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Isolation of high-quality total rna from leaves of myrciaria dubia Camu Camu
Isolation of high-quality total rna from leaves of myrciaria dubia Camu Camu
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Fecha
2013
Autores
Gómez J.C.C.
Reátegui A.D.C.E.
Flores J.T.
Saavedra R.R.
Ruiz M.C.
Correa S.A.I.
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Taylor & Francis Online
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Abstracto
Myrciaria dubia is a main source of vitamin C for people in the Amazon region. Molecular studies of M. dubia require high-quality total RNA from different tissues. So far, no protocols have been reported for total RNA isolation from leaves of this species. The objective of this research was to develop protocols for extracting high-quality total RNA from leaves of M. dubia. Total RNA was purified following two modified protocols developed for leaves of other species (by Zeng and Yang, and by Reid et al.) and one modified protocol developed for fruits of the studied species (by Silva). Quantity and quality of purified total RNA were assessed by spectrophotometric and electrophoretic analysis. Additionally, quality of total RNA was evaluated with reverse-transcription polymerase chain reaction (RT-PCR). With these three modified protocols we were able to isolate high-quality RNA (A260nm/A280nm > 1.9 and A260nm/A230nm > 2.0). Highest yield was produced with the Zeng and Yang modified protocol (384 ± 46 µg ARN/g fresh weight). Furthermore, electrophoretic analysis showed the integrity of isolated RNA and the absence of DNA. Another proof of the high quality of our purified RNA was the successful cDNA synthesis and amplification of a segment of the M. dubia actin 1 gene. We report three modified protocols for isolation total RNA from leaves of M. dubia. The modified protocols are easy, rapid, low in cost, and effective for high-quality and quantity total RNA isolation suitable for cDNA synthesis and polymerase chain reaction.
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This work was supported by grants from Consejo Nacional de Ciencia y Tecnología from Perú (contract 280-2010-CONCYTEC-OAJ), and Universi-dad Nacional de la Amazonía Peruana (RR number 2010-UNAP). We also thank to Dr. Jorge L. Marapara for his help with the infrastructure and equipment of Unidad Especializada de Biotecnología and Instituto Nacional de Innovación Agraria (INIA)–San Roque-Iquitos for access to the germ-plasm collection of Myrciaria dubia. We also thank Alexander Cerdeira for his technical assistance. The authors are very grateful to Antje Trukenmüller for critical reading of the article.
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