A Simple Method of RNA Extraction from Apple Stem and Leaf Tissues via Heating

가열에 의한 사과줄기 및 잎조직으로부터의 RNA 간편 추출

  • Duong, Thanh Van (Department of Biotechnology, Catholic University of Daegu) ;
  • Shin, Dong-Il (Department of Biotechnology, Catholic University of Daegu) ;
  • Park, Hee-Sung (Department of Biotechnology, Catholic University of Daegu)
  • 즈엉반탄 (대구가톨릭대학교 생명공학과) ;
  • 신동일 (대구가톨릭대학교 생명공학과) ;
  • 박희성 (대구가톨릭대학교 생명공학과)
  • Received : 2010.05.25
  • Accepted : 2010.10.19
  • Published : 2010.10.31

Abstract

Apple, one of the most important fruit crops world-widely, is required for rapid, cost-effective and sensitive virus detection for its better productivity. RT-PCR is extensively employed for apple virus diagnosis, but the technique requires complete tissue homogenization which is time consuming and laborious. In this study, heating-based RNA extraction method was developed and proven to effectively work for stem tissues slightly better than for leaf tissues. In RT-PCR, almost identical results were generated from the use of RNA extracts from both tissues.

사과는 전 세계적으로 대표적 과수의 하나로서 우량 사과의 생산을 위하여 신속하고 경제적이며 정확한 사과바이러스 진단이 요구되고 있다. RT-PCR은 사과바이러스 진단을 위한 중요한 기술로서 우선 시료조직의 분쇄 및 균질화를 통한 양질의 RNA 추출이 필수적이다. 그러나 분쇄작업은 다량의 시료의 경우 많은 시간과 노동이 요구된다. 본 연구에서는 조직 분쇄과정이 없이 단순 가열에 의한 RNA 추출을 시도하였으며 줄기조직이 잎조직보다 약간 더 적합함을 보여주었다. 그러나 RT-PCR에 의한 사과바이러스 진단에서는 모두 동일한 결과를 나타냈다. 이로써 사과 조직에 대한 단순가열로써 매우 간편하게 양질의 RNA추출이 가능함을 제시하였다.

Keywords

References

  1. Besancon, B., A. Bencsik, B. Voutsinos, M. F. Belin, and M. Fevre-Montange. 1995. Rapid in situ hybridization using digoxigenin probe and microwave oven. Cell Mol. Biol. 41: 975–977.
  2. Boom, R., C. J. Sol, M. M. Salimans, C. L. Jansen, P. M. Wertheim-van Dillen, and J. van der Noordaa. 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28: 495-503.
  3. Cieslinska, M. 2004. Detection of strawberry mottle virus (SMov) using RT-PCR - comparison of two RNA extraction methods. J. Fru. Orna. Plant Res. 12: 17- 22.
  4. Ekuni, D., J. D. Firth, and E. E. Putnins. 2006. RNA integrity and in situ RT-PCR in dento-alveolar tissues after microwave accelerated demineralization. Arch. Oral Biol. 51: 164-169. https://doi.org/10.1016/j.archoralbio.2005.06.010
  5. EPPO. 1999. Certification schemes - Pathogen-tested material of Malus, Pyrus and Cydonia. EPPO Bull. 29: 239-252. https://doi.org/10.1111/j.1365-2338.1999.tb00828.x
  6. John, M. E. 1992. An efficient method for isolation of RNA and DNA from plants containing polyphenolics. Nucleic Acids Res. 20: 2381. https://doi.org/10.1093/nar/20.9.2381
  7. Lazarowitz, S. G. and R. N. Beachy. 1999. Viral movement proteins as probes for intracellular and intercellular trafficking in plants. Plant Cell 11: 535-548.
  8. Menzel, W., W. Jelkmann, and E. Maiss. 2002. Detection of four apple viruses by mutiplex RT-PCR assays with coamplification of plant mRNA as internal control. J. Virol. Methods. 99: 81-92 https://doi.org/10.1016/S0166-0934(01)00381-0
  9. Mink, G. I. 1989. Apple chlorotic leaf spot. In Virus and Virus-like Diseases of Pome Fruits and Simulating Noninfectious Disorders. Cooperative Extension College of Agriculture and Home Economics, Washington State University. Pullman, WA.
  10. Nemeth, M. 1986. In Virus, Mycoplasma and Ricketsia Diseases of Fruit Trees. Kluwer Academic Publishers, Boston.
  11. Picard, C., C. Ponsonnet, E. Paget, X. Nesme, and P. Simonet. 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Appl. Environ. Microbiol. 58: 2717-2722.
  12. Ryu, K. K. and W. M. Park. 1995. Rapid detection and identification of odontoglossum ringspot virus by polymerase chain reaction amplification. FEMS Microbiol. Lett. 133: 265-269. https://doi.org/10.1111/j.1574-6968.1995.tb07895.x
  13. Saini, H. S., M. Shepherd, and R. J. Henry. 1999. Microwave extraction of total genomic DNA from barley grains for use in PCR. J. Inst. Brew. 105: 185-190. https://doi.org/10.1002/j.2050-0416.1999.tb00018.x
  14. Stouffer, R. F. 1989. Apple stem pitting. In: Virus and Virus-like Diseases of Pome Fruits and Simulating Non-infectious Disorders. Cooperative Extension College of Agriculture and Home Economics, Washington State University, Pullman, WA.