Assessing cellular and circulating miRNA recovery: the impact of the RNA isolation method and the quantity of input material
Reads0
Chats0
TLDR
Compared the efficiency of commonly used RNA extraction kits in miRNA recovery from cells, plasma and urine/plasma-derived exosomes, using single-gene RT-qPCR and miRNA profiling, it is shown that miRNAs were preferentially or exclusively isolated by either of the methods.Abstract:
MicroRNAs (miRNAs) have emerged as promising cancer biomarkers. However, exploiting their informative potential requires careful optimization of their detection. Here, we compared the efficiency of commonly used RNA extraction kits in miRNA recovery from cells, plasma and urine/plasma-derived exosomes, using single-gene RT-qPCR and miRNA profiling. We used increasing amounts of starting material to investigate the impact of the input material size on miRNA extraction. We showed that miRNA recovery was largely influenced by the isolation method and by the amount of input material. In particular, the miRCURY™ kit provided highly pure RNA. However, its columns poorly recovered miRNAs from limiting amounts of cells and plasma, and rapidly saturated by large RNA species and plasma components, thus impeding miRNA recovery from high input amounts. Overall, the miRNeasy® kit permitted a better miRNA detection despite a less pure extracted RNA. Nevertheless, some miRNAs were preferentially or exclusively isolated by either of the methods. Trizol® LS resulted in very low purity RNA which affected RT-qPCR efficiency. In general, miRCURY™ biofluids kit efficiently extracted miRNAs from plasma. A careful selection of the RNA isolation method and the consideration of the type and size of input material are highly recommended to avoid biased results.read more
Citations
More filters
Journal ArticleDOI
A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents
Inas Helwa,Jingwen Cai,Michelle Drewry,Arthur D. Zimmerman,Michael B. Dinkins,Mariam Lotfy Khaled,Mutsa Seremwe,W. Michael Dismuke,Erhard Bieberich,W. Daniel Stamer,Mark W. Hamrick,Yutao Liu,Yutao Liu +12 more
TL;DR: Comparing the exosomes extracted by three different exosome isolation kits and differential ultracentrifugation using six different volumes of a non-cancerous human serum shows that three kits are viable alternatives to UC, even when limited amounts of biological samples are available.
Journal ArticleDOI
Clinical utility of circulating non-coding RNAs — an update
TL;DR: A compendium of miRNAs and long ncRNAs that have been reported in the literature to be present in human body fluids and that have the potential to be used as diagnostic and prognostic cancer biomarkers is provided.
Journal ArticleDOI
Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum.
Yue-Ting Tang,Yiyao Huang,Lei Zheng,Sihua Qin,Xu-Ping Xu,Taixue An,Yong Xu,Ying-Song Wu,Xiumei Hu,Bao-Hong Ping,Qian Wang +10 more
TL;DR: It was found that combinations of the TEI and TER methods resulted in high extraction efficiency and purity of small RNA obtained using CCM, and different methods may introduce variations in the concentration, purity and size of exosomes and exoRNA.
Journal ArticleDOI
Nucleic acid hybridization on an electrically reconfigurable network of gold-coated magnetic nanoparticles enables microRNA detection in blood
Roya Tavallaie,Joshua A. McCarroll,Marion Le Grand,Nicholas Ariotti,Wolfgang Schuhmann,Eric Bakker,Richard D. Tilley,David Brynn Hibbert,Maria Kavallaris,J. Justin Gooding +9 more
TL;DR: An ultrasensitive miRNA sensor based on gold-coated magnetic nanoparticles modified with redox-labelled probe DNA is capable of detecting miRNA at a concentration of 10 aM to 1 nM in unprocessed blood, and following tumour-induced variation in miRNA levels.
Journal ArticleDOI
Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next-generation sequencing.
Dominik Buschmann,Benedikt Kirchner,Stefanie Hermann,Melanie Märte,Christine Wurmser,Florian Brandes,Stefan Kotschote,Michael Bonin,Ortrud K. Steinlein,Michael W. Pfaffl,Gustav Schelling,Marlene Reithmair +11 more
TL;DR: Five different methods of EV isolation in combination with two RNA extraction methods are compared regarding their suitability for biomarker discovery-focused miRNA sequencing as well as biological characteristics of captured vesicles to reveal striking method-specific differences.
References
More filters
Journal ArticleDOI
Circulating microRNAs as stable blood-based markers for cancer detection
Patrick S. Mitchell,Rachael K. Parkin,Evan M. Kroh,Brian R. Fritz,Brian R. Fritz,Stacia K. Wyman,Era L. Pogosova-Agadjanyan,Amelia Peterson,Jennifer Noteboom,Kathy O'Briant,April Allen,Daniel W. Lin,Daniel W. Lin,Daniel W. Lin,Nicole Urban,Charles W. Drescher,Beatrice S. Knudsen,Derek L. Stirewalt,Robert Gentleman,Robert L. Vessella,Robert L. Vessella,Peter S. Nelson,Daniel Martin,Daniel Martin,Muneesh Tewari +24 more
TL;DR: It is shown here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity and established the measurement of tumor-derived mi RNAs in serum or plasma as an important approach for the blood-based detection of human cancer.
Journal ArticleDOI
Identification and proteomic profiling of exosomes in human urine
TL;DR: The results indicate that exosome isolation may provide an efficient first step in biomarker discovery in urine and identify numerous protein components of MVBs and of the endosomal pathway in general.
Journal ArticleDOI
Circulating MicroRNAs in Patients With Coronary Artery Disease
Stephan Fichtlscherer,Salvatore De Rosa,Henrik Fox,Thomas Schwietz,Ariane Fischer,Christoph Liebetrau,Michael Weber,Christian W. Hamm,Tino Röxe,Marga Muller-Ardogan,Angelika Bonauer,Andreas M. Zeiher,Stefanie Dimmeler +12 more
TL;DR: Circulating levels of vascular and inflammation-associated microRNAs are significantly downregulated in patients with coronary artery disease.
Journal ArticleDOI
Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR).
TL;DR: The procedure for qRT-PCR analysis of circulating miRNAs as biomarkers is described, and relevant issues of sample preparation, experimental design and data analysis are discussed.
Journal ArticleDOI
Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood
TL;DR: This study demonstrates thatExosomal RNA is protected by RNaseA treatment and that exosomes provide a consistent source of miRNA for disease biomarker detection.
Related Papers (5)
Circulating microRNAs as stable blood-based markers for cancer detection
Patrick S. Mitchell,Rachael K. Parkin,Evan M. Kroh,Brian R. Fritz,Brian R. Fritz,Stacia K. Wyman,Era L. Pogosova-Agadjanyan,Amelia Peterson,Jennifer Noteboom,Kathy O'Briant,April Allen,Daniel W. Lin,Daniel W. Lin,Daniel W. Lin,Nicole Urban,Charles W. Drescher,Beatrice S. Knudsen,Derek L. Stirewalt,Robert Gentleman,Robert L. Vessella,Robert L. Vessella,Peter S. Nelson,Daniel Martin,Daniel Martin,Muneesh Tewari +24 more
Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases
Xi Chen,Yi Ba,Lijia Ma,Lijia Ma,Xing Cai,Yuan Yin,Kehui Wang,Jigang Guo,Yujing Zhang,Jiangning Chen,Xing Guo,Qibin Li,Qibin Li,Xiaoying Li,Wenjing Wang,Yan Zhang,Jin Wang,Xueyuan Jiang,Yang Xiang,Chen Xu,Pingping Zheng,Juanbin Zhang,Ruiqiang Li,Hongjie Zhang,Xiaobin Shang,Ting Gong,Guang Ning,Jun Wang,Jun Wang,Ke Zen,Junfeng Zhang,Chen-Yu Zhang +31 more