Display options
Cite
Hashemi SH, Nematzadeh G, Ahmadian G, et al. Identification and validation of Aeluropus littoralis reference genes for Quantitative Real-Time PCR Normalization. J Biol Res (Thessalon). 2016;23:18doi: 10.1186/s40709-016-0053-8.
Hashemi, S. H., Nematzadeh, G., Ahmadian, G., Yamchi, A., & Kuhlmann, M. (2016). Identification and validation of Aeluropus littoralis reference genes for Quantitative Real-Time PCR Normalization. Journal of biological research (Thessalonike, Greece), 2318. https://doi.org/10.1186/s40709-016-0053-8
Hashemi, Seyyed Hamidreza, et al. "Identification and validation of Aeluropus littoralis reference genes for Quantitative Real-Time PCR Normalization." Journal of biological research (Thessalonike, Greece) vol. 23 (2016): 18. doi: https://doi.org/10.1186/s40709-016-0053-8
Hashemi SH, Nematzadeh G, Ahmadian G, Yamchi A, Kuhlmann M. Identification and validation of Aeluropus littoralis reference genes for Quantitative Real-Time PCR Normalization. J Biol Res (Thessalon). 2016 Jul 19;23:18. doi: 10.1186/s40709-016-0053-8. eCollection 2016 Dec. PMID: 27437194; PMCID: PMC4950632.
Copy Download .nbib Format: NLM
Share it on

J Biol Res (Thessalon). 2016 Jul 19;23:18. doi: 10.1186/s40709-016-0053-8. eCollection 2016 Dec.

Identification and validation of Aeluropus littoralis reference genes for Quantitative Real-Time PCR Normalization.

Journal of biological research (Thessalonike, Greece)

Seyyed Hamidreza Hashemi, Ghorbanali Nematzadeh, Gholamreza Ahmadian, Ahad Yamchi, Markus Kuhlmann

Affiliations

  1. Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University, PO Box 578, Sari, Iran.
  2. National Institute of Genetics Engineering and Biotechnology (NIGEB), Tehran, Iran.
  3. Department of Plant Breeding and Biotechnology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran.
  4. Research Group Abiotic Stress Genomics, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK Gatersleben), OT Gatersleben Corrensstraße 3, 06466 Stadt Seeland, Germany.

PMID: 27437194 PMCID: PMC4950632 DOI: 10.1186/s40709-016-0053-8

Abstract

BACKGROUND: The use of stably expressed genes as normalizers has crucial role in accurate and reliable expression analysis estimated by quantitative real-time polymerase chain reaction (qPCR). Recent studies have shown that, the expression levels of common housekeeping genes are varying in different tissues and experimental conditions. The genomic DNA contamination in RNA samples is another important factor that also influence the interpretation of the data obtained from qPCR. It is estimated that the gDNA contamination in gene expression analysis lead to an overestimation of the RNA transcript level. The aim of this study was to validate the most stably expressed reference genes in two different tissues of Aeluropus littoralis-halophyte grass at salt stress and recovery condition. Also, a qPCR-based approach for monitoring contamination with gDNA was conducted.

RESULTS: Ten candidate reference genes participating in different biological processes were analyzed in four groups of samples including root and leaf tissues, salt stress and recovery condition. To determine the most stably expressed reference genes, three statistical methods (geNorm, NormFinder and BestKeeper) were applied. According to results obtained, ten candidate reference genes were ranked based on the stability of their expression. Here, our results show that a set of four housekeeping genes (HKGs) e.g. RPS3, EF1A, GTF and RPS12 could be used as general reference genes for the all selected conditions and tissues. Also, four set of reference genes were proposed for each tissue and condition including: RPS3, EF1A and UBQ for salt stress and root samples; RPS3, EF1A, UBQ as well as GAPDH for recovery condition; U2SURP and GTF for leaf samples. Additionally, for assessing DNA contamination in RNA samples, a set of unique primers were designed based on the conserved region of ribosomal DNA (rDNA). The universality, specificity and sensitivity of these primer pairs were also evaluated in Poaceae.

CONCLUSIONS: Overall, the sets of reference genes proposed in this study are ideal normalizers for qPCR analysis in A. littoralis transcriptome. The novel reference gene e.g. RPS3 that applied this study had higher expression stability than commonly used housekeeping genes. The application of rDNA-based primers in qPCR analysis was addressed.

Keywords: Aeluropus littoralis; DNA contamination; Halophyte; Recovery condition; Reference genes; Salt stress; qPCR; rDNA; rRNA

References

  1. J Comput Biol. 2000 Feb-Apr;7(1-2):203-14 - PubMed
  2. J Mol Endocrinol. 2000 Oct;25(2):169-93 - PubMed
  3. Genome Biol. 2002 Jun 18;3(7):RESEARCH0034 - PubMed
  4. Neurosci Lett. 2003 Mar 13;339(1):62-6 - PubMed
  5. Mol Phylogenet Evol. 2003 Dec;29(3):417-34 - PubMed
  6. Biotechnol Lett. 2004 Mar;26(6):509-15 - PubMed
  7. Cell Cycle. 2004 Jul;3(7):880-3 - PubMed
  8. Biotechniques. 2004 Jul;37(1):112-4, 116, 118-9 - PubMed
  9. Cancer Res. 2004 Aug 1;64(15):5245-50 - PubMed
  10. Expert Rev Mol Diagn. 2005 Mar;5(2):221-30 - PubMed
  11. Plant Physiol. 2005 Sep;139(1):5-17 - PubMed
  12. Biochem Biophys Res Commun. 2006 Jun 30;345(2):646-51 - PubMed
  13. Nat Protoc. 2006;1(3):1559-82 - PubMed
  14. Nat Rev Mol Cell Biol. 2007 Jul;8(7):574-85 - PubMed
  15. J Plant Res. 2007 Jul;120(4):529-37 - PubMed
  16. Plant Methods. 2007 Jun 08;3:7 - PubMed
  17. Protoplasma. 2008 Nov;233(3-4):195-202 - PubMed
  18. Plant Cell. 2008 Jul;20(7):1734-5 - PubMed
  19. Nucleic Acids Res. 2009 Apr;37(6):e45 - PubMed
  20. Clin Chem. 2009 Apr;55(4):611-22 - PubMed
  21. Plant Mol Biol. 2010 Jan;72(1-2):171-90 - PubMed
  22. RNA. 2010 Feb;16(2):450-61 - PubMed
  23. BMC Plant Biol. 2010 Apr 19;10:71 - PubMed
  24. Transgenic Res. 2011 Oct;20(5):1003-18 - PubMed
  25. Biochem Biophys Res Commun. 2012 Aug 3;424(3):398-403 - PubMed
  26. Planta. 2013 Mar;237(3):873-89 - PubMed
  27. Oecologia. 1993 May;94(2):204-209 - PubMed

Publication Types