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Immobilization of microalgae cells in alginate facilitates isolation of DNA and RNA.


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Científica

Tipología

Investigación y estudios

Medio de publicación

Impreso: Revista indexada

Resumen

Isolation of nucleic acids from Chlorella is difcult, given the chemically complexnatureoftheir cell walls and variable production of metabolites. Immobilization of microalgae in polymers adds additional difculty. Here, we modied, amended, and standardized methods for isolation of nucleic acids and compared the yield of DNA and RNA from free-living and encapsulated microalgaemC. sorokiniana. Isolation of nucleic acids from immobilized cells requiredtwosteps indissolving the alginate matrix, releasing thecells,and mechanical disruptionwith glass beads. For DNA extraction, we used modified versions of a commercial kit along with the hexadecyltrimethylammonium bromide (CTAB) method. For RNA extraction, we used the commercial TRI reagent procedure and the CTAB-dithiotreitol method. Quantity and quality of nucleic acids in extracts varied with growth conditions, isolation procedures, and time of incubation of the original culture. There were consistently higher amounts of DNA and RNA in extracts from immobilized cells. Quantitatively, the modified procedure with the commercial Promega kit was the most reliable procedure for isolating DNA and a modified commercial TRI reagent procedure was the choice for isolating RNA. All four procedures eliminated proteins eficiently and had low levelsofcontamination from residualpolysaccharidesfrom the matrices and/ormetabolites naturally produced by the microalgae. All DNA extracts under both growth conditions, time of incubation, and two isolation methods successfully amplified the 18S ribosomal RNA by PCR and quantitative reverse transcription (RT-qPCR)

Autores

Blanca R. Lopez
Juan-Pablo Hernandez
Yoav Bashan
Luz E. de-Bashan

SNIES Área

Environmental Science

SNIES Categoría

Applied Microbiology and Biotechnology

Fecha de publicación 04 de abril de 2017
Fecha de aceptación 20 de febrero de 2017
Medio indexado (nombre)

Journal of Microbiological Methods

English information
Title

Immobilization of microalgae cells in alginate facilitates isolation of DNA and RNA.

Abstract

Isolation of nucleic acids from Chlorella is difcult, given the chemically complexnatureoftheir cell walls and variable production of metabolites. Immobilization of microalgae in polymers adds additional difculty. Here, we modied, amended, and standardized methods for isolation of nucleic acids and compared the yield of DNA and RNA from free-living and encapsulated microalgaemC. sorokiniana. Isolation of nucleic acids from immobilized cells requiredtwosteps indissolving the alginate matrix, releasing thecells,and mechanical disruptionwith glass beads. For DNA extraction, we used modified versions of a commercial kit along with the hexadecyltrimethylammonium bromide (CTAB) method. For RNA extraction, we used the commercial TRI reagent procedure and the CTAB-dithiotreitol method. Quantity and quality of nucleic acids in extracts varied with growth conditions, isolation procedures, and time of incubation of the original culture. There were consistently higher amounts of DNA and RNA in extracts from immobilized cells. Quantitatively, the modified procedure with the commercial Promega kit was the most reliable procedure for isolating DNA and a modified commercial TRI reagent procedure was the choice for isolating RNA. All four procedures eliminated proteins eficiently and had low levelsofcontamination from residualpolysaccharidesfrom the matrices and/ormetabolites naturally produced by the microalgae. All DNA extracts under both growth conditions, time of incubation, and two isolation methods successfully amplified the 18S ribosomal RNA by PCR and quantitative reverse transcription (RT-qPCR)

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