Abstract
Qualitative and quantitative changes in its chemical composition make it difficult to use any single procedure for isolating good-quality RNA from fruits at various ripening stages. Although the CTAB method has eliminated some specific problems, e.g., low pH in raw fruit or high levels of polysaccharides, oligosaccharides and phenolics in raw and ripe fruits, the total time required is long and unsuitable for high throughput. Here, we successfully modified this CTAB protocol to isolate good-quality RNA from (i) fleshy fruits, especially raw and ripe mangos; (ii) the leaves of a succulent air plant; and (iii) an oligosaccharide-rich onion epidermis. This RNA proved useful for downstream transcriptomic applications, where RT-PCR followed by RACE yielded the complete open reading frame of the (mango) terpene synthase gene. We also extended the utility of this protocol to co-isolate good-quality genomic DNA from the supernatant that remained after RNA precipitation. This preparation was useful for the arbitrary primer multilocus amplification of genomic DNA as well as for single locus diversity marker amplifications of the ctDNA and mtDNA.
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Abbreviations
- ßME:
-
2-mercaptoethanol
- DEPC:
-
diethyl pyrocarbonate
- EtBr:
-
ethidium bromide
- EtOH:
-
ethanol
- PVPF:
-
polyvinylpolypyrrolidone
- RACE:
-
rapid amplification of cDNA ends
- RTPCR:
-
reverse transcription polymerase chain reaction
- TE:
-
Tris (10 mM) with EDTA (1 mM)
Literature Cited
Aljanabi SM, Forget L, Dookun A (1999) An improved and rapid protocol for the isolation of polysaccharide- and polyphenolfree sugarcane DNA. Plant Mol Biol Rep17: 1–8
Alvarez M, Juusola J, Ballantyne J (2004) An mRNA and DNA coisolation method for forensic casework samples. Anal Biochem335: 289–298
Asif MH, Dhawan P, Nath P (2000) A simple procedure for the isolation of high quality RNA from ripening banana fruit. Plant Mol Biol Rep18: 109–115
Bandopadhyay C, Gholap AS (1973) Changes in fatty acids in ripening mango pulp (variety Alphonso). J Agric Food Chem21: 496–497
Breeden L (2006) mRNA Purification. Fred Hutchinson Cancer Research Center, http://wvwv.fhcrc.org/science/labs/ breeden/Methods/mRNA_purification.html
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem162: 156–159
Gehrig HH, Winter K, Cushman J, Borland A, Taybi T (2000) An improved RNA isolation method for succulent plant species rich in polyphenols and polysaccharides. Plant Mol Biol Rep18: 369–376
Germain P, Linden G (1981) Activités enzymatiques,In B Deymier, JL Multon, D Simon, eds, Analyse des Constituants Alimentaires: Techniques d’Analyse et de Contrôle Dans les Industries Agro-Alimentaires, Vol 4, Tec. et Doc Lavoisier, Paris, pp 211–244
Kansci G, Koubala BB, Lape IM (2003) Effect of ripening on the composition and the suitability for jam processing of different varieties of mango (Mangifera indica). Afric J Biotechnol2: 301–306
La Claire II JW, Herrin DL (1997) Co-isolation of high-quality DNA and RNA from coenocytic green algae. Plant Mol Biol Rep15: 263–272
Levi A, Galau GA, Wetzstein HY (1992) A rapid procedure for the isolation of RNA from high phenolic-containing tissues of pecan. HortScience27: 1316–1318
Liu JJ, Goh CJ, Loh CS, Liu P, Pua EC (1998) A method for isolation of total RNA from fruit tissues of banana. Plant Mol Biol Rep16: 1–6
Logemann J, Schell J, Willmitzer L (1987) Improved method for the isolation of RNA from plant tissues. Anal Biochem163: 16–20
Lopez-Gomez R, Gomez-Lim MA (1992) A method for extraction of intact RNA from fruits rich in polysaccharides using ripe mango mesocarp. HortScience27: 440–442
Manning K (1990) Isolation of nucleic acids from plants by differential solvent precipitation. Anal Biochem195: 45–50
Saby JK, Jagan MRL, Bhat SG, Prasada RUJS (1999) Characterization of aroma components of sap from different Indian mango varieties. Phytochemistry52: 891–894
Sambrook T, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York
Tel-Zur N, Abbo S, Myslabodski D, Mizrahi Y (1999) Modified CTAB procedure for DNA isolation from epiphytic cacti of the generaHylocereus andSeienicereus (cactaceae). Plant Mol Biol Rep17: 249–254
Zhou J, Pesacreta TC, Brown RC (1999) RNA isolation without gel formation from oligosaccharide-rich onion epidermis. Plant Mol Biol Rep17: 397–407
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Pandit, S.S., Mitra, S.S., Giri, A.P. et al. A quick method for isolating rna from raw and ripe fleshy fruits as well as for co-isolating DNA and RNA from polysaccharideand polyphenol-rich leaf tissues. J. Plant Biol. 50, 60–64 (2007). https://doi.org/10.1007/BF03030601
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DOI: https://doi.org/10.1007/BF03030601