Advertisement

Journal of Forestry Research

, Volume 27, Issue 1, pp 33–40 | Cite as

Comparison of methods for extracting high-throughput sequencing RNA from Korean pine seeds

  • Yan Liang
  • Hai-long Shen
  • Chun-ping Liu
  • Ling Yang
  • Peng Zhang
ORIGINAL PAPER
  • 171 Downloads

Abstract

Korean pine (Pinus koraiensis Sieb. et Zucc.) is an ecologically and economically important tree species in East Asia. Molecular studies of seed development of this species are limited due to the lack of effective RNA extraction protocols. This study aimed to obtain an effective method to extract high-quality RNA from Korean pine seeds. The TRIzol kit and CTAB methods were used to extract the total RNA from Korean pine seeds at different developmental stages. The bands of RNA extracted by CTAB were not clear, whereas the bands of RNA extracted by the TRIzol kit were brighter and clearer, indicating higher quality and integrity of the RNA products extracted by the TRIzol kit. The 28S rRNA band was approximately 1.5- to 2-fold brighter than the 18S rRNA band on the agarose gel electrophoresis. The absorbance value A 260/280 was 1.8–2.0, and the absorbance value A 260/230 was >1.9. The Bioanalyzer RNA integrity results showed that the RNA integrity number of the RNA extracted using the TRIzol kit was acceptable for high-throughput sequencing. Therefore, the total RNA extracted using the TRIzol kit method can be used for high-throughput sequencing and other molecular biology experiments.

Keywords

Pinus koraiensis Seeds RNA extraction CTAB TRIzol kit 

Notes

Acknowledgments

We thank the staff at Weihe Seed Orchard in Heilongjiang Province for their help with cone collection.

References

  1. Balbuena TS, Silveira V, Junqueira M, Dias LL, Santa-Catarina C, Shevchenko A, Floh E (2009) Changes in the 2-DE protein profile during zygotic embryogenesis in the Brazilian Pine (Araucaria angustifolia). J Proteomics 72:337–352PubMedCrossRefGoogle Scholar
  2. Berendzen K, Searle I, Ravenscroft D, Koncz C, Batschauer A, Coupland G, Somssich IE, Ulker B (2005) A rapid and versatile combined DNA/RNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsis thaliana ecotypes Col-0 and Landsberg erecta. Algorithms Mol Biol 1(4):1–15Google Scholar
  3. Chang SJ, Puryear J, Cainey J (1993) A simple and efficient method for RNA isolation from pine trees. Plant Mol Biol Report 11(2):113–116CrossRefGoogle Scholar
  4. Chen MM, Feng FJ, Sui X, Li MH, Zhao D, Han SJ (2010) Construction of a framework map for Pinus koraiensis Sieb. et Zucc. using SRAP. SSR and ISSR markers. Trees 24(4):685–693CrossRefGoogle Scholar
  5. Chen XP, Zhu W, Azam S, Li HY, Zhu FH, Li HF, Hong YB, Liu HY, Zhang E, Wu H, Yu SL, Zhou GY, Li SX, Zhong N, Wen SJ, Li XG, Knapp SJ, Ozias-Akins P, Varshney RK (2013) Deep equencing analysis of the transcriptomes of peanut aerial and subterranean young pods identifies candidate genes related to early embryo abortion. Plant Biotechnol J 11:115–127PubMedCrossRefGoogle Scholar
  6. Chomczynski P, Sacchi N (2006) The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on. Nat Protoc 1:581–585PubMedCrossRefGoogle Scholar
  7. Davidson RM, Hansey CN, Gowda M, Childs KL, Lin H, vaillancourt B, Sekhon RS, Leon N, Kaeppler SM, Jiang N, Buell R (2011) Utility of RNA sequencing for analysis of maize reproductive transcriptome. Plant Genome 4:191–203CrossRefGoogle Scholar
  8. Gayral P, Weinert L, Chiari Y, Tsagkogeorga G (2011) Next-generation sequencing of transcriptomes: a guide to RNA isolation in nonmodel animals. Mol Ecol Resour 11:650–661PubMedCrossRefGoogle Scholar
  9. Guo B, Hou SY, Huang KS (2013) Different methods for extrating total RNA and their application in gene cloning and gene expression analysis in soybean (Glycine max). Mol Plant Breed 1(2):255–261Google Scholar
  10. Hao FL, Liu YL, Wang YJ (2005) Extracting methods of the total RNA in the petals of Lilium flowers. Acta Bot Boreali-Occident Sin 25(6):1143–1147Google Scholar
  11. Hillyard G, Clark MS (2012) RNA preservation of antarctic marine invertebrates. Polar Biol 35:633–636CrossRefGoogle Scholar
  12. Huang C, Picimbon JF, Li HQ, Li Z, Liu Q, Liu W (2012) An efficient method of total RNA extraction from peanut seeds. J Peanut Sci 59(1):127–131Google Scholar
  13. Kim ZS, Huang JW, Lee SW, Yang C, Gorovoy PG (2005) Genetic variation of korean pine (Pinus koraiensis Sieb. et Zucc.) at allozyme and RAPD markers in Korea, China and Russia. Silvae Genet 54:4–5Google Scholar
  14. Klimaszewska K, Morency F, Jones-Overton C, Cooke J (2004) Accumulation pattern and identification of seed storage proteins in zygotic embryos of Pinus strobus and in somatic embryos from different maturation treatments. Physiol Plant 12:1682–1690Google Scholar
  15. Kolosova N, Miller B, Ralph S, Ellis BE, Douglas C, Ritland K, Bohlmann J (2004) Isolation of high-quality RNA from gymnosperm and angiosperm trees. Biotechniques 35:821–824Google Scholar
  16. Lippert D, Yuen M, Bohlmann J (2009) Spruce proteome DB: a resource for conifer proteomics research. Tree Genet Genomes 5:723–727CrossRefGoogle Scholar
  17. Liu H, Yang ZL, Yang MF (2011) The differential proteome of endosperm and embryo from mature seed of Jatropha curcas. Plant Sci 181:660–666PubMedCrossRefGoogle Scholar
  18. Perez-Portela R, Riesgo A (2013) Optimizing preservation protocols to extract high-quality RNA from different tissues of echinoderms for next-generation sequencing. Mol Ecol Resour 13:884–889PubMedCrossRefGoogle Scholar
  19. Rajakani R, Narnoliya L, Sangwan NS, Sangwan RS, Gupta V (2013) Activated charcoal-mediated RNA extraction method for Azadirachta indicaand plants highly rich in polyphenolics, polysaccharides and other complex secondary compounds. BMC Res Notes 6:125PubMedPubMedCentralCrossRefGoogle Scholar
  20. Riesgo A, Andrade SCS, Sharma PP, Novo M, Pérez-Porro AR, Vahtera V, González VL, Kawauchi GY, Giribet G (2012a) Comparative description of ten transcriptomes of newly sequenced invertebrates and efficiency estimation of genomic sampling in non-model taxa. Front Zool 9:33–56PubMedPubMedCentralCrossRefGoogle Scholar
  21. Riesgo A, Peiez-Porro AR, Carmona S, Leys S, Giribet G (2012b) Optimization of preservation and storage time of sponge tissues to obtain quality mRNA for next-generation sequencing. Mol Ecol Resour 12:312–322PubMedCrossRefGoogle Scholar
  22. Ruan MB, Li WB, Yu XL, Peng M (2011) Rapid isolation of high-quality RNA from high phenol-polysaccharide plants. Chin J Trop Crops 32(9):1704–1707Google Scholar
  23. Salter MG, Conlon HE (2007) Extraction of plant RNA. Methods Mol Biol 362:309–314PubMedCrossRefGoogle Scholar
  24. Shi J, Zhen Y, Zheng RH (2010) Proteome profiling of early seed development in Cunninghamia lanceolata (Lamb.) Hook. J Exp Bot 61:2367–2381PubMedPubMedCentralCrossRefGoogle Scholar
  25. Sun HB, Wang ZH, Liu XQ, Huang JY, Dong CH, Liu SHY (2012) Modified RNA extraction method for seeds of rape seed, soybean, peanut and sesame. Chin J Oil Cropsci 34(4):353–358Google Scholar
  26. Tong ZG, Qu SC, Zhang JY, Wang F, Tao JM, Gao ZH, Zhang Z (2012) A Modified protocol for RNA extraction from different peach tissues suitable for gene isolation and real-time PCR analysis. Mol Biotechnol 50:229–236PubMedCrossRefGoogle Scholar
  27. Wan YF, Poole RL, Huttly AK, Toscano-Underwood C, Feeney K, Welham S, Gooding MJ, Mills C, Edwards KJ, Shewry PR, Mitchell RA (2008) Transcriptome analysis of grain development in hexaploid wheat. BMC Genom 9:1471–2164Google Scholar
  28. Yan Z, Zhao ZZ, Zheng RH, Shi J (2012) Proteomic analysis of early seed development in Pinus massoniana L. Plant Physiol Biochem 54:97–104CrossRefGoogle Scholar
  29. Yin DM, Liu HY, Zhang XG, Cui DQ (2011) A protocol for extraction of high-quality RNA and DNA from peanut plant tissues. Mol Biotechnol 49:187–191PubMedCrossRefGoogle Scholar
  30. Zhao BH, Zheng CX, Xu LL, Zhao N, Xu GJ (2010) Extraction Method of RNA from Pinus tabularformis carr Mature Embryo. J Anhui Agric Sci 38(6):2812–2814Google Scholar

Copyright information

© Northeast Forestry University and Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Yan Liang
    • 1
    • 2
  • Hai-long Shen
    • 1
  • Chun-ping Liu
    • 1
  • Ling Yang
    • 1
  • Peng Zhang
    • 1
  1. 1.State Key Laboratory of Forest Genetics and BreedingNortheast Forestry UniversityHarbinChina
  2. 2.School of Life Sciences and A & FQiqihar UniversityQiqiharChina

Personalised recommendations