threshold and higher sensitivity than other sequencing technologies (Wang et al., 2009).”
BackgroundInfluentialRecent Advances in Molecular Diagnosis of Pulmonary Fibrosis for Precision Medicine
Jeong, M. H., Han, H., Lagares, D., & Im, H. (2022) · ACS Pharmacology & Translational Science · doi:10.1021/acsptsci.2c00028 · Cited by 9 · see citing works
Choi, S., Sathe, A. A., Mathé, E. A., Xing, C., & Pan, Z. (2021) · Frontiers in Oncology · doi:10.3389/fonc.2021.724687 · Cited by 7 · see citing works
“To evaluate if these EGFR-upstream regions indeed generate ncRNAs, we synthesized cDNAs from RNAs with double-DNase treatments from each cell line and performed ddPCR known to be capable of detecting very low expressed genes (33).”
MethodKhouzam, R. A., Molinari, C., Salvi, S., Marabelli, M., Molinaro, V., Orioli, D., Saragoni, L., Morgagni, P., Calistri, D., & Ranzani, G. N. (2016) · Oncotarget · doi:10.18632/oncotarget.13401 · Cited by 6 · see citing works
“dPCR is a method for sensitive measurement and quantification of nucleic acids; it improves precision and reproducibility with respect to real-time quantitative PCR (qPCR) and provides an alternative approach for detection of gene expression in settings where the target RNA is limited or present in quantity that appro…”
MethodDroplet Digital PCR for Quantitation of Extracellular RNA
Yan, I. K., Lohray, R., & Patel, T. C. (2018) · Methods in molecular biology · doi:10.1007/978-1-4939-7652-2_12 · Cited by 5 · see citing works
Self-citationQuantitation of Long Noncoding RNA Using Digital PCR
Ota, Y., Yan, I. K., & Patel, T. C. (2021) · Methods in molecular biology · doi:10.1007/978-1-0716-1581-2_7 · Cited by 3 · see citing works
Self-citationZhang, Q., & Bansal, A. K. (2020) · Digestive Diseases and Sciences · doi:10.1007/s10620-020-06250-1 · Cited by 3 · see citing works
Mauger, F., & Deleuze, J. (2019) · Elsevier eBooks · doi:10.1016/b978-0-12-814259-2.00003-0 · Cited by 3 · see citing works
Technical Aspects for the Evaluation of Exosomes and Their Content
Fontana, S., Giallombardo, M., & Alessandro, R. (2017) · Current clinical pathology · doi:10.1007/978-3-319-55661-1_7 · Cited by 2 · see citing works
A Developed Accurate Digital PCR Detection Technology in Food Safety
Xu, W. (2016) · Springer eBooks · doi:10.1007/978-981-10-1618-9_7 · Cited by 2 · see citing works
Bae, S. J., Lee, S. J., & Im, D. J. (2023) · Small · doi:10.1002/smll.202309062 · Cited by 3 · see citing works
Droplet Gene Analysis – Digital PCR
Gegevicius, E., Goda, K., & Mažutis, L. (2020) · Royal Society of Chemistry eBooks · doi:10.1039/9781839162855-00089 · Cited by 2 · see citing works
Çarhan, A., Ercan, E., & Yalçınkaya, T. (2016) · Turkish Bulletin of Hygiene and Experimental Biology · doi:10.5505/turkhijyen.2016.48902 · Cited by 1 · see citing works
“Reaksiyon PZR‘nin son noktasına ulaştığında; damlalar ilgili hedef dizinin kopyalarını içermeyebilir ya da bazı kopyalarını içerir (27).”
InfluentialZaborowski, M. P., Lai, C. P., Sammarco, A., György, B., Zhang, X., Lee, K., Na, Y. J., Ceppi, L., Breyne, K., Champagne, E. M., et al. (2026) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2026.102970 · Cited by 1 · see citing works
“37,38 Some circRNAs released in EVs may be unique to cancer tissue.”
Borelli, B., Crucitta, S., Boccaccino, A., et al. (2023) · Tumori Journal · doi:10.1177/03008916221147944 · Cited by 1 · see citing works
“77 No 33 (24) 18 (22) 15 (26) Primary tumor site Right 46 (33) 24 (30) 22 (38) 0.”
InfluentialTranslational Opportunities of Extracellular Vesicles in Biomedicine
Politis, M., Im, H., Carter, B. S., Skog, J., Balaj, L., Castro, C. M., & Lee, H. (2023) · Current cancer research · doi:10.1007/978-3-031-22903-9_4 · Cited by 1 · see citing works
Extracellular vesicle RNA in liver disease
Patel, T. C., & Haga, H. (2015) · doi:10.1002/9781118663387.ch9
Self-citationYngve, S. (2015) · University Library of Skövde (University of Skövde)
TBHEB 2016-2 Vol 73 Full Printed Journal
DUMAN, M. (2016) · Turkish Bulletin of Hygiene and Experimental Biology · doi:10.5505/turkhijyen.2016.80008
Brown, V. (2019) · KU ScholarWorks (The University of Kansas)
Onukwugha, N., McEacheron, H., Smith, S. M., et al. (2025) · Biosensors and Bioelectronics X · doi:10.1016/j.biosx.2025.100652
A Developed Accurate Digital PCR Detection Technology in Food Safety
Xu, W. (2025) · Springer eBooks · doi:10.1007/978-981-96-9603-1_11
Isolation of Extracellular Vesicles and the Analysis of Their RNA Cargo
Nwachukwu, F., Gordon, M. E., Witek, M. A., & Soper, S. A. (2025) · Springer eBooks · doi:10.1007/978-3-031-89270-7_7
Vitale, S. R., Helmijr, J. A., Gerritsen, M., et al. (2021) · Figshare · doi:10.6084/m9.figshare.14302707
Vitale, S. R., Helmijr, J. A., Gerritsen, M., et al. (2021) · Figshare · doi:10.6084/m9.figshare.14302716
Vitale, S. R., Helmijr, J. A., Gerritsen, M., et al. (2021) · Figshare · doi:10.6084/m9.figshare.14302710
Vitale, S. R., Helmijr, J. A., Gerritsen, M., et al. (2021) · Figshare · doi:10.6084/m9.figshare.14302713
Recent Progress in Extracellular Vesicles Therapeutics for Atopic Dermatitis Treatment
Jeong, S., Kim, H. J., Oh, M. J., Song, Y., Kim, J., Kim, T. G., Shin, Y. J., Cho, S. H., Park, J. M., Park, M., et al. (2026) · BioChip Journal · doi:10.1007/s13206-026-00273-y
“First, exosomal RNA consists of miRNA, mRNA fragments, lncRNA, circRNA, etc [150, 151].”
Self-citation
- Authors
- Takahashi, K., Yan, I. K., Kim, C., Kim, J., & Patel, T.
- Affiliations
- Mayo Clinic in Florida (US)
- Corresponding author
- Patel, Tushar
- Venue
- Frontiers in Oncology
- Publisher
- Frontiers Media SA
- Published
- 2014-06-04
- Volume
- 4
- Pages
- 129-129
- ISSN
- 2234-943X (ISSN-L)
- PMC
- PMC4044516
- OpenAlex
- W2171278955
- Semantic Scholar
- 10693866e84d021bf93218492b640ed5eee819e4
- Cited by
- 51
- References
- 16
- Citations by year
- 2026: 2 · 2025: 4 · 2024: 1 · 2023: 3 · 2022: 1 · 2021: 15 · 2020: 5 · 2019: 3 · 2018: 3 · 2017: 1 · 2016: 6 · 2015: 7
- FWCI
- 1.70
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by
- Locations
- Frontiers in Oncology (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (submittedVersion, cc-by)
- PubMed Central (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- National Institutes of Health (UH2TR000884)
- NIH Office of the Director (UH2TR000884)
- National Institute of Diabetes and Digestive and Kidney Diseases (DK-069370, R01DK069370)
- National Center for Advancing Translational Sciences (UH2 TR000884)
- Common Fund
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
- Keywords
- extracellular RNA, digital PCR, exosomes, intercellular communication, RNA detection, cancer cell biology, extracellular vesicles
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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
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