Efficacy of long-term coral tissue storage in ethanol for genotyping studies

With climate change threatening the future of coral reefs, there is an urgent need for effective coral tissue preservation and repositories from which DNA can be extracted. Most collections use 95 % ethanol as the storage medium, but its efficacy for long-term storage for short-fragment DNA use remains poorly documented. We conducted an accelerated DNA aging trial on three species of coral to ascertain whether ethanol-stored tissue and skeleton samples could yield fit-for-purpose DNA at time scales of 100+ yrs. We conclude that even using a crude DNA extraction technique, samples kept at 40 °C for 20 months yielded DNA of sufficient quality for Symbiodinium and coral host genotyping. If stored at ?20 °C, these samples are likely to still yield useable DNA after 100 yrs. Ethanol-stored samples compared favorably in terms of DNA quality, quantity and sample integrity with those stored in an analogue of the commercial storage buffer RNAlater ^®.     

Impact of tissue preservation on collagen fiber architecture

Microarchitectural features of collagen-rich extracellular matrices provide the mechanical foundation for tissue function and exhibit topographical cues that influence cellular behavior including proliferation, migration and protein expression. Preservation of tissue microarchitecture is required for accurate evaluation of tissue characteristics and pathology. It is unclear whether common tissue preservation methods possess equal ability to preserve microarchitecture. We investigated collagen microarchitecture in samples that had been flash frozen, fixed in formalin or preserved in RNAlater®, and which contained both collagen-rich and collagen-sparse regions. Fibrillar collagen organization was characterized using picrosirius red staining and second harmonic generation (SHG) microscopy. Maintenance of collagen fiber characteristics compared to the gold standard of flash freezing depended on both the method of preservation and the local collagen content of the tissue. Both formalin fixation and RNAlater® preserved collagen fiber characteristics similar to flash freezing in collagen-rich areas of the tissue, but not in collagen-sparse regions. Analysis using picrosirius red staining indicated preservation-dependent changes in overall tissue architecture and suprafibrillar organization. Together with considerations of cost, ease of use, storage conditions and ability to use the preserved tissue for RNA or protein analysis, our quantitative characterization of the effects of preservation method on collagen microarchitecture may help investigators select the most appropriate preservation approach for their needs.     

Preservation of RNA and DNA from mammal samples under field conditions

Ecological and conservation genetics require sampling of organisms in the wild. Appropriate preservation of the collected samples, usually by cryostorage, is key to the quality of the genetic data obtained. Nevertheless, cryopreservation in the field to ensure RNA and DNA stability is not always possible. We compared several nucleic acid preservation solutions appropriate for field sampling and tested them on rat (Rattus rattus) blood, ear and tail tip, liver, brain and muscle. We compared the efficacy of a nucleic acid preservation (NAP) buffer for DNA preservation against 95% ethanol and Longmire buffer, and for RNA preservation against RNAlater (Qiagen) and Longmire buffer, under simulated field conditions. For DNA, the NAP buffer was slightly better than cryopreservation or 95% ethanol, but high molecular weight DNA was preserved in all conditions. The NAP buffer preserved RNA as well as RNAlater. Liver yielded the best RNA and DNA quantity and quality; thus, liver should be the tissue preferentially collected from euthanized animals. We also show that DNA persists in nonpreserved muscle tissue for at least 1 week at ambient temperature, although degradation is noticeable in a matter of hours. When cryopreservation is not possible, the NAP buffer is an economical alternative for RNA preservation at ambient temperature for at least 2 months and DNA preservation for at least 10 months.     

Assessment of sample handling practices on microbial activity in sputum samples from patients with cystic fibrosis

Aim: The aim of this study was to quantitatively and qualitatively assess the effect of sample storage on the metabolically active microbial community found in sputum samples from patients with cystic fibrosis (CF). Methods: Sputum samples were collected and split in two equal aliquots one of which was immersed in RNAlater and refrigerated immediately, the second stored at room temperature for 24 h and RNAlater was subsequently added. mRNA was extracted, and RT‐PCR‐DGGE and qPCR analysis of the bacterial and fungal communities was carried out. Results: Significant differences in the bacterial communities between the two protocols were observed but there were no significant difference seen in the fungal community analyses. Analysis by qPCR demonstrated that room temperature storage gave statistically significant increases in eubacteria and Pseudomonas spp. and a statistically significant decrease in those of Haemophilus influenzae. Conclusions: The analysis of metabolically active microbial communities from CF sputum using molecular techniques indicated that samples should be stored at 4°C upon addition of RNAlater to obtain an accurate depiction of the CF lung microbiota. Also, storing respiratory samples at room temperature may cause an over representation of Pseudomonas aeruginosa and mask the presence of other clinically significant organisms.     

Method Optimization for Extracting High-Quality RNA From the Human Pancreas Tissue

Nucleic acid sequencing is frequently used to determine the molecular basis of diseases. Therefore, proper storage of biological specimens is essential to inhibit nucleic acid degradation. RNA isolated from the human pancreas is generally of poor quality because of its high concentration of endogenous RNase. In this study, we optimized the method for extracting high quality RNA from paired tumor and normal pancreatic tissues obtained from eight pancreatic cancer patients post-surgery. RNA integrity number (RIN) was checked to evaluate the integrity of RNA, we tried to extract the RNA with an RIN value of 8 or higher that allows for the latest genetic analysis. The effect of several parameters, including the method used for tissue lysis, RNAlater treatment, tissue weight at storage, and the time to storage after surgical resection, on the quantity and quality of RNA extracted was examined. Data showed that the highest quantity of RNA was isolated using a combination of manual and mechanical methods of tissue lysis. Additionally, sectioning the tissues into small pieces (<100 mg) and treating them with RNAlater solution prior to storage increased RNA stability. Following these guidelines, high quality RNA was obtained from 100% (8/8) of tumor tissues and 75% (6/8) of normal tissues. High-quality RNA was still stable under repeated freezing and thawing. The application of these results during sample handling and storage in clinical settings will facilitate the genetic diagnosis of diseases and their subsequent treatment.     

Dynamics of organic acid occurrence under flooding stress in the rhizosphere of three plant species from the water fluctuation zone of the Three Gorges Reservoir, P.R. China

The effects of flooding on rhizospheric organic acid concentrations of three abundant flooding tolerant plant species (Alternanthera philoxeroides Mart., Arundinella anomala Steud., Salix variegata Franch.) from the water fluctuation zone of the Three Gorges Reservoir (TGR, Yangtze River) were investigated. Soil solution samples of eight low molecular weight organic acids were obtained from rhizotrons using micro suction cups during 3 weeks of waterlogging, after 6 weeks flooding and after a 1 week recovery. To estimate the contribution of water temperature and microbial community, plants in sterile glass bead substrate and original Yangtze sediment were submerged in laboratory at +10°, +20° and +30°C. Waterlogged plants did seldom express a significantly different pattern of rhizospheric organic acid (OA) composition compared to control plants. Flooding caused no burst of organic acid concentration in soil solution: All species express a silencing strategy. Average OA levels were higher in A. anomala rhizosphere than in the other two species, but increased again after resurfacing in all species. Temperature had a stronger influence in sediment than in sterile setup. In contrast to field measurements, succinate, malate and citrate were detected in the sterile setup. Microbial contribution appeared to have great influence on increasing OA occurrence.     

Preservation of nucleic acid integrity in guanidine thiocyanate lysates of whole blood

High-molecular-mass RNA and DNA have been shown to retain their integrity for three days at room temperature, no less than two weeks at +4°C, and more than a year at ?20°C when whole blood samples are stored as lysates containing 4 M guanidine thiocyanate. Storage time at room temperature can be prolonged at least up to 14 days if nucleic acids were precipitated by two volumes of isopropanol. This preservation technique allows storage and transportation of samples at ambient temperature and is completely compatible with the procedure of subsequent isolation of nucleic acids.     

Bacterial Diversity and Bioprospecting for Cold-Active Lipases,Amylases and Proteases,from Culturable Bacteria of Kongsfjorden and Ny-Ålesund,Svalbard, Arctic

Culturable bacterial diversity of seven marine sediment samples of Kongsfjorden and a sediment and a soil sample from Ny-Ålesund, Svalbard, Arctic was studied. The bacterial abundance in the marine sediments of Kongsfjorden varied marginally (0.5 × 10³–1.3 × 10⁴ cfu/g sediment) and the bacterial number in the two samples collected from the shore of Ny-Ålesund also was very similar (0.6 × 10⁴ and 3.4 × 10⁴, respectively). From the nine samples a total of 103 bacterial isolates were obtained and these isolates could be grouped in to 47 phylotypes based on the 16S rRNA gene sequence belonging to 4 phyla namely Actinobacteria, Bacilli, Bacteroidetes and Proteobacteria. Representatives of the 47 phylotypes varied in their growth temperature range (4–37°C), in their tolerance to NaCl (0.3–2 M NaCl) and growth pH range (2–11). Representatives of 26 phylotypes exhibited amylase and lipase activity either at 5 or 20°C or at both the temperatures. A few of the representatives exhibited amylase and/or lipase activity only at 5°C. None of the phylotypes exhibited protease activity. Most of the phylotypes (38) were pigmented. Fatty acid profile studies indicated that short chain fatty acids, unsaturated fatty acids, branched fatty acids, the cyclic and the cis fatty acids are predominant in the psychrophilic bacteria.     

Effects of storage type and time on DNA amplification success in tropical ungulate faeces

The present study compares the effect of three storage media (silica, RNAlater®, ethanol) and time to extraction (1 week, 1 month and 3 months) on mitochondrial and nuclear marker amplification success in faecal DNA extracts from a sympatric community of small to medium‐sized Central African forest ungulates (genera Cephalophus, Tragelaphus, Hyemoschus). The effect of storage type and time on nuclear DNA concentrations, genotyping errors and percentage recovery of consensus genotypes was also examined. Regardless of storage method, mitochondrial and nuclear amplification success was high in DNA extracted within the first week after collection. Over longer storage periods, RNAlater yielded better amplification success rates in the mitochondrial assay. However, samples stored on silica showed (i) highest nuclear DNA concentrations, (ii) best microsatellite genotyping success, (iii) lowest genotyping errors, and (iv) greatest percentage recovery of the consensus genotype. The quantity of nuclear DNA was generally a good predictor of microsatellite performance with 83% amplification success or greater achieved with sample DNA concentrations of ≥ 50 pg/µL. If faecal DNA samples are to be used for nuclear microsatellite analyses, we recommend silica as the best storage method. However, for maximum mitochondrial amplification success, RNAlater appears to be the best storage medium. In contrast, ethanol appeared inferior to the other two methods examined here and should not be used to store tropical ungulate faeces. Regardless of storage method, samples should be extracted as soon as possible after collection to ensure optimal recovery of DNA.     

An effective method for isolation of DNA from pig faeces and comparison of five different methods

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and β-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp^® DNA Stool Mini Kit.     

Isolation,selection, and biological characterization research of highly effective electricigens from MFCs for phenol degradation

The microbial fuel cells (MFCs) are recognized to be highly effective for the biodegradation of phenol. For isolating the phenol-degrading bacteria, the sample containing 500 mg/L phenol was collected from the MFCs. The strain (WL027) was identified basing on the 16S rRNA gene analysis and phylogenetic analysis as Bacillus cereus. The effects of pH, temperature, concentrations of phenol, heavy metal ions, and salt on the growth of strain as well as the degradation of phenol have been carefully studied. The WL027-strain exhibited favorable tolerance for the metal cations including Cr²⁺, Co²⁺, Pb²⁺, and Cu²⁺ with the concentration of 0.2 mg/L and NaCl solution with a high concentration of 30 g/L. In 41 h, 86.44% of 500 mg/L phenol has been degraded at the initial pH at 6 and the temperature of 30 °C. The strain was highly active electrogenesis bacteria and the coulombic efficiency reached 64.25%, which showed significant advantage on the efficient energy conversion. Therefore, due to the highly efficient degradation of phenol, WL027-strain could be used in the treatment of phenol-containing wastewater.     

Screening of a beta-cypermethrin-degrading bacterial strain <Emphasis Type="Italic">Brevibacillus parabrevis</Emphasis> BCP-09 and its biochemical degradation pathway

A novel beta-cypermethrin (Beta-CP)-degrading strain isolated from activated sludge was identified as Brevibacillus parabrevis BCP-09 based on its morphological and physio-biochemical characteristics, and 16S rRNA gene analysis. Strain BCP-09 could effectively degrade Beta-CP at pH 5.0–9.0, 20–40 °C, and 10–500 mg L^?1 Beta-CP. Under optimal conditions (pH 7.41, 38.9 °C, 30.9 mg L^?1 Beta-CP), 75.87% Beta-CP was degraded within 3 days. Beta-CP degradation (half-life, 33.45 h) and strain BCP-09 growth were respectively described using first-order-kinetic and logistic-kinetic models. Seven metabolites were detected by high-performance liquid chromatography and gas chromatography-mass spectrometry- methyl salicylate, catechol, phthalic acid, salicylic acid, 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-phenoxybenzaldehyde, and 3-phenoxybenzoic acid (3-PBA). The major Beta-CP metabolite, 3-PBA was further degraded into phenol, benzoic acid, and 4-methylhexanoic acid. BCP-09 also degraded aromatic compounds such as phenol, catechol, and protocatechuic acid. Beta-CP appears to be mainly degraded into 3-PBA, which is continuously degraded into smaller benzene or chain compounds. Thus, strain BCP-09 could form a complete degradation system for Beta-CP and might be considered a promising strain for application in the bioremediation of environments and agricultural products polluted by Beta-CP.     

Modeling studies on mono and binary component biosorption of phenol and cyanide from aqueous solution onto activated carbon derived from saw dust

Biosorption is an effective treatment method for the removal of phenol and cyanide from aqueous solution by saw dust activated carbon (SDAC). Batch experiments were achieved as a function of several experimental parameters, i.e. influence of biosorbent dose (5–60 g/L) contact time (2–40 h), pH (4–12), initial phenol concentration (100–1000 mg/L) and initial cyanide concentration (10–100 mg/L) and temperature (20–40 °C). The biosorption capacities of the biosorbent were detected as 178.85 mg/g for phenol with 300 mg/L of initial concentration and 0.82 mg/g for cyanide with 30 mg/L of initial concentration. The optimum pH is found to be 8 for phenol and 9 for cyanide biosorption. The mono component biosorption equilibrium data for both phenol and cyanide were well defined by Redlich–Peterson model and binary component adsorption equilibrium data well fitted by extended Freundlich model. The percentage removal of phenol and cyanide using SDAC was 66.67% and 73.33%, respectively. Equilibrium established within 30 h for phenol and 28 h for cyanide. Kinetic studies revealed that biosorption of phenol followed pseudo second order indicating adsorption through chemisorption and cyanide followed pseudo first order kinetic model indicating adsorption through physisorption. Thermodynamic studies parameters, i.e., enthalpy (Δh₀), entropy (ΔS₀) and Gibb’s free energy (ΔG₀) have also been considered for the system. Thermodynamic modeling studies revealed that the process of cyanide biosorption was endothermic and phenol biosorption was exothermic in nature.     

Use of RNAlater® as a preservation method for parasitic coprology studies in wild-living chimpanzees

We evaluated the use of an RNA stabilisation buffer, RNAlater® (Ambion, Austin, Texas), as a preservation medium for parasitic coprology analysis of faecal samples collected from chimpanzees living in the wild (Pan troglodytes troglodytes). Thirty faecal samples collected in the forests of south-east Cameroon (Mambele area) from 2003 to 2011 were preserved in RNAlater® at −80 °C and analysed for their parasite content. We identified and counted parasitic elements and assessed their shape, size and morphology in relation to the storage time of the samples. We found that parasite elements were identifiable in RNAlater® preserved samples after as many as 7 years, showing that RNAlater® could be an effective and reliable preservation medium for coprology. Thus, its use could be an interesting way to optimise sample collection for several types of studies (parasitology and bacteriology/virology) at once, especially considering the logistically challenging and time-consuming field campaigns needed to obtain these faecal samples.     

The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol–chloroform–isoamyl alcohol DNA extraction

Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2‐week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol–chloroform–isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction.