Exosomes molecular diagnostics: Direct conversion of exosomes into the cDNA for gene amplification by two-step polymerase chain reaction
Main Article Content
Keywords
Exosome, cDNA, PCR, single cell PCR, Exo-cDNA, exosomal mRNA transcript
Abstract
Exosomes are cell derived lipid nanoparticle with a size of 30–100 nm in diameter, found in almost all biological fluids. The composition of the exosomes is mainly lipid, proteins, RNA, DNA, and non-coding RNAs. Currently, most available methods and commercial kits for exosomal-RNA (Exo-RNA) isolation have limitations and shortcomings. Small starting volume of exosomes and the use of extraction/filtration columns results usually insufficient yield of exosomal RNA after isolation. The majority of RNA contained in purified exosomes range in size from 15–500 nucleotides. Some RNA isolation kits are well suited for small RNA transcripts isolation but larger mRNA transcripts are hard to detect. For all of the kits, the cost prize per sample analyzed is very high. Our current method provides a novel way for direct conversion of exosomes into cDNA synthesis (Exo-cDNA) and subsequent gene detection by polymerase chain reaction (PCR). This method has several advantages compared to established available kits. No extraction column is utilized in this procedure which means total recovery of exosomal RNA with maximal yield. In addition, this method is fast and uses a minimal amount of lab supplies, thereby reducing the overall working costs. Our findings suggest that direct conversion of exosomes into cDNA and subsequent gene amplification by two step PCR is a most efficient and reproducible technique. This novel method can be applied to and is useful to advance molecular research of exosomes by solving the problem of low molecular yields.
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References
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2. Thakur BK, Zhang H, Becker A, Matei I, Huang Y, Costa-Silva B, Zheng Y, Hoshino A, Brazier H, Xiang J, Williams C, Rodriguez-Barrueco R, Silva JM, Zhang W, Hearn S, Elemento O, Paknejad N, Manova-Todorova K, Welte K, Bromberg J, Peinado H, Lyden D: Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res 2014, 24(6):766-9.
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6. Rabinowits G, Gerçel-Taylor C, Day JM, Taylor DD, Kloecker GH: Exosomal microRNA: a diagnostic marker for lung cancer. Clin Lung Cancer 2009, 10(1):42-6.
7. Hegmans JP, Bard MP, Hemmes A, Luider TM, Kleijmeer MJ, Prins JB, Zitvogel L, Burgers SA, Hoogsteden HC, Lambrecht BN: Proteomic analysis of exosomes secreted by human mesothelioma cells. Am J Pathol 2004, 164(5):1807-15.
8. Théry C, Amigorena S, Raposo G, Clayton A: Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 2006, Chapter 3: Unit 3.22.
9. Enderle D, Spiel A, Coticchia CM, Berghoff E, Mueller R, Schlumpberger M, Sprenger-Haussels M, Shaffer JM, Lader E, Skog J, Noerholm M: Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method. PLoS One 2015, 10(8):e0136133.
10. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987, 162(1):156-9.
11. Haque S, Yan XJ, Rosen L, McCormick S, Chiorazzi N, Mongini PK: Effects of prostaglandin E2 on p53 mRNA transcription and p53 mutagenesis during T-cell-independent human B-cell clonal expansion. FASEB J 2014, 28(2):627-43.
12. György B, Szabó TG, Pásztói M, Pál Z, Misják P, Aradi B, László V, Pállinger E, Pap E, Kittel A, Nagy G, Falus A, Buzás EI: Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci 2011, 68(16):2667-88.
13. Vaiselbuh SR: Exosomes in Cancer Research. Cancer Research Frontiers 2015; 1(1): 11-24.
14. Revenfeld AL, Bæk R, Nielsen MH, Stensballe A, Varming K, Jørgensen M: Diagnostic and prognostic potential of extracellular vesicles in peripheral blood. Clin Ther 2014, 36(6):830-46.
15. Wills QF, Livak KJ, Tipping AJ, Enver T, Goldson AJ, Sexton DW, Holmes C: Single-cell gene expression analysis reveals genetic associations masked in whole-tissue experiments. Nat Biotechnol 2013, 31(8):748-52.
16. Kobayashi M, Rice GE, Tapia J, Mitchell MD, Salomon C: Exosomes are fingerprints of originating cells: potential biomarkers for ovarian cancer. Research and Reports in Biochemistry 2015, 101–109.
17. Stevanato L, Thanabalasundaram L, Vysokov N, Sinden JD: Investigation of Content, Stoichiometry and Transfer of miRNA from Human Neural Stem Cell Line Derived Exosomes. PLoS One 2016, 11(1):e0146353.
1. Théry C, Zitvogel L, Amigorena S: Exosomes: composition, biogenesis and function. Nat Rev Immunol 2002, 2(8):569-79.
2. Thakur BK, Zhang H, Becker A, Matei I, Huang Y, Costa-Silva B, Zheng Y, Hoshino A, Brazier H, Xiang J, Williams C, Rodriguez-Barrueco R, Silva JM, Zhang W, Hearn S, Elemento O, Paknejad N, Manova-Todorova K, Welte K, Bromberg J, Peinado H, Lyden D: Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res 2014, 24(6):766-9.
3. Yáñez-Mó M, Siljander PR, Andreu Z, Zavec AB, Borràs FE, Buzas EI, Buzas K, Casal E, Cappello F, Carvalho J, Colás E, Cordeiro-da Silva A, Fais S, Falcon-Perez JM, Ghobrial IM, Giebel B, Gimona M, Graner M, Gursel I, Gursel M, Heegaard NH, Hendrix A, Kierulf P, Kokubun K, Kosanovic M, Kralj-Iglic V, Krämer-Albers EM, Laitinen S, Lässer C, Lener T, Ligeti E, LinÄ“ A, Lipps G, Llorente A, Lötvall J, Manček-Keber M, Marcilla A, Mittelbrunn M, Nazarenko I, Nolte-'t Hoen EN, Nyman TA, O'Driscoll L, Olivan M, Oliveira C, Pállinger É, Del Portillo HA, Reventós J, Rigau M, Rohde E, Sammar M, Sánchez-Madrid F, Santarém N, Schallmoser K, Ostenfeld MS, Stoorvogel W, Stukelj R, Van der Grein SG, Vasconcelos MH, Wauben MH, De Wever O: Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 2015, 4:27066.
4. Admyre C, Johansson SM, Qazi KR, Filén JJ, Lahesmaa R, Norman M, Neve EP, Scheynius A, Gabrielsson S: Exosomes with immune modulatory features are present in human breast milk. J Immunol 2007, 179(3):1969-78.
5. Nilsson J, Skog J, Nordstrand A, Baranov V, Mincheva-Nilsson L, Breakefield XO, Widmark A: Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer. Br J Cancer 2009, 100(10):1603-7.
6. Rabinowits G, Gerçel-Taylor C, Day JM, Taylor DD, Kloecker GH: Exosomal microRNA: a diagnostic marker for lung cancer. Clin Lung Cancer 2009, 10(1):42-6.
7. Hegmans JP, Bard MP, Hemmes A, Luider TM, Kleijmeer MJ, Prins JB, Zitvogel L, Burgers SA, Hoogsteden HC, Lambrecht BN: Proteomic analysis of exosomes secreted by human mesothelioma cells. Am J Pathol 2004, 164(5):1807-15.
8. Théry C, Amigorena S, Raposo G, Clayton A: Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 2006, Chapter 3: Unit 3.22.
9. Enderle D, Spiel A, Coticchia CM, Berghoff E, Mueller R, Schlumpberger M, Sprenger-Haussels M, Shaffer JM, Lader E, Skog J, Noerholm M: Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method. PLoS One 2015, 10(8):e0136133.
10. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987, 162(1):156-9.
11. Haque S, Yan XJ, Rosen L, McCormick S, Chiorazzi N, Mongini PK: Effects of prostaglandin E2 on p53 mRNA transcription and p53 mutagenesis during T-cell-independent human B-cell clonal expansion. FASEB J 2014, 28(2):627-43.
12. György B, Szabó TG, Pásztói M, Pál Z, Misják P, Aradi B, László V, Pállinger E, Pap E, Kittel A, Nagy G, Falus A, Buzás EI: Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci 2011, 68(16):2667-88.
13. Vaiselbuh SR: Exosomes in Cancer Research. Cancer Research Frontiers 2015; 1(1): 11-24.
14. Revenfeld AL, Bæk R, Nielsen MH, Stensballe A, Varming K, Jørgensen M: Diagnostic and prognostic potential of extracellular vesicles in peripheral blood. Clin Ther 2014, 36(6):830-46.
15. Wills QF, Livak KJ, Tipping AJ, Enver T, Goldson AJ, Sexton DW, Holmes C: Single-cell gene expression analysis reveals genetic associations masked in whole-tissue experiments. Nat Biotechnol 2013, 31(8):748-52.
16. Kobayashi M, Rice GE, Tapia J, Mitchell MD, Salomon C: Exosomes are fingerprints of originating cells: potential biomarkers for ovarian cancer. Research and Reports in Biochemistry 2015, 101–109.
17. Stevanato L, Thanabalasundaram L, Vysokov N, Sinden JD: Investigation of Content, Stoichiometry and Transfer of miRNA from Human Neural Stem Cell Line Derived Exosomes. PLoS One 2016, 11(1):e0146353.
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Published
Jul 25, 2018
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Haque S, Vaiselbuh SR. Exosomes molecular diagnostics: Direct conversion of exosomes into the cDNA for gene amplification by two-step polymerase chain reaction. J Biol Methods [Internet]. 2018 Jul. 25 [cited 2024 Apr. 20];5(3):e96. Available from: https://jbmethods.org/index.php/jbm/article/view/249
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Author Biography
Shabirul Haque, The Feinstein Institute for Medical Research, Northwell Health
Assistant Professor, The Karches Center for Oncology, The Feinstein Institute of Medical Research Director, Pediatric Hemato/Oncology, Children
