Plasma microrna quantification protocol

Abstract

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate translation and are involved in many pathological processes. They have emerged as promising biomarkers for diagnosis of conditions such as aortic aneurysm disease. Quantifying miRNAs in plasma is uniquely challenging because of the lack of standardized reproducible protocols. To facilitate the independent verification of conclusions, it is necessary to provide a thorough disclosure of all pertinent experimental details. In this technical note, we present a comprehensive protocol for quantifying plasma miRNAs using droplet digital PCR. We detail the entire workflow, including blood collection, plasma processing, cryo-storage, miRNA isolation, reverse transcription, droplet generation, PCR amplification, fluorescence reading, and data analysis. We offer comprehensive guidance regarding optimization, assay conditions, expected results, and insight into the troubleshooting of common issues. The stepwise normalization and detailed methodological guide enhance reproducibility. Moreover, multiple portions of this protocol may be automated. The data provided in this technical note is demonstrative of the values typically obtained when following its steps. To facilitate standardization in data reporting, we include a table of expected aortic aneurysm-related miRNA levels in healthy human plasma. This versatile protocol can be easily adapted to quantify most circulating miRNAs in plasma, making it a valuable resource for diagnostic development.

Keywords

MicroRNA / plasma / quantification / aortic aneurysm / ddPCR

References

[1] Sessa F,Esposito M.New insight into mechanisms of cardiovascular diseases: an integrative analysis approach to identify TheranoMiRNAs.Int J Mol Sci2023;24:6781 PMCID:PMC10095439

[2] Sessa F,Esposito M,Pomara C.miRNA dysregulation in cardiovascular diseases: current opinion and future perspectives.Int J Mol Sci2023;24:5192 PMCID:PMC10048938

[3] Ikonomidis JS,Wheeler JB.Plasma biomarkers for distinguishing etiologic subtypes of thoracic aortic aneurysm disease.J Thorac Cardiovasc Surg2013;145:1326-33 PMCID:PMC4057430

[4] Hindson CM,Briggs HA.Absolute quantification by droplet digital PCR versus analog real-time PCR.Nat Methods2013;10:1003-5 PMCID:PMC4118677

[5] Campomenosi P,Noonan DM.A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer.BMC Biotechnol2016;16:60 PMCID:PMC4991011

[6] Ferracin M,Salamon I.Absolute quantification of cell-free microRNAs in cancer patients.Oncotarget2015;6:14545-55 PMCID:PMC4546486

[7] Ferracin M,Lupini L,Sabbioni S.Circulating MicroRNA quantification using DNA-binding dye chemistry and droplet digital PCR.J Vis Exp2016;26:54102 PMCID:PMC4993260

[8] Miotto E,Lupini L,Negrini M.Quantification of circulating miRNAs by droplet digital PCR: comparison of EvaGreen- and TaqMan-based chemistries.Cancer Epidemiol Biomarkers Prev2014;23:2638-42

[9] El-Khoury V,Kaoma T,Berchem G.Assessing cellular and circulating miRNA recovery: the impact of the RNA isolation method and the quantity of input material.Sci Rep2016;6:19529 PMCID:PMC4726450

[10] Quackenbush J.Microarray data normalization and transformation.Nat Genet2002;32:496-501

[11] Davis FM,Daugherty A.Mechanisms of aortic aneurysm formation: translating preclinical studies into clinical therapies.Heart2014;100:1498-505

[12] Heron M.Deaths: leading causes for 2014.Natl Vital Stat Rep2016;65:1-96

[13] Akerman AW,Stroud RE.Elevated wall tension leads to reduced miR-133a in the thoracic aorta by exosome release.J Am Heart Assoc2019;8:e010332 PMCID:PMC6405702

[14] Jones JA,O'Quinn EC.Selective microRNA suppression in human thoracic aneurysms: relationship of miR-29a to aortic size and proteolytic induction.Circ Cardiovasc Genet2011;4:605-13 PMCID:PMC3246193

[15] García ME,Caballero J.Anticoagulants interfere with PCR used to diagnose invasive aspergillosis.J Clin Microbiol2002;40:1567-8 PMCID:PMC140326

[16] Permenter J,Rounsavall A.Quantitative analysis of genomic DNA degradation in whole blood under various storage conditions for molecular diagnostic testing.Mol Cell Probes2015;29:449-53

[17] Kondratov K,Popov M.Heparinase treatment of heparin-contaminated plasma from coronary artery bypass grafting patients enables reliable quantification of microRNAs.Biomol Detect Quantif2016;8:9-14 PMCID:PMC4906134

[18] Mompeón A,Vidal-Gómez X.Disparate miRNA expression in serum and plasma of patients with acute myocardial infarction: a systematic and paired comparative analysis.Sci Rep2020;10:5373 PMCID:PMC7096393

[19] Wan Azman WN, Omar J, Koon TS, Tuan Ismail TS. Hemolyzed specimens: major challenge for identifying and rejecting specimens in clinical laboratories.Oman Med J2019;34:94-8 PMCID:PMC6425048

[20] Shabihkhani M,Wei B.The procurement, storage, and quality assurance of frozen blood and tissue biospecimens in pathology, biorepository, and biobank settings.Clin Biochem2014;47:258-66 PMCID:PMC3982909

[21] Matias-Garcia PR,Mussack V.Impact of long-term storage and freeze-thawing on eight circulating microRNAs in plasma samples.PLoS One2020;15:e0227648 PMCID:PMC6959605

[22] Akerman AW,Peterson AR.miR-133a replacement attenuates thoracic aortic aneurysm in mice.J Am Heart Assoc2021;10:e019862 PMCID:PMC8475064

[23] Khetan D,Chaudhary R.Comparison of UV spectrometry and fluorometry-based methods for quantification of cell-free DNA in red cell components.Asian J Transfus Sci2019;13:95-9 PMCID:PMC6910032

[24] Wright K,Purdie AC.Comparison of methods for miRNA isolation and quantification from ovine plasma.Sci Rep2020;10:825 PMCID:PMC6972740

[25] Garcia-Elias A,Puigdecanet E.Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs.Sci Rep2017;7:7725 PMCID:PMC5552704

[26] Silver N,Proctor G,Paterson KL.Selection of housekeeping genes for gene expression studies in the adult rat submandibular gland under normal, inflamed, atrophic and regenerative states.BMC Mol Biol2008;9:64 PMCID:PMC2492873

[27] Rice J,Rai SN.Housekeeping genes for studies of plasma microRNA: a need for more precise standardization.Surgery2015;158:1345-51

[28] dMIQE Group, Huggett JF. The digital MIQE guidelines update: minimum information for publication of quantitative digital PCR experiments for 2020.Clin Chem2020;66:1012-29