Skip to main content
SpringerLink
Log in

Evaluation of appropriate reference gene for normalization of microRNA expression by real-time PCR in Lablab purpureus under abiotic stress conditions

  • Published:
Biologia Aims and scope Submit manuscript

    We’re sorry, something doesn't seem to be working properly.

    Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.

Abstract

MicroRNAs (miRNAs) play key roles in plant responses to biotic and abiotic stresses by modulating their own expression and a wide array of target mRNAs. Reverse transcription quantitative real-time PCR is a sensitive and widely used method to study miRNA expression profile. Accurate analysis and interpretation of results require the selection of an appropriate reference gene. Reference genes selected should have a constant expression level under different stress conditions. Six reference candidates, including two miRNAs (miRNA 156 and miRNA 172), an rRNA (5S rRNA), a snRNA (U6) and two protein coding genes (actin and protein phosphatase 2A), were selected for normalization of miRNA expression in Lablab purpureus. The expression stability of candidate reference genes was investigated in ten samples and analysed using NormFinder, BestKeeper and hkgFinder softwares. The analyses suggested that miRNA 156 is the appropriate reference gene, as it had better expression stability than protein-coding genes, and other non-coding RNAs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

Cq:

quantification cycle

miRNA:

microRNA

PP2A:

protein phosphatase 2A

RT-qPCR:

reverse transcription quantitative PCR

SD:

standard deviation

References

  • Andersen C.L., Jensen J.L. & Orntoft T.F. 2004. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 64: 5245–5250.

    Article  CAS  Google Scholar 

  • Borowski J.M., Galli V., Messias R.S., Perin E.C., Buss J.H., dos Anjos e Silva S.D. & Rombaldi C.V. 2014. Selection of candidate reference genes for real-time PCR studies in lettuce under abiotic stresses. Planta 239: 1187–1200.

    CAS  PubMed  Google Scholar 

  • Brunner A.M., Yakovlev I.A. & Strauss S.H. 2004. Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol. 4: 14.

    Article  Google Scholar 

  • Bustin S.A. 2002. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems, J. Mol. Endocrinol. 29: 23–39.

    Article  CAS  Google Scholar 

  • Bustin S.A., Benes V., Garson J.A., Hellemans J., Huggett J., Kubista M., Mueller R., Nolan T., Pfaffl M.W., Shipley G.L., Vandesompele J. & Wittwer C.T. 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55: 611–622.

    Article  CAS  Google Scholar 

  • Chen C., Ridzon D.A., Broomer A.J., Zhou Z., Lee D.H., Nguyen J.T., Barbisin M., Xu N.L., Mahuvakar V.R., Andersen M.R., Lao K.Q., Livak K.J. & Guegler K.J. 2005. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 33: e179.

    Article  Google Scholar 

  • Chen X., Zhang Z., Liu D., Zhang K., Li A. & Mao L. 2010. SQUAMOSA promoter-binding protein-like transcription factors: star players for plant growth and development. J. Integr. Plant Biol. 52: 946–951.

    Article  CAS  Google Scholar 

  • Cushman J.C. & Bohnert H.J. 2000. Genomic approaches to plant stress tolerance. Curr. Opin. Plant Biol. 3: 117–124.

    Article  CAS  Google Scholar 

  • Czechowski T., Stitt M., Altmann T., Udvardi M.K. & Scheible W.R. 2005. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 139: 5–17.

    Article  CAS  Google Scholar 

  • D’Souza M.R. & Devaraj V.R. 2010. Biochemical responses of hyacinth bean (Lablab purpureus) to salinity stress. Acta Physiol. Plant. 32: 341–353.

    Article  Google Scholar 

  • D’Souza M.R. & Devaraj V.R. 2011. Specific and non-specific responses of hyacinth bean (Dolichos lablab) to drought stress. Indian J. Biotechnol. 10: 130–139.

    Google Scholar 

  • Feng H., Huang X., Zhang Q., Wei G., Wang X. & Kang Z. 2012. Selection of suitable inner reference genes for relative quantification expression of microRNA in wheat. Plant Physiol. Biochem. 51: 116–122.

    Article  CAS  Google Scholar 

  • Fuller D.Q. 2003. African crops in prehistoric South Asia: a critical review, pp. 239–271. In: Neumann K., Butler A. & Kahlheber S. (eds), Food, Fuel and Fields; Progress in Africa Archaeobotany. Heinrich-Barth-Institut, Cologne.

    Google Scholar 

  • Gutierrez L., Mauriat M., Guenin S., Pelloux J., Lefebvre J.F., Louvet R., Rusterucci C., Moritz T., Guerineau F., Bellini C. & Wuytswinkel O.V. 2008. The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription polymerase chain reaction (RT-PCR) analysis in plants. Plant Biotechnol. 6: 609–618.

    Article  CAS  Google Scholar 

  • Guenin S., Mauriat M., Pelloux J., Wuytswinkel O.V., Bellini C. & Gutierrez L. 2009 Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditionsspecific, validation of references. J. Exp. Bot. 60: 487–493.

    Article  CAS  Google Scholar 

  • Kokila S. 2015. Molecular characterization of transcripts induced under drought and salt stress from Lablab purpureus (hyacinth bean). PhD Thesis, Department of Biochemistry, Central College Campus, Bangalore University, Bangalore, India.

    Google Scholar 

  • Kong W., Zhao J.J., He L. & Cheng J.Q. 2009. Strategies for profiling microRNA expression. J. Cell. Physiol. 218: 22–25.

    Article  CAS  Google Scholar 

  • Kou S.J., Wu X.M., Liu Z., Liu Y.L., Xu Q. & Guo W.W. 2012. Selection and validation of suitable reference genes for miRNA expression normalization by quantitative RT-PCR in citrus somatic embryogenic and adult tissues. Plant Cell Rep. 31: 2151–2163.

    Article  CAS  Google Scholar 

  • Kulcheski F.R., Marcelino-Guimaraes F.C., Nepomuceno A.L., Abdelnoor R.V. & Margis R. 2010. The use of microRNAs as reference genes for quantitative polymerase chain reaction in soybean. Anal. Biochem. 406: 185–192.

    Article  CAS  Google Scholar 

  • Li Q.Q., Skinner J. & Bennett J.E. 2012. Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment. BMC Mol. Biol. 13: 22.

    Article  Google Scholar 

  • Libault M., Thibivilliers S., Bilgin D., Radwan O., Benitez M., Clough S.J. & Stacey G. 2008. Identification of four soybean reference genes for gene expression normalization. Plant Genome 1: 44–54.

    Article  CAS  Google Scholar 

  • Lin Y.L. & Lai Z.X. 2013. Evaluation of suitable reference genes for normalization of microRNA expression by real-time reverse transcription PCR analysis during longan somatic embryogenesis. Plant Physiol. Biochem. 66: 20–25.

    Article  CAS  Google Scholar 

  • Livak K.J. & Schmittgen T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-δδCT method. Methods 25: 402–408.

    Article  CAS  Google Scholar 

  • Luo X., Shi T., Sun H., Song J., Ni Z. & Gao Z. 2014. Selection of suitable inner reference genes for normalisation of microRNA expression response to abiotic stresses by RT-qPCR in leaves, flowers and young stems of peach. Scientia Horticulturae 165: 281–287.

    Article  CAS  Google Scholar 

  • Maass B.L., Knox M.R., Venkatesha S.C., Angessa T.T., Ramme S. & Pengelly B.C. 2010. Lablab purpureus — a crop lost for Africa? Tropical Plant Biol. 3: 123–135.

    Article  Google Scholar 

  • Peltier H.J. & Latham G.J. 2008. Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitable reference RNA targets in normal and cancerous human solid tissues. RNA 14: 844–852.

    Article  CAS  Google Scholar 

  • Devaraj Pfaffl M.W., Tichopad A., Prgomet C. & Neuvians T.P. 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excelbased tool using pair-wise correlations. Biotechnol. Lett. 26: 509–515.

    Article  Google Scholar 

  • Rodriguez M., Canales E. & Borras-Hidalgo O. 2005. Molecular aspects of abiotic stress in plants. Biotecnologia Aplicada 22: 1–10.

    CAS  Google Scholar 

  • Schmittgen T.D. & Zakrajsek B.A. 2000. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J. Biochem. Biophys. Methods 46: 69–81.

    Article  CAS  Google Scholar 

  • Selvey S., Thompson E.W., Matthaei K., Lea R.A., Irving M.G. & Griffiths L.R. 2001. ß-Actin — an unsuitable internal control for RT-PCR. Mol. Cell. Probes 15: 307–311.

    Article  CAS  Google Scholar 

  • Shi R. & Chiang V.L. 2005. Facile means for quantifying microRNA expression by real-time PCR. Biotechniques 39: 519–525.

    Article  CAS  Google Scholar 

  • Sunkar R., Chinnusamy V., Zhu J. & Zhu J.K. 2007. Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci. 12: 301–309.

    Article  CAS  Google Scholar 

  • Thellin O., Zorzi W., Lakaye B., Borman B.D., Coumans B., Hennen G., Grisar T., Igout A. & Heinen E. 1999. Housekeeping genes as internal standards: use and limits. J. Biotechnol. 75: 291–295.

    Article  CAS  Google Scholar 

  • Udvardi M.K., Czechowski T. & Scheible W.R. 2008. Eleven golden rules of quantitative RT-PCR. Plant Cell 20: 1736–1737.

    Article  CAS  Google Scholar 

  • Varkonyi-Gasic E., Wu R., Wood M., Walton E.F. & Hellens R.P. 2007. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3: 12.

    Article  Google Scholar 

  • Yao L.M., Wang B., Cheng L.J. & Wu T.L. 2013. Identification of key drought stress-related genes in the hyacinth bean. PLoS One 8: e58108.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry, Central College Campus, Bangalore University, Bangalore, 560 001, India

    Adikeshavan Thilagavathy & Varadahally R. Devaraj

Authors
  1. Adikeshavan Thilagavathy
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Varadahally R. Devaraj
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Varadahally R. Devaraj.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Thilagavathy, A., Devaraj, V.R. Evaluation of appropriate reference gene for normalization of microRNA expression by real-time PCR in Lablab purpureus under abiotic stress conditions. Biologia 71, 660–668 (2016). https://doi.org/10.1515/biolog-2016-0091

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1515/biolog-2016-0091

Key words

Search

Navigation