A novel, simplified technique for preservation and rapid isolation of total RNA from the toxic dinoflagellate<i>Alexandrium catenella</i>(Dinophyceae)

Harlow, Lucy D.; Koutoulis, Anthony; Hallegraeff, Gustaaf M.

doi:10.2216/05-29.1

Cited by 14 publications

(9 citation statements)

References 34 publications

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“…Several cell disruption methods have been tested (bead beating, grinding using a micropestle and sonication) against dinoflagellates Alexandrium catenella [46]. Cell disruption using micropestle were reported the most efficient method to disrupt the dinoflagellates cells.…”

Section: Isolation Of Rnamentioning

confidence: 99%

“…However, a recent report demonstrated that mRNA degradation may occur rapidly with some genes start to degrade as early as 2 min [76]. Utilizing RNA stabilization solution such as RNAlater and RNAlater ICE seems effective to stop the dinoflagellates gene expression without compromising RNA integrity [46].…”

Section: Challenges and Consideration In Dinoflagellates Transcriptommentioning

confidence: 99%

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RNA-Seq as an Emerging Tool for Marine Dinoflagellate Transcriptome Analysis: Process and Challenges

et al. 2018

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Abstract:Dinoflagellates are the large group of marine phytoplankton with primary studies interest regarding their symbiosis with coral reef and the abilities to form harmful algae blooms (HABs). Toxin produced by dinoflagellates during events of HABs cause severe negative impact both in the economy and health sector. However, attempts to understand the dinoflagellates genomic features are hindered by their complex genome organization. Transcriptomics have been employed to understand dinoflagellates genome structure, profile genes and gene expression. RNA-seq is one of the latest methods for transcriptomics study. This method is capable of profiling the dinoflagellates transcriptomes and has several advantages, including highly sensitive, cost effective and deeper sequence coverage. Thus, in this review paper, the current workflow of dinoflagellates RNA-seq starts with the extraction of high quality RNA and is followed by cDNA sequencing using the next-generation sequencing platform, dinoflagellates transcriptome assembly and computational analysis will be discussed. Certain consideration needs will be highlighted such as difficulty in dinoflagellates sequence annotation, post-transcriptional activity and the effect of RNA pooling when using RNA-seq.

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Section: Isolation Of Rnamentioning

confidence: 99%

Section: Challenges and Consideration In Dinoflagellates Transcriptommentioning

confidence: 99%

RNA-Seq as an Emerging Tool for Marine Dinoflagellate Transcriptome Analysis: Process and Challenges

et al. 2018

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“…2). Various approaches for cell homogenizations were tested here as previously used for different dinoflagellate species such as vortexing (Lin et al 2002), micropestle (Harlow et al 2006), or glass beads (Takishita et al 2003). Interestingly, the highest yield was obtained when applying vigorous cell homogenization with glass beads using the bead beater at a speed of 4,500 rpm for 1.5 to 6 min ( Table 2).…”

Section: Rna Extraction and Rna Qualitymentioning

confidence: 99%

“…), total RNA was isolated using TriReagent (Karako-Lampert et al 2006), then Stratagene RNA RT-PCR Miniprep kit and glass beads for homogenization step (Takishita et al 2003) or Qiagen RNeasy Plant Mini-kit (Takahashi et al 2008). In toxic dinoflagellate Alexandrium catenella, RNA isolation was carried out using Qiagen column-based RNeasy Plant Minikit and cell disruption by grinding in a micropestle (Harlow et al 2006). Consequently, the objectives of this work were to evaluate various RNA extraction methods with respect to RNA recovery and RNA quality and to explore their use in downstream applications such as complementary DNA (cDNA) synthesis, reverse transcriptase polymerase chain reaction (RT-PCR) and qPCR.…”

Section: Introductionmentioning

confidence: 99%

A method for extracting a high-quality RNA from Symbiodinium sp.

Rosic

Høegh-Guldberg

2009

J Appl Phycol

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Good quality RNA is essential for a range of analyses including microarray and gene expression studies. A number of methods for RNA extraction from symbiotic dinoflagellates were assessed for their ability to recover a high-quality RNA applicable for evaluation of gene expression profiles. The recovery and quality of the obtained RNA were evaluated with respect to UV light absorbance profiles and automated microcapillary electrophoretic RNA separation. A modified RNA extraction procedure that combines two existing commercial kits, Trizol and Qiagen RNeasy kits, was efficiently employed for the recovery of a high-quality RNA under specific homogenization conditions. Cell homogenization using glass beads at the speed of 4,500 rpm for up to 6 min resulted in a good RNA recovery and preserved RNA integrity. A high-quality RNA obtained following the described procedure was successfully applied in reverse transcriptase-polymerase chain reaction (PCR) and in quantitative PCR studies. Gene expression profiles were changed with RNA extraction procedure, with the highest transcript numbers at precise conditions of cell homogenization. RNA samples with RNA integrity number values from 6 and above were recommended for downstream applications. This sequence of RNA isolation and RNA evaluation represents a methodological improvement required for functional genomic studies in dinoflagellates.

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“…This supersaturated ammonium sulfate solution has been shown to adequately preserve both RNA and DNA for lengthy periods of time (from 6 weeks to 4 months) in a variety of animal tissues as well as whole, relatively small organisms, such as arachnids (Vink et al 2005), bacteria (Bachoon et al 2001), and zooplankton (Gorokhova 2005). RNAlater has consequently gained popularity as a preservation method for nucleic acid analyses (e.g., Harlow et al 2006;Höök et al 2008;MacLean et al 2008).…”

mentioning

confidence: 99%

Length Reduction of Larval Yellow Perch and Freshwater Amphipods in RNAlater Solution

Foley

Ryan

Höök

2010

N American J Fish Manag

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Use of RNAlater (Ambion, Inc.) solution to preserve whole organisms for nucleic acid analysis is gaining popularity, in part because it effectively preserves both RNA and DNA without the need for freezing the organism. Similar preservatives have been shown to cause short‐term changes in animal length, and such changes must be accounted for as analyses progress. We examined the rates and degrees of shrinkage for freshwater amphipods Diporeia spp. and larval yellow perch Perca flavescens after preservation in RNAlater solution in comparison with more common preservatives. We found that after 2 months of preservation, Diporeia shrank by 3% in RNAlater versus gaining length by 5% in diluted ethanol and by 1% in diluted formaldehyde. After 5.5 months of preservation, larval yellow perch lengths decreased by 23% in RNAlater, 10% in diluted ethanol, 12% in diluted formaldehyde, and 19% when frozen in water. Overall length reduction for Diporeia and larval yellow perch was significantly greater in RNAlater than in ethanol or formalin. Since accurate measures of length are necessary for many nucleic acid analyses, researchers utilizing RNAlater solution should attempt to measure organisms while they are alive or should develop preservation‐specific correction functions to overcome biases associated with shrinkage.

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A novel, simplified technique for preservation and rapid isolation of total RNA from the toxic dinoflagellateAlexandrium catenella(Dinophyceae)

Cited by 14 publications

References 34 publications

RNA-Seq as an Emerging Tool for Marine Dinoflagellate Transcriptome Analysis: Process and Challenges

RNA-Seq as an Emerging Tool for Marine Dinoflagellate Transcriptome Analysis: Process and Challenges

A method for extracting a high-quality RNA from Symbiodinium sp.

Length Reduction of Larval Yellow Perch and Freshwater Amphipods in RNAlater Solution

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