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Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex Embryophyta.

ABSTRACT: Here we present a series of protocols for RNA extraction across a diverse array of plants; we focus on woody, aromatic, aquatic, and other chemically complex taxa. Ninety-one taxa were subjected to RNA extraction with three methods presented here: (1) TRIzol/TURBO DNA-free kits using the manufacturer's protocol with the addition of sarkosyl; (2) a combination method using cetyltrimethylammonium bromide (CTAB) and TRIzol/sarkosyl/TURBO DNA-free; and (3) a combination of CTAB and QIAGEN RNeasy Plant Mini Kit. Bench-ready protocols are given. After an iterative process of working with chemically complex taxa, we conclude that the use of TRIzol supplemented with sarkosyl and the TURBO DNA-free kit is an effective, efficient, and robust method for obtaining RNA from 100 mg of leaf tissue of land plant species (Embryophyta) examined. Our protocols can be used to provide RNA of suitable stability, quantity, and quality for transcriptome sequencing.

SUBMITTER: Jordon-Thaden IE

PROVIDER: S-EPMC4435465 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex Embryophyta.

Jordon-Thaden Ingrid E IE Chanderbali Andre S AS Gitzendanner Matthew A MA Soltis Douglas E DE

Applications in plant sciences 20150513 5

<h4>Premise of the study</h4>Here we present a series of protocols for RNA extraction across a diverse array of plants; we focus on woody, aromatic, aquatic, and other chemically complex taxa.<h4>Methods and results</h4>Ninety-one taxa were subjected to RNA extraction with three methods presented here: (1) TRIzol/TURBO DNA-free kits using the manufacturer's protocol with the addition of sarkosyl; (2) a combination method using cetyltrimethylammonium bromide (CTAB) and TRIzol/sarkosyl/TURBO DNA-f ...[more]

PMID: 25995975

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Project description:The usage of cladocerans as non-model organisms in ecotoxicological and risk assessment studies has intensified in recent years due to their ecological importance in aquatic ecosystems. The molecular assessment such as gene expression analysis has been introduced in ecotoxicological and risk assessment to link the expression of specific genes to a biological process in the cladocerans. The validity and accuracy of gene expression analysis depends on the quantity, quality and integrity of extracted ribonucleic acid (RNA) of the sample. However, the standard methods of RNA extraction from the cladocerans are still lacking. This study evaluates the extraction of RNA from tropical freshwater cladocerans Moina micrura using two methods: the phenol-chloroform extraction method (QIAzol) and a column-based kit (Qiagen Micro Kit). Glycogen was introduced in both approaches to enhance the recovery of extracted RNA and the extracted RNA was characterised using spectrophotometric analysis (NanoDrop), capillary electrophoresis (Bioanalyzer). Then, the extracted RNA was analysed with reverse transcription polymerase chain reaction (RT-PCR) to validate the RNA extraction method towards downstream gene expression analysis. The results indicate that the column-based kit is most suitable for the extraction of RNA from M. micrura, with the quantity (RNA concentration = 26.90 ± 6.89 ng/μl), quality (A260:230 = 1.95 ± 0.15, A280:230 = 1.85 ± 0.09) and integrity (RNA integrity number, RIN = 7.20 ± 0.16). The RT-PCR analysis shows that the method successfully amplified both alpha tubulin and actin gene at 33-35 cycles (i.e. Ct = 32.64 to 33.48). The results demonstrate that the addition of glycogen is only suitable for the phenol-chloroform extraction method. RNA extraction with high and comprehensive quality control assessment will increase the accuracy and reliability of downstream gene expression, thus providing more ecotoxicological data at the molecular biological level on other freshwater zooplankton species.

Dataset Information

Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex Embryophyta.

Publications

Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex Embryophyta.

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