A convenient and efficient protocol for isolating high-quality RNA from latex of Hevea brasiliensis (para rubber tree)

Isolating high-quality RNA from latex of H. brasiliensis is a prerequisite to elucidating the molecular mechanisms of rubber biosynthesis and its regulation. Here, an improved protocol was developed for latex collection, transportation, storage, and RNA isolation. Compared with existing ones, our protocol eliminated liquid nitrogen for latex collection and subsequent low-temperature (− 70 °C) condition for latex storage, making it more convenient and feasible when latex was collected in remote sampling sites, and latex storage and RNA isolation were conducted in poorly-equipped laboratories. Different methods (UV absorbance scans, denaturing gel electrophoresis, autoradiograph monitoring of cDNA synthesis) were used to confirm the high quality of the RNA prepared with this protocol, whose usefulness was further verified by several practical applications, including construction of one high-quality cDNA library, cloning of the full-length cDNAs of 3 novel Hevea sucrose transporter genes, and semi-quantitative RT-PCR analysis of two rubber-biosynthesis essential genes and one sucrose transporter gene.     

Resolving the intra-specific succession within Cochlodinium polykrikoides populations in southern Korean coastal waters via use of quantitative PCR assays

While the toxic dinoflagellate Cochlodinium polykrikoides is known to form blooms that are maintained for extended periods, the genetic differentiation of these blooms are currently unknown. To assess this, we developed a real-time PCR assay to quantify C. polykrikoides at the intra-specific level, and applied this assay to field samples collected in Korean coastal waters from summer through fall. Assays were successfully developed to target the large-subunit ribosomal RNA region of the three major ribotypes of C. polykrikoides: Philippines, East Asian, and American/Malaysian. Significant linear relationships (r² ≥ 0.995) were established between C_t and the log of the copy number for each ribotype qPCR assay. Using these assays, C. polykrikoides blooms in Korean coastal waters were found to be comprised of Philippines and East Asian ribotypes but not the American/Malaysian ribotype. The Philippines ribotype was found to be highly abundant during summer bloom initiation and peak, whereas the East Asian ribotype became the dominant ribotype in the fall. As such, this newly developed qPCR assay can be used to quantify the cryptic ecological succession of sub-populations of C. polykrikoides during blooms that light microscopy and previously developed qPCR assays cannot resolve.     

Identification of the dinoflagellate community during Cochlodinium polykrikoides (Dinophyceae) blooms using amplified rDNA melting curve analysis and real-time PCR probes

The dinoflagellate community present during blooms of the fish killing dinoflagellate Cochlodinium polykrikoides was characterized by DNA melting curve analysis and direct sequencing of the SSU rDNA amplified from environmental sample extracts. PCR amplification of genomic DNA from Gaedo water samples using dinoflagellate-specific SSU rDNA primers yielded 280 clones, which were screened by closed tube PCR-melting curve analysis targeting a region of the SSU rDNA, enabling high throughput analysis. Twenty-eight clones producing distinct melting curve patterns were sequenced, and their phylogenetic information revealed that C. polykrikoides co-occurred with morphologically similar species including Gymnodinium impudicum and Gymnodinium catenatum. Temporal variations of C. polykrikoides and G. impudicum abundances in South Sea were also examined by species-specific real-time TaqMan-based PCR probes developed in this study. C. polykrikoides- and G. impudicum-specific real-time PCR probes were designed targeting the internal transcribed spacer 2 ribosomal DNA region. The probe specificity was confirmed by testing against related dinoflagellates and verified by sequencing PCR products from environmental samples. The real-time PCR assays showed that C. polykrikoides cell densities peaked in August at 16,928 cells mL^?1, while G. impudicum was present at low abundances (below 25 cells mL^?1). Our amplified rDNA melting curve protocol provides a facile method for the characterization of the dinoflagellate community, and the real-time PCR assay could be an alternative method for rapid and sensitive enumeration of harmful dinoflagellates in the marine environment.     

Development of real-time RT-PCR for detecting viable Cochlodinium polykrikoides (Dinophyceae) cysts in sediment

Morphological observations have confirmed that cysts are produced by dinoflagellates. However, finding a seed bed or unknown cysts in field samples by microscopy is extremely time consuming. Real-time PCR has been used to facilitate the detection of dinoflagellate cysts in sediment. However, DNA from dead vegetative cells remaining on the surface sediment may persist for a long period of time, which can cause false positive DNA detection. In this study, a non-quantitative RNA targeted probe using real-time RT-PCR was developed for detection of viable cysts in sediment. Large-subunit rRNA was used to develop a species-specific RNA targeted probe for the ichthyotoxic dinoflagellate Cochlodinium polykrikoides. The sediment samples were sieved and incubated at 30 °C for 3 h prior to RNA extraction to remove RNA from dead cells remaining in the sediment. Nested-PCR was conducted to maximize assay sensitivity. A field survey to determine the distribution of cysts at 155 sampling stations in the western and southern part of the Korean peninsula showed that C. polykrikoides cysts were detected at five sampling stations.     

Post-Mortem Tissue Biopsies Obtained at Minimally Invasive Autopsy: An RNA-Quality Analysis

IntroductionBereaved relatives often refuse to give consent for post-mortem investigation of deceased cancer patients, mainly because of the mutilation due to conventional autopsy (CA). Minimally invasive autopsy (MIA) may be a more acceptable alternative and, if implemented in clinical practice, creates an opportunity to more often obtain post-mortem tissue samples of (recurred) primary tumors and metastases for molecular research. As a measure for tissue quality for molecular studies, we hereby present a feasibility study, comparing the RNA quality of MIA and CA samples, and fresh frozen samples as reference.ResultsRIN values in MIA samples were significantly higher than those in CA samples. GAPDH was expressed significantly higher in MIA samples than in CA samples and 530 bp PCR products could be measured in all cases. GAPDH expression was significantly lower in samples with PMI >15 hours. As expected, the samples of the fresh frozen reference standard performed best in all analyses.ConclusionMIA samples showed better RNA quality than CA samples, probably due to shorter PMI. Both had lower RNA quality and expression levels than fresh frozen tissue, however, remaining GAPDH RNA was still sufficiently intact. Therefore, other highly expressed genes are most likely also detectable. Gene array analysis should be performed to gain insight into the quality of entire post-mortem genomes. Reducing PMI will further improve the feasibility of demanding molecular research on post-mortem tissues, this is most likely more feasible with MIA than CA.     

Improved RNA isolation from Laminaria japonica Aresch (Laminariaceae, Phaeophyta)

RNA isolation is difficult in some plants and algae because phenolics, polysaccharides, or other compounds can bind or co-precipitate with RNA, and because the success of RNA isolation can be strain-specific and species-specific. To create an improved RNA isolation protocol for Laminaria japonica Aresch (Laminariaceae, Phaeophyta), four methods for extracting RNA were tested. A cetyltrimethylammonium bromide (CTAB)-based RNA extraction protocol was developed that clearly showed 28S and 18S ribosomal RNA bands and produced RNA with high yield (68 μg g⁻¹ fresh weight) and high quality (A _260/280 ratio 1.96 ± 0.05). The isolated RNA was intact, and RT-PCR analysis confirmed that further molecular application is feasible.     

Elimination of oxalate contamination in RNA isolation from<Emphasis Type="Italic">Rumex obtusifolius</Emphasis>

Many plant RNA isolation techniques aim to prevent contamination by means of secondary phenolics, carbohydrates, RNase, and other chemicals. However, when applied in our laboratory to the isolation of RNA fromRumex obtusifolius, these protocols failed to produce good quality RNA. A major problem was contamination of the RNA samples with the secondary metabolite oxalate. The relative quantities of guanidine isothiocyanate extraction buffer to plant tissue used in the protocol had significant effects on oxalate contamination. An increase in extraction buffer, from 1.5 mL in the original method to 15 mL per 200–300 mg of tissue in our protocol, removed the oxalate from the RNA. This RNA was of a good quality and was suitable for molecular biology applications.     

Improved real-time PCR method for quantification of the abundance of all known ribotypes of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides by comparing 4 different preparation methods

Red tides by the ichthyotoxic dinoflagellate Cochlodinium polykrikoides have caused large scaled mortality of fish and great loss in aquaculture industry in many countries. Detecting and quantifying the abundance of this species are the most critical step in minimizing the loss. The conventional quantitative real-time PCR (qPCR) method has been used for quantifying the abundance of this species. However, when analyzing > 500 samples collected during huge C. polykrikoides red tides in South Sea of Korea in 2014, this conventional method and the previously developed specific primer and probe set for C. polykrikoides did not give reasonable abundances when compared with cell counting data. Thus improved qPCR methods and a new specific primer and probe set reflecting recent discovery of 2 new ribotypes have to be developed. A new species-specific primer and probe set for detecting all 3 ribotypes of C. polykrikoides was developed and provided in this study. Furthermore, because the standard curve between cell abundance and threshold cycle value (Ct) is critical, the efficiencies of 4 different preparation methods used to determine standard curves were comparatively evaluated. The standard curves were determined by using the following 4 different preparations: (1) extraction of DNA from a dense culture of C. polykrikoides followed by serial dilution of the extracted DNA (CDD method), (2) extraction of DNA from each of the serially diluted cultures with different concentrations of C. polykrikoides cultures (CCD method), (3) extraction of DNA from a dense field sample of C. polykrikoides collected from natural seawater and then dilution of the extracted DNA in serial (FDD method), and (4) extraction of DNA from each of the serially diluted field samples having different concentrations of C. polykrikoides (FCD method). These 4 methods yielded different results. The abundances of C. polykrikoides in the samples collected from the coastal waters of South Sea, Korea, in 2014–2015, obtained using the standard curves determined by the CCD and the FCD methods, were the most similar (0.93–1.03 times) and the second closest (1.16–1.33 times) to the actual cell abundances obtained by enumeration of cells. Thus, our results suggest that the CCD method is a more effective tool to quantify the abundance of C. polykrikoides than the conventional method, CDD, and the FDD and FCD methods.     

Spermatozoa input concentrations and RNA isolation methods on RNA yield and quality in bull (Bos taurus)

Sperm RNA can be used to understand the past spermatogenic process, future successful fertilization, and embryo development. To study the sperm RNA composition and function, isolation of good quality RNA with sufficient quantity is essential. The objective of this study was to assess the influence of sperm input concentrations and RNA isolation methods on RNA yield and quality in bull sperm. The fresh semen samples from bulls (n = 6) were snap-frozen in liquid nitrogen and stored at −80 °C. The sperm RNA was isolated using membrane-based methods combined with TRIzol (RNeasy + TRIzol and PureLink + TRIzol) and conventional methods (TRIzol, Double TRIzol, and RNAzol RT). Based on fluorometric quantification, combined methods resulted in significantly (P < 0.05) higher total RNA yields (800–900 ng/30–40 × 10⁶) as compared with other methods and yielded 20 to 30 fg of RNA/spermatozoon. The quality of RNA isolated by membrane-based methods was superior to that isolated by conventional methods. The sperm RNA was observed to be intact as well as fragmented (50–2000 bp). The study revealed that the membrane-based methods with a cocktail of lysis solution and an optimal input concentration of 30 to 40 million sperm were optimal for maximum recovery of RNA from bull spermatozoa.     

Alteration of plankton communities and biogeochemical cycles by harmful Cochlodinium polykrikoides (Dinophyceae) blooms

Cochlodinium polykrikoides is a globally distributed, ichthyotoxic, bloom-forming dinoflagellate. Blooms of C. polykrikoides manifest themselves as large (many km²) and distinct patches with cell densities exceeding 10³ ml⁻¹ while water adjacent to these patches can have low cell densities (<100 cells ml⁻¹). While the effect of these blooms on fish and shellfish is well-known, their impacts on microbial communities and biogeochemical cycles are poorly understood. Here, we investigated plankton communities and the cycling of carbon, nitrogen, and B-vitamins within blooms of C. polykrikoides and compared them to areas in close proximity (<100 m) with low C. polykrikoides densities. Within blooms, C. polykrikoides represented more than 90% of microplankton (>20 μm) cells, and there were significantly more heterotrophic bacteria and picoeukaryotic phytoplankton but fewer Synechococcus. Terminal restriction fragment length polymorphism analysis of 16S and 18S rRNA genes revealed significant differences in community composition between bloom and non-bloom samples. Inside the bloom patches, concentrations of vitamin B₁₂ were significantly lower while concentrations of dissolved oxygen were significantly higher. Carbon fixation and nitrogen uptake rates were up to ten times higher within C. polykrikoides bloom patches. Ammonium was a more important source of nitrogen, relative to nitrate and urea, for microplankton within bloom patches compared to non-bloom communities. While uptake rates of vitamin B₁ were similar in bloom and non-bloom samples, vitamin B₁₂ was taken up at rates five-fold higher (>100 pmol⁻¹ L⁻¹ d⁻¹) in bloom samples, resulting in turn-over times of hours during blooms. This high vitamin demand likely led to the vitamin B₁₂ limitation of C. polykrikoides observed during nutrient amendment experiments conducted with bloom water. Collectively, this study revealed that C. polykrikoides blooms fundamentally change microbial communities and accelerate the cycling of carbon, some nutrients, and vitamin B₁₂.     

A reliable and efficient method for total rna isolation from various members of spurge family (Euphorbiaceae)

Introduction – It is prerequisite and crucial to extract RNA with high quality and integrity in order to carry out molecular biology studies in any plant species of a family. Euphorbiaceae members are known for high levels of their waxes, oils with polysaccharides, polyphenolics and secondary metabolites. These conditions are recognised to interfere unfavourably with various methodologies of RNA isolation. Objective – To develop a simple, rapid and reproducible cetyltrimethylamonium bromide (CTAB)‐based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA extracted from various recalcitrant Euphorbiaceae member plant tissues such as from tree leaves (Hevea brasilensis), woody shrubs leaves (Ricinus communis, Jatropha curcas, Manihot esculenta) and storage root tissue (M. esculenta). Methodology – Simple modifications and fast steps were introduced to the original CTAB protocol. All centrifugation steps were carried out at 4°C at 12000 rpm for 10 min, the sample weight was decreased and usage of spermidine and LiCl was omitted, reducing incubation time prior to RNA precipitation. This rapid CTAB protocol was compared with various RNA isolation methods intended for use with plants rich in polysaccharides and secondary metabolites. Results – The procedure can be completed within 2 h and many samples can be processed at the same time. RNA of high quality could be isolated from all the tissues of species that we tried. The isolated RNA from different species served as a robust template for RT‐PCR analysis. Conclusion – The study has shown that the improvement of a CTAB‐based protocol allows the rapid isolation of high‐quality RNA from various recalcitrant Euphorbiaceae members. Copyright © 2010 John Wiley & Sons, Ltd.     

Mapping the Distribution of Cysts from the Toxic Dinoflagellate Cochlodinium polykrikoides in Bloom-Prone Estuaries by a Novel Fluorescence In Situ Hybridization Assay

Cochlodinium polykrikoides is a cosmopolitan dinoflagellate that is notorious for causing fish-killing harmful algal blooms (HABs) across North America and Asia. While recent laboratory and ecosystem studies have definitively demonstrated that Cochlodinium forms resting cysts that may play a key role in the dynamics of its HABs, uncertainties regarding cyst morphology and detection have prohibited even a rudimentary understanding of the distribution of C. polykrikoides cysts in coastal ecosystems. Here, we report on the development of a fluorescence in situ hybridization (FISH) assay using oligonucleotide probes specific for the large subunit (LSU) ribosomal DNA (rDNA) of C. polykrikoides. The LSU rDNA-targeted FISH assay was used with epifluorescence microscopy and was iteratively refined to maximize the fluorescent reaction with C. polykrikoides and minimize cross-reactivity. The final LSU rDNA-targeted FISH assay was found to quantitatively recover cysts made by North American isolates of C. polykrikoides but not cysts formed by other common cyst-forming dinoflagellates. The method was then applied to identify and map C. polykrikoides cysts across bloom-prone estuaries. Annual cyst and vegetative cell surveys revealed that elevated densities of C. polykrikoides cysts (>100 cm⁻³) during the spring of a given year were spatially consistent with regions of dense blooms the prior summer. The identity of cysts in sediments was confirmed via independent amplification of C. polykrikoides rDNA. This study mapped C. polykrikoides cysts in a natural marine setting and indicates that the excystment of cysts formed by this harmful alga may play a key role in the development of HABs of this species.     

Control of ichthyotoxic Cochlodinium polykrikoides using the mixotrophic dinoflagellate Alexandrium pohangense: A potential effective sustainable method

Red tides dominated by Cochlodinium polykrikoides often lead to great economic losses and some methods of controlling these red tides have been developed. However, due to possible adverse effects and the short persistence of their control actions, safer and more effective sustainable methods should be developed. The non-toxic dinoflagellate Alexandrium pohangense is known to grow well mixotrophically feeding on C. polykrikoides, and populations are also maintained by photosynthesis. Thus, compared with other methods, the use of mass-cultured A. pohangense is safer and the effects can be maintained in the long term. To develop an effective method, the concentrations of A. pohangense cells and culture filtrate resulting in the death of C. polykrikoides cells were determined by adding the cells or filtrates to cultured and natural populations of C. polykrikoides. Cultures containing 800 A. pohangense cells ml⁻¹ eliminated almost all cultured C. polykrikoides cells at a concentration of 1000 cells ml⁻¹ within 24 h. Furthermore, the addition of A. pohangense cultures at a concentration of 800 cells ml⁻¹ to C. polykrikoides populations from a red-tide patch resulted in the death of most C. polykrikoides cells (99.8%) within 24 h. This addition of A. pohangense cells also lowered the abundances of total phototrophic dinoflagellates excluding C. polykrikoides, but did not lower the abundance of total diatoms. Filtrate from 800 cells ml⁻¹ A. pohangense cultures reduced the population of cultured C. polykrikoides by 80% within 48 h. This suggests that A. pohangense cells eliminate C. polykrikoides by feeding and releasing extracellular compounds. Over time, A. pohangense concentrations gradually increased when incubated with C. polykrikoides. Thus, an increase in the concentration of A. pohangense by feeding may lead to A. pohangense cells eliminating more C. polykrikoides cells in larger volumes. Based on the results of this study, a 1 m³ stock culture of A. pohangense at 4000 cells ml⁻¹ is calculated to remove all C. polykrikoides cells in ca. 200 m³ within 6 days. Furthermore, maintenance of A. pohangense populations through photosynthesis prepared A. pohangense to eliminate C. polykrikoides cells in future red-tide patches. Moreover, incubation of A. pohangense at 2000 cells ml⁻¹ with juvenile olive flounder Paralichthys olivaceus for 3 days did not result in the death of fish. Therefore, the method developed in this study is a safe and effective way of controlling C. polykrikoides populations and can be easily applied to aqua-tanks on land.     

Preservation of algal and higher plant ribosomal RNA integrity during extraction and electrophoretic quantitation

Isolation of high molecular weight ribosomal RNA from the wall-less alga Olisthodiscus luteus and the angiospermous plant Sauromatum guttatum is described. It has been found that a buffer which contains magnesium must be used to successfully isolate Olisthodiscus rRNA whereas the isolation of intact Sauromatum rRNA requires a buffer system containing a high amount of the chelator EDTA, Sauromatum but not Olisthodiseus extracts were contaminated with ribonuclease unless the inhibitor diethylpyrocarbonate was used during the ribonucleic acid extraction procedure. Nuclease levels were monitored by coincubating [³H]-labeled Escherichia coli ribosomal RNA with the experimental RNA samples. The effects of detergents on the isolation and quantitation of RNA are presented, and methods to avoid loss of highly thermolabile plant ribosomal RNA species are discussed.     

Dynamics of bacterial community structure during blooms of Cochlodinium polykrikoides (Gymnodiniales,Dinophyceae) in Korean coastal waters

Recent studies of dinoflagellates have reported that blooms can be closely related to the characteristics of the associated bacteria, but studies of the correlation between the toxic dinoflagellate, Cochlodinium polykrikoides and their associated bacterial community composition has not been explored. To understand this correlation, changes in bacterial community structure through the evolution of a C. polykrikoides bloom in Korean coastal waters via clone library analysis were investigated. Although there were no apparent changes in physio-chemical factors during the onset of the C. polykrikoides bloom, the abundance of bacteria bourgeoned in parallel with C. polykrikoides densities. Alpha-, gamma-proteobacteria and Flavobacteria were found to be dominant phyletic groups during C. polykrikoides blooms. The proportion of gamma-proteobacteria was lower (11.8%) during peak of the bloom period compared to the post-bloom period (26.2%). In contrast, alpha-proteobacteria increased in dominance during blooms. Among the alpha-proteobacteria, members of Rhodobacterales abruptly increased from 38% of the alpha-proteobacteria before the bloom to 74% and 56% during the early bloom and peak bloom stages, respectively. Moreover, multiple sites concurrently hosting C. polykrikoides blooms also contained high portions of Rhodobacterales and principal component analysis (PCA) demonstrated that Rhodobacterales had a positive, significant correlation with C. polykrikoides abundances (p ≤ 0.01, Pearson correlation coefficients). Collectively, this study reveals the specific clades of bacteria that increase (Rhodobacterales) and decrease (gamma-proteobacteria) in abundance C. polykrikoides during blooms.     

Controls on the initiation and development of blooms of the dinoflagellate Cochlodinium polykrikoides Margalef in lower Chesapeake Bay and its tributaries

Massive blooms of the dinoflagellate Cochlodinium polykrikoides occur annually in the Chesapeake Bay and its tributaries. The initiation of blooms and their physical transport has been documented and the location of bloom initiation was identified during the 2007 and 2008 blooms. In the present study we combined daily sampling of nutrient concentrations and phytoplankton abundance at a fixed station to determine physical and chemical controls on bloom formation and enhanced underway water quality monitoring (DATAFLOW) during periods when blooms are known to occur. While C. polykrikoides did not reach bloom concentrations until late June during 2009, vegetative cells were present at low concentrations in the Elizabeth River (4 cells ml⁻¹) as early as May 27. Subsequent samples collected from the Lafayette River documented the increase in C. polykrikoides abundance in the upper branches of the Lafayette River from mid-June to early July, when discolored waters were first observed. The 2009 C. polykrikoides bloom began in the Lafayette River when water temperatures were consistently above 25 °C and during a period of calm winds, neap tides, high positive tidal residuals, low nutrient concentrations, and a low dissolved inorganic nitrogen (DIN) to dissolved inorganic phosphorous (DIP) ratio. The pulsing of nutrients associated with intense but highly localized storm activity during the summer months when water temperatures are above 25 °C may play a role in the initiation of C. polykrikoides blooms. The upper Lafayette River appears to be an important area for initiation of algal blooms that then spread to other connected waterways.