Advertisement

Isolation and Detection Methods of Plant miRNAs

  • Pedro Fernando Vera-Hernández
  • Stefan de Folter
  • Flor de Fátima Rosas-CárdenasEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1932)

Abstract

Small RNAs (sRNAs) are RNAs of low abundance in organisms. Among sRNAs, miRNAs are included and represent approximately 10% of the total number of sRNAs. The isolation of sRNAs is critical for miRNA detection and analysis. The precipitation of low-molecular-weight (LMW) RNAs from total RNA extracts has allowed enrichment of sRNAs. Here, we describe a simple method to isolate sRNAs from different plant species. The main advantage of this method is that it does not need first an extraction of total RNA and it is not based on TRIzol® reagent. This method has been successfully used for miRNA analyses by Northern blot assay and RT-qPCR (these techniques are as well described in this chapter), as well as sRNA library preparation.

Key words

sRNA extraction Plant miRNAs miRNA analysis miRNA detection 

Notes

Acknowledgments

We thank the Mexican National Council of Science and Technology (CONACyT) for the fellowship of PPVH. This work was financed by CONACyT grant CB-2013-221522 and SIP grants 20170477 and 20180545. Work in the SDF laboratory was financed by the CONACyT grants CB-2012-177739 and FC-2015-2/1061.

References

  1. 1.
    Carra A, Gambino G, Schubert A (2007) A cetyltrimethylammonium bromide-based method to extract low-molecular-weight RNA from polysaccharide-rich plant tissues. Anal Biochem 360:318–320CrossRefGoogle Scholar
  2. 2.
    Carra A, Mica E, Gambino G, Pindo M, Moser C, Pè ME, Schubert A (2009) Cloning and characterization of small non-coding RNAs from grape. Plant J 59:750–763CrossRefGoogle Scholar
  3. 3.
    Chappell L, Baulcombe D, Molnár A (2006) Isolation and Cloning of Small RNAs from virus-infected Plants. Curr Protoc Microbiol 16:16H.2Google Scholar
  4. 4.
    Pilcher RL, Moxon S, Pakseresht N, Moulton V, Manning K, Seymour G, Dalmay T (2007) Identification of novel small RNAs in tomato (Solanum lycopersicum). Planta 226:709–717CrossRefGoogle Scholar
  5. 5.
    Chavez Montes RA, de Fatima Rosas-Cardenas F, De Paoli E, Accerbi M, Rymarquis LA, Mahalingam G, Marsch-Martinez N, Meyers BC, Green PJ, de Folter S (2014) Sample sequencing of vascular plants demonstrates widespread conservation and divergence of microRNAs. Nat Commun 5:3722CrossRefGoogle Scholar
  6. 6.
    de Fátima Rosas-Cárdenas F, Duran-Figueroa N, Vielle-Calzada JP, Cruz-Hernandez A, Marsch-Martinez N, de Folter S (2011) A simple and efficient method for isolating small RNAs from different plant species. Plant Methods 7:4CrossRefGoogle Scholar
  7. 7.
    Rosas-Cardenas Fde F, Escobar-Guzman R, Cruz-Hernandez A, Marsch-Martinez N, de Folter S (2015) An efficient method for miRNA detection and localization in crop plants. Front Plant Sci 6:99PubMedGoogle Scholar
  8. 8.
    Khandal H, Parween S, Roy R, Meena MK, Chattopadhyay D (2017) MicroRNA profiling provides insights into post-transcriptional regulation of gene expression in chickpea root apex under salinity and water deficiency. Sci Rep 7:4632CrossRefGoogle Scholar
  9. 9.
    Qing-Tian Z, Shu-Tian F, Yi-Ming Y, Pei-Lei X, Jun A (2016) De novo transcriptome assembly of Schisandra chinensis Turcz. (Baill.). Genomics data 10:153–154CrossRefGoogle Scholar
  10. 10.
    Shchennikova AV, Beletsky AV, Shulga OA, Mazur AM, Prokhortchouk EB, Kochieva EZ, Ravin NV, Skryabin KG (2016) Deep-sequence profiling of miRNAs and their target prediction in Monotropa hypopitys. Plant Mol Biol 91:441–458CrossRefGoogle Scholar
  11. 11.
    Xie M, Huang Y, Zhang Y, Wang X, Yang H, Yu O, Dai W, Fang C (2013) Transcriptome profiling of fruit development and maturation in Chinese white pear (Pyrus bretschneideri Rehd). BMC Genomics 14:823CrossRefGoogle Scholar
  12. 12.
    Huang H, Roh J, Davis CD, Wang TT (2017) An improved method to quantitate mature plant microRNA in biological matrices using modified periodate treatment and inclusion of internal controls. PLoS One 12:e0175429CrossRefGoogle Scholar
  13. 13.
    Wong QW, Sun MA, Lau SW, Parsania C, Zhou S, Zhong S, Ge W (2017) Identification and characterization of a specific 13-miRNA expression signature during follicle activation in the zebrafish ovary. Biol Reprod 98:42–53CrossRefGoogle Scholar
  14. 14.
    Pall GS, Hamilton AJ (2008) Improved northern blot method for enhanced detection of small RNA. Nat Protoc 3:1077–1084CrossRefGoogle Scholar
  15. 15.
    Kang K, Zhang X, Liu H, Wang Z, Zhong J, Huang Z, Peng X, Zeng Y, Wang Y, Yang Y (2012) A novel real-time PCR assay of microRNAs using S-Poly (T), a specific oligo (dT) reverse transcription primer with excellent sensitivity and specificity. PLoS One 7:e48536CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Pedro Fernando Vera-Hernández
    • 1
  • Stefan de Folter
    • 2
  • Flor de Fátima Rosas-Cárdenas
    • 1
    Email author
  1. 1.Centro de Investigación en Biotecnología Aplicada del Instituto Politécnico Nacional (CIBA-IPN)Ex-Hacienda San Juan MolinoTlaxcalaMexico
  2. 2.Unidad de Genómica Avanzada, Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO)Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN)IrapuatoMexico

Personalised recommendations