title: Comparative evaluation of total RNA extraction methods in Theobroma cacao using shoot apical meristems.
abstract: Theobroma cacao is a species of great economic importance with its beans used for chocolate production. The tree has been a target of various molecular studies. It contains many polyphenols, which complicate the extraction of nucleic acids with the extraction protocols requiring a large amount of plant material. These issues, therefore, necessitate the optimization of the protocols. The aim of the present study was to evaluate different methods for extraction of total RNA from shoot apical meristems of T. cacao 'CCN 51' and to assess the influence of storage conditions for the meristems on the extraction. The study also aimed to identify the most efficient protocol for RNA extraction using a small amount of plant material. Four different protocols were evaluated for RNA extraction using one shoot apical meristem per sample. Among these protocols, one that was more efficient was then tested to extract RNA using four different numbers of shoot apical meristems, subjected to three different storage conditions. The best protocol was tested for cDNA amplification using reverse transcription-polymerase chain reaction; the cDNA quality was determined to be satisfactory for molecular analyses. The study revealed that with the best RNA extraction protocol, one shoot apical meristem was sufficient for extraction of high-quality total RNA. The results obtained might enable advances in genetic analyses and molecular studies using reduced amount of plant material.
authors.affiliation: Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Estadual de Santa Cruz, Ilhéus, BA, Brasil.
authors.forename: D V
authors.initials: DV
authors.lastname: Silva
authors.affiliation: Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Estadual de Santa Cruz, Ilhéus, BA, Brasil.
authors.forename: S M J
authors.initials: SM
authors.lastname: Branco
authors.affiliation: Departamento de Ciências Vegetais, Universidade Federal Rural do Semi-Árido, Mossoró, RN, Brasil.
authors.forename: I S A
authors.initials: IS
authors.lastname: Holanda
authors.affiliation: Mars Center for Cocoa Science, Barro Preto, BA, Brasil.
authors.forename: S
authors.initials: S
authors.lastname: Royaert
authors.affiliation: MARS Inc. c/o USDA-ARS, Subtropical Horticulture Research Station, Miami, FL, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Motamayor
authors.affiliation: MARS Inc. c/o USDA-ARS, Subtropical Horticulture Research Station, Miami, FL, USA.
authors.forename: J P
authors.initials: JP
authors.lastname: Marelli
authors.affiliation: Departamento de Ciências Biológicas, Universidade Estadual de Santa Cruz, Ilhéus, BA, Brasil.
authors.forename: R X
authors.initials: RX
authors.lastname: Corrêa
mesh_terms: D002099:Cacao / Q000378:metabolism*; D005591:Chemical Fractionation / Q000379:methods*; D018076:DNA, Complementary; D018519:Meristem / Q000378:metabolism*; D012333:RNA, Messenger / Q000302:isolation & purification*
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger