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Sample Collection Protocol Effects on Quantification of Gene Expression in Potato Leaf Tissue

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Abstract

New platforms allow quantification of gene expression from large, replicated experiments but current sampling protocols for plant tissue using immediate flash freezing in liquid nitrogen are a barrier to these high-throughput studies. In this study, we compared four sampling methods for RNA extraction for gene expression analysis: (1) the standard sampling method of flash freezing whole leaves in liquid nitrogen immediately upon removal from the plant; (2) incubation of excised leaf disks for 2 min at field temperature followed by flash freezing; (3) incubation of excised leaf disks for 1 h on ice followed by flash freezing; and (4) incubation of excised leaf disks for 1 h at field temperature followed by flash freezing. Gene expression analysis was done for 23 genes using nCounter, and normalization of the data was done using the geometric mean of five housekeeping genes. Quality of RNA was highest for protocol A and lowest for protocol D. Despite some differences in RNA quality, gene expression was not significantly different among protocols A, B, and C for any of the 23 genes. Expression of some genes was significantly different between protocol D and the other protocols. This study demonstrates that when sampling leaf disks for gene expression analysis, the time between tissue removal from the plant and flash freezing in liquid nitrogen can be extended. This increase in time allowable during sampling provides greater flexibility in sampling large replicated field experiments for statistical analysis of gene expression data.

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References

  • Arnis D, Elena P, Zewei L, Ning J, Xinwei C, Mike K, Robbie W (2010) Expression quantitative trait loci analysis in plants. Plant Biotechnol J 8(1):10–27

    Article  Google Scholar 

  • Cheang MCU, van de Rijn M, Nielsen TO (2008) Gene expression profiling of breast cancer. Annu Rev Pathol: Mech Dis 3(1):67–97. doi:10.1146/annurev.pathmechdis.3.121806.151505

    Article  CAS  Google Scholar 

  • Forde BG (2000) Nitrate transporters in plants: structure, function and regulation. Biochim Biophys Acta 1:1–2

    Google Scholar 

  • Forrest AR, Carninci P (2009) Whole genome transcriptome analysis. RNA Biol 6(2):107–112. doi:7931

    Article  PubMed  CAS  Google Scholar 

  • Fu X, Deng S, Su G, Zeng Q, Shi S (2004) Isolating high-quality rna from mangroves without liquid nitrogen. Plant Mol Biol Report 22(2):197

    Article  Google Scholar 

  • Geiss GK, Bumgarner RE, Birditt B, Dahl T, Dowidar N, Dunaway DL, Fell HP, Ferree S, George RD, Grogan T, James JJ, Maysuria M, Mitton JD, Oliveri P, Osborn JL, Peng T, Ratcliffe AL, Webster PJ, Davidson EH, Hood L, Dimitrov K (2008) Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol 26(3):317–325

    Article  PubMed  CAS  Google Scholar 

  • Krupp G (2005) Stringent rna quality control using the agilent 2100 bioanalyzer. Agilent Technologies Application Notes

  • Lacombe B, Becker D, Hedrich R, DeSalle R, Hollmann M, Kwak JM, Schroeder JI, Le Novere N, Nam HG, Spalding EP, Tester M, Turano FJ, Chiu J, Coruzzi G (2001) The identity of plant glutamate receptors. Science 292(5521):1486–1487

    Article  PubMed  CAS  Google Scholar 

  • Li X-Q, Sveshnikov D, Zebarth B, Tai H, De Koeyer D, Millard P, Haroon M, Singh M (2010) Detection of nitrogen sufficiency in potato plants using gene expression markers. Am J Potato Res. doi:citeulike-article-id:5976537

    Google Scholar 

  • Lopez MM, Bertolini E, Olmos A, Caruso P, Gorris MT, Llop P, Penyalver R, Cambra M (2003) Innovative tools for detection of plant pathogenic viruses and bacteria. Int Microbiol 6(4):233–243. doi:10.1007/s10123-003-0143-y

    Article  PubMed  CAS  Google Scholar 

  • Nakane E, Kawakita K, Doke N, Yoshioka H (2003) Elicitation of primary and secondary metabolism during defense in the potato. J Gen Plant Pathol 69(6):378–384

    Article  CAS  Google Scholar 

  • Nicot N, Hausman J-F, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time rt-pcr normalization in potato during biotic and abiotic stress. J Exp Bot 56(421):2907–2914. doi:10.1093/jxb/eri285

    Article  PubMed  CAS  Google Scholar 

  • Nielsen KL (ed) (2008) Serial analysis of gene expression (sage) methods and protocols. Humana Press, Totowa, NJ, USA

  • Orsel M, Krapp A, Daniel-Vedele F (2002) Analysis of the nrt2 nitrate transporter family in arabidopsis. Structure and gene expression. Plant Physiol 129(2):886–896

    Article  PubMed  CAS  Google Scholar 

  • Pitt D (1975) Changes in activity of lysosomal ribonuclease following mechanical damage to leaves of solanum tuberosum l. Planta 123(2):125–136

    Article  CAS  Google Scholar 

  • Quinones V, Zanlungo S, Holuigue L, Litvak S, Jordana X (1995) The cox1 initiation codon is created by rna editing in potato mitochondria. Plant Physiol 108(3):1327–1328

    Article  PubMed  CAS  Google Scholar 

  • Schachtman DP, Shin R (2007) Nutrient sensing and signaling: Npks. Annu Rev Plant Biol 58:47–69

    Article  PubMed  CAS  Google Scholar 

  • Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, Menzel W, Granzow M, Ragg T (2006) The rin: an rna integrity number for assigning integrity values to rna measurements. BMC Mol Biol 7(1):3

    Article  PubMed  Google Scholar 

  • Sugiharto B, Suzuki I, Burnell JN, Sugiyama T (1992) Glutamine induces the n-dependent accumulation of mrnas encoding phosphoenolpyruvate carboxylase and carbonic anhydrase in detached maize leaf tissue. Plant Physiol 100(4):2066–2070

    Article  PubMed  CAS  Google Scholar 

  • Tai H, Conn G, Davidson C, Platt H (2009) Arbitrary multi-gene reference for normalization of real-time pcr gene expression data. Plant Mol Biol Report 27(3):315–320

    Article  CAS  Google Scholar 

  • Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative rt-pcr data by geometric averaging of multiple internal control genes. Genome Biology 3 (7):research0034.0031–research0034.0011

    Google Scholar 

  • Wan CY, Wilkins TA (1994) A modified hot borate method significantly enhances the yield of high-quality rna from cotton (gossypium hirsutum l.). Anal Biochem 223(1):7–12. doi:10.1006/abio.1994.1538

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

Funding was provided by the SAGES Program of Agriculture and Agri-Food Canada and by the Canada-China AAFC-MOE Program. Technical assistance was provided by Muhammed Haroon, Kraig Worrall, and Karen Terry.

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Authors and Affiliations

  1. Institute of Horticulture, Hunan Academy of Agricultural Sciences, Changsha, Hunan, 410125, China

    Sainan Luo & Xingyao Xiong

  2. Potato Research Centre, Agriculture and Agri-Food Canada, PO Box 20280, Fredericton, NB E3B 4Z7, Canada

    Sainan Luo, Helen Tai, Bernie Zebarth, Xiu-Qing Li & David De Koeyer

  3. Macaulay Institute, Craigiebuckler, Aberdeen, AB15 8QH, UK

    Pete Millard

Authors
  1. Sainan Luo
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  2. Helen Tai
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  3. Bernie Zebarth
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  4. Xiu-Qing Li
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  5. Pete Millard
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  6. David De Koeyer
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  7. Xingyao Xiong
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Correspondence to Helen Tai.

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Luo, S., Tai, H., Zebarth, B. et al. Sample Collection Protocol Effects on Quantification of Gene Expression in Potato Leaf Tissue. Plant Mol Biol Rep 29, 369–378 (2011). https://doi.org/10.1007/s11105-010-0239-4

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