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Protocols for Isolation of Genetic Materials from RNA Viruses

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Protocols for the Diagnosis of Pig Viral Diseases

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Abstract

The isolation of viral RNA with purity and integrity is a critical element for the overall success of viral diagnosis. The era of classical virology has transcended way beyond the labor-intensive manual method of RNA extraction to the modern-age efficient and simpler protocols. With an aim to obtain a RNA material free from carry over contaminants such as protein, unwarranted cellular genome, and chemicals, etc. there are three major techniques followed worldwide such as organic extraction viz phenol-guanidine isothiocyanate (GITC)-based solutions, silica-membrane-based spin column technology, and paramagnetic particle technology. The method of extraction and the flow of processes within a particular method would vary with the type of material being handled. The major considerations while extracting RNA from tissue sample would be eliminating endogenous RNase that would compromise RNA integrity. The final step of RNA extraction is the storage of the isolated genome, which solely depend upon the purpose with which the extraction was carried out. If the sample is not intended for immediate application, then several commercially available formulations such as FORMAzol and RNA stable have been found suitable for long-term storage.

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References

  1. Wink M (2006) An introduction to molecular biotechnology: molecular fundamentals, methods and application in modern biotechnology. Wiley-VCH, Weinheim

    Google Scholar 

  2. Tan SC, Yiap BC (2009) DNA, RNA, and protein extraction: the past and the present. J Biomed Biotechnol

    Google Scholar 

  3. Cseke LJ, Kaufman PB, Podila GK, Tsai C-J (2004) Handbook of molecular and cellular methods in biology and medicine, 2nd edn. CRC Press, Boca Raton, FL

    Google Scholar 

  4. Buckingham L, Flaws ML (2007) Molecular diagnostics: fundamentals, methods, & clinical applications. F.A. Davis, Philadelphia, PA

    Google Scholar 

  5. Amani P, Hofmann A (2018) Basic principles. In: Hofmann A, Clokie S (eds) Wilson and Walker's principles and techniques of biochemistry and molecular biology. Cambridge University Press, Cambridge, pp 8–39. https://doi.org/10.1017/9781316677056.004

    Chapter  Google Scholar 

  6. Farrell RE (2017) RNA isolation strategies. In: RNA methodologies, 5th edn. Academic Press, pp 75–115. https://doi.org/10.1016/B978-0-12-804678-4.00003-8

    Chapter  Google Scholar 

  7. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299

    Article  CAS  Google Scholar 

  8. Cox RA (1968) The use of guanidinium chloride in the isolation of nucleic acids. In: Grossman L, Moldave K (eds) Methods in enzymology, vol 12. Academic Press, Orlando, FL, pp 120–129, part B

    Google Scholar 

  9. Gordon JA (1972) Denaturation of globular proteins. Interaction of guanidinium salts with three proteins. Biochemistry 11:1862–1970

    Article  CAS  Google Scholar 

  10. Nozaki Y, Tanford C (1970) The solubility of amino acids, diglycine, and triglycine in aqueous guanidinium hydrochloride solutions. J Biol Chem 245:1648–1652

    Article  CAS  Google Scholar 

  11. Chomczynski P (1993) A reagent for the single-step simultaneous isolation of RNA, DNA, and proteins from cell and tissue samples. BioTechniques 15:532–536

    CAS  PubMed  Google Scholar 

  12. Majumdar D, Avissar YJ, Wyche JH (1991) Simultaneous and rapid isolation of bacterial and eukaryotic DNA and RNA: a new approach for isolating DNA. BioTechniques 11(1):94–101

    CAS  PubMed  Google Scholar 

  13. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159

    Article  CAS  Google Scholar 

  14. Ott T, Krusell L, Udvardi M (2005) RNA isolation using CsCl gradients. In: Lotus japonicus handbook. Springer, Dordrecht, pp 125–128

    Chapter  Google Scholar 

  15. Lindberg U, Persson T (1974) Messenger RNA isolation with poly(U) agarose. Methods Enzymol 34:496–499

    Article  CAS  Google Scholar 

  16. Squire KRE, Chuang RY, Osburn BI, Knudson DL, Doi RH (1983) Rapid methods for comparing the double-stranded RNA genome profiles of bluetongue virus. Vet Microbiol 8(6):543–553

    Article  CAS  Google Scholar 

  17. Barlow JJ, Mathias AP, Williamson R, Gammack DB (1963) A simple method for the quantitative isolation of undegraded high molecular weight ribonucleic acid. Biochem Biophys Res Commun 13:61–66

    Article  CAS  Google Scholar 

  18. Cathala G, Savouret J, Mendez B, West BL, Karin M, Martial JA, Baxter JD (1983) A method for isolation of intact, translationally active ribonucleic acid. DNA 2:329–335

    Article  CAS  Google Scholar 

  19. Attoui H, Billoir F, Cantaloube JF, Biagini P, de Micco P, de Lamballerie X (2000) Strategies for the sequence determination of viral dsRNA genomes. J Virol Methods 89(1–2):147–158

    Article  CAS  Google Scholar 

  20. Potgieter AC, Steele AD, Van Dijk AA (2002) Cloning of complete genome sets of six dsRNA viruses using an improved cloning method for large dsRNA genes. J Gen Virol 83(9):2215–2223

    Article  CAS  Google Scholar 

  21. Singh KP, Maan S, Samuel AR, Rao S, Meyer AJ, Mertens PPC (2004) Phylogenetic analysis of bluetongue virus genome segment 6 (encoding VP5) from different serotypes. Vet Ital 40(4):479–483

    CAS  PubMed  Google Scholar 

  22. Potgieter AC, Page NA, Liebenberg J, Wright IM, Landt O, Van Dijk AA (2009) Improved strategies for sequence-independent amplification and sequencing of viral double-stranded RNA genomes. J Gen Virol 90(6):1423–1432

    Article  CAS  Google Scholar 

  23. Maan S, Rao S, Maan NS, Anthony SJ, Attoui H, Samuel AR, Mertens PP (2007) Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses. J Virol Methods 143(2):132–139

    Google Scholar 

  24. Herring AJ et al (1982) Rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in silver-stained polyacrylamide gels. J Clin Microbiol 16(3):473–477

    Article  CAS  Google Scholar 

  25. Chomczynski P (1992) Solubilization in formamide protects RNA from degradation. Nucleic Acids Res 20:3791–3792

    Article  CAS  Google Scholar 

  26. Biswas SK, Chand K, Pandey AB, Mondal B (2016) Experimental protocol for quick extraction of double-stranded rna (dsRNA) of bluetongue virus (BTV) from infected BHK-21 cells with ribonuclease T1 (RNAse T1) at different concentrations. Anim Sci 10(1)

    Google Scholar 

  27. Smith LM, Burgoyne LA (2004) Collecting, archiving and processing DNA from wildlife samples using FTA® databasing paper. BMC Ecol. 4, 4

    Google Scholar 

  28. Cardona-Ospina JA, Villalba-Miranda MF, Palechor-Ocampo LA, Mancilla LI, Sepúlveda-Arias JC (2019) A systematic review of FTA cards® as a tool for viral RNA preservation in fieldwork: Are they safe and effective?. Prev Vet Med 172:104772

    Google Scholar 

  29. Stevenson HS, Wang Y, Muller R, Edelman DC (2015) Long-term stability of total RNA in RNAstable® as evaluated by expression microarray. Biopreserv Biobank 13(2):114–122. https://doi.org/10.1089/bio.2014.0068

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Author information

Authors and Affiliations

  1. CCS-National Institute of Animal Health (NIAH), Baghpat, Uttar Pradesh, India

    Nihar Nalini Mohanty & Vikas Gupta

  2. National Dairy Development Board, Research and Development Laboratory, Hyderabad, Telangana, India

    Laxmi Narayan Sarangi

  3. Department of Biotechnology, University of Agricultural Science, GKVK, Bengaluru, Karnataka, India

    Rohini Bhat

  4. ICAR-National Institute of Veterinary Epidemiology and Disease Informatics (ICAR-NIVEDI), Bengaluru, Karnataka, India

    Sathish B. Shivachandra

Authors
  1. Nihar Nalini Mohanty
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  2. Vikas Gupta
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  3. Laxmi Narayan Sarangi
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  4. Rohini Bhat
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  5. Sathish B. Shivachandra
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Editor information

Editors and Affiliations

  1. National Research Centre on Pig, Indian Council of Agricultural Research, Guwahati, Assam, India

    Rajib Deb

  2. National Research Centre on Pig, Indian Council of Agricultural Research, Guwahati, Assam, India

    Ajay Kumar Yadav

  3. National Research Centre on Pig, Indian Council of Agricultural Research, Guwahati, Assam, India

    Swaraj Rajkhowa

  4. College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India

    Yashpal Singh Malik

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Mohanty, N.N., Gupta, V., Sarangi, L.N., Bhat, R., Shivachandra, S.B. (2022). Protocols for Isolation of Genetic Materials from RNA Viruses. In: Deb, R., Yadav, A.K., Rajkhowa, S., Malik, Y.S. (eds) Protocols for the Diagnosis of Pig Viral Diseases. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2043-4_4

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  • DOI: https://doi.org/10.1007/978-1-0716-2043-4_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2042-7

  • Online ISBN: 978-1-0716-2043-4

  • eBook Packages: Springer Protocols

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