reagent storage
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Freezing is considered to be the best method for long-term storage of bacterial DNA from feces; however this method cannot be usually applied for samples of wild primates collected in the challenging conditions of the tropical forest. In order to find an alternative conservation method of fecal samples from wild great apes, we compared freezing with other fixation methods. Fecal samples from 11 captive gorillas (Gorilla gorilla gorilla) from three Czech Zoos were stored using freezing, RNA Stabilization Reagent (RNAlater), and 96% ethanol. Subsequently, the samples were examined using culture-independent methods (PCR-DGGE, and Real-time PCR) to qualitatively and quantitatively assess fecal microbiota composition and to compare differences among the storage methods. Noticeably, freezing samples resulted in the highest recoveries of DNA. No significant differences in DNA recovery were found between freezing and using RNAlater; however, significantly lower DNA concentrations were recovered from samples stored in 96% ethanol. Using PCR-DGGE we found that either 96% ethanol, RNAlater or freezing were suitable for preserving bacterial DNA; however fingerprints obtained from RNAlater storage were more similar to those obtained from the frozen method; in comparison to the patterns resulting from storing samples in ethanol. Using qPCR, frozen samples yielded the highest values of bacterial counts, with the exception of Enterobacteriaceae, which showed the highest numbers using samples stored in ethanol. Sequences of amplicons obtained from PCR-DGGE belonged to the families Clostridiaceae, Lactobacillaceae, Staphylococcaceae, and Lachnospiraceae, phylum Firmicutes; however most amplicons showed sequence similarity to previously uncultured microorganisms. Bacteria belonging to the phylum Firmicutes were the most frequently identified species in the fecal bacterial communities of captive western gorillas. The study showed that RNAlater is an optimal storage method when freezing is not possible.
The pH of the guts of mites strongly affects their digestive processes. This study was carried out to determine the pH in the guts of 12 species of stored product and house dust mites. Eighteen pH indicators were chosen and offered to the mites in the feeding biotest. Based on the color changes of the indicators, the gut contents of acaridid mites were determined to be within a pH range of 4 to neutral. The gut contents showed a gradient in pH from the anterior to the posterior part. The anterior midgut (ventriculus and caeca) of most species had a pH ranging from 4.5 to 5, or slightly more alkaline for most of the species, while the middle midgut (intercolon/colon) had a pH of 5 to 6. Finally, the pH of the posterior midgut (postcolon) was between 5.5 and 7. Except for Dermatophagoides spp., no remarkable differences in the pH of the gut were observed among the tested species. Dermatophagoides spp. had a more acidic anterior midgut (a pH of 4 to 5) and colon (a pH of 5) with postcolon (a pH of below 6). The results characterizing in vivo conditions in the mite gut offer useful information to study the activity of mite digestive enzymes including their inhibitors and gut microflora.
In this paper, we present the ImmunoDisk, a fully automated sample-to-answer centrifugal microfluidic cartridge, integrating a heterogeneous, wash-free, magnetic- and fluorescent bead-based immunoassay (bound-free phase detection immunoassay/BFPD-IA). The BFPD-IA allows the implementation of a simple fluidic structure, where the assay incubation, bead separation and detection are performed in the same chamber. The system was characterized using a C-reactive protein (CRP) competitive immunoassay. A parametric investigation on air drying of protein-coupled beads for pre-storage at room temperature is presented. The key parameters were buffer composition, drying temperature and duration. A protocol for drying two different types of protein-coupled beads with the same temperature and duration using different drying buffers is presented. The sample-to-answer workflow was demonstrated measuring CRP in 5 μL of human serum, without prior dilution, utilizing only one incubation step, in 20 min turnaround time, in the clinically relevant concentration range of 15-115 mg/L. A reproducibility assessment over three disk batches revealed an average signal coefficient of variation (CV) of 5.8 ± 1.3%. A CRP certified reference material was used for method verification with a concentration CV of 8.6%. Our results encourage future testing of the CRP-ImmunoDisk in clinical studies and its point-of-care implementation in many diagnostic applications.
- MeSH
- C-reaktivní protein * MeSH
- imunoanalýza metody MeSH
- indikátory a reagencie MeSH
- lidé MeSH
- mikrofluidika * MeSH
- reprodukovatelnost výsledků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
A little is known about the link between the macromolecular architecture of dialdehyde polysaccharides (DAPs), their crosslinking capabilities, and the properties of resulting hydrogels. Here, DAPs based on cellulose, dextrin, dextran, and hyaluronate were compared as crosslinkers for poly(vinyl alcohol), PVA. The swelling, network parameters, viscoelastic properties, porosity, and cytotoxicity of PVA/DAP hydrogels were investigated concerning the crosslinker structure, molecular weight, aldehyde group density per macromolecule, and the size of spontaneously formed crosslinker nano-assemblies. Generally, crosslinkers based on linear polysaccharides (cellulose, hyaluronate) performed more reliably, while the presence of branching could be both beneficial (dextran) but also detrimental (dextrin) at lower crosslinker concentrations. For example, the hydrogel swelling differed by up to one-third (600 vs. 400%) and storage modulus even by up to one half (~7000 vs. ~3500 Pa) depending on crosslinker structure and properties. These differences were rationalized by variances in crosslinking modes derived based on obtained data.
- MeSH
- buňky NIH 3T3 MeSH
- hydrogely chemie farmakologie MeSH
- myši MeSH
- polysacharidy chemie farmakologie MeSH
- polyvinylalkohol chemie farmakologie MeSH
- reagencia zkříženě vázaná chemie farmakologie MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
The key to obtaining an optimum performance of an enzyme is often a question of devising a suitable enzyme and optimisation of conditions for its immobilization. In this study, laccases from the native isolates of white rot fungi Fomes fomentarius and/or Trametes versicolor, obtained from Czech forests, were used. From these, cross-linked enzyme aggregates (CLEA) were prepared and characterised when the experimental conditions were optimized. Based on the optimization steps, saturated ammonium sulphate solution (75 wt.%) was used as the precipitating agent, and different concentrations of glutaraldehyde as a cross-linking agent were investigated. CLEA aggregates formed under the optimal conditions showed higher catalytic efficiency and stabilities (thermal, pH, and storage, against denaturation) as well as high reusability compared to free laccase for both fungal strains. The best concentration of glutaraldehyde seemed to be 50 mM and higher efficiency of cross-linking was observed at a low temperature 4 °C. An insignificant increase in optimum pH for CLEA laccases with respect to free laccases for both fungi was observed. The results show that the optimum temperature for both free laccase and CLEA laccase was 35 °C for T. versicolor and 30 °C for F. fomentarius. The CLEAs retained 80% of their initial activity for Trametes and 74% for Fomes after 70 days of cultivation. Prepared cross-linked enzyme aggregates were also investigated for their decolourisation activity on malachite green, bromothymol blue, and methyl red dyes. Immobilised CLEA laccase from Trametes versicolor showed 95% decolourisation potential and CLEA from Fomes fomentarius demonstrated 90% decolourisation efficiency within 10 h for all dyes used. These results suggest that these CLEAs have promising potential in dye decolourisation.
- MeSH
- azosloučeniny chemie MeSH
- barva MeSH
- barvicí látky chemie MeSH
- bromthymolová modř chemie MeSH
- enzymy imobilizované chemie MeSH
- glutaraldehyd chemie MeSH
- katalýza MeSH
- lakasa chemie MeSH
- Polyporales enzymologie MeSH
- reagencia zkříženě vázaná chemie MeSH
- rosanilinová barviva chemie MeSH
- síran amonný chemie MeSH
- teplota MeSH
- Trametes enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Elevated levels of pteridines can indicate the activation of cellular immune system by certain diseases. No work dealing with the simultaneous determination of urinary neopterin, biopterin and their reduced forms has been published. Therefore, a new SPE-UHPLC-FD method for the analysis of these compounds has been developed. The main emphasis was put on the stability of dihydroforms during the sample processing and storage. As a stabilizing agent, dithiothreitol, at various concentrations, and various pH values (3.8-9.8) of working solutions were tested. Chromatographic separation was performed under HILIC isocratic conditions on BEH Amide column. The method was linear for the calibration standard solutions in the range of 10-10,000 ng/ml (dihydroforms) and 0.5-1000 ng/ml (oxidized forms), and for real samples in the range of 25-1000 ng/ml (dihydroforms) and 1-100 ng/ml (oxidized forms). The development of a new SPE sample preparation method was carried out on different types of sorbents (based on a mixed-mode cation exchange, porous graphitic carbon and a polymer comprising hydrophilic and hydrophobic components). Final validation was performed on a MCAX SPE column. Method accuracy ranged from 76.9 to 121.9%. The intra- and inter-day precision did not exceed 10.7%. The method provided high sensitivity for the use in routine clinical measurements of urine (LLOQ 1 ng/ml for oxidized forms and 25 ng/ml for dihydroforms). Average concentrations of biopterin, neopterin, and dihydrobiopterin found in urine of healthy persons were related to the mol of creatinine (66.8, 142.3, and 257.3 μmol/mol of creatinine, respectively) which corresponded to the literature data. The concentration of dihydroneopterin obtained using our method was 98.8 μmol/mol of creatinine.
INTRODUCTION: Nowadays, on-a-chip capillary electrophoresis is a routine method for the detection of PCR fragments. The Agilent 2100 Bioanalyzer was one of the first commercial devices in this field. Our project was designed to study the characteristics of Agilent DNA 1000 kit in PCR fragment analysis as a part of circulating tumour cell (CTC) detection technique. Despite the common use of this kit a complex analysis of the results from a long-term project is still missing. MATERIALS AND METHODS: A commercially available Agilent DNA 1000 kit was used as a final step in the CTC detection (AdnaTest) for the determination of the presence of PCR fragments generated by Multiplex PCR. Data from 30 prostate cancer patients obtained during two years of research were analyzed to determine the trueness and precision of the PCR fragment size determination. Additional experiments were performed to demonstrate the precision (repeatability, reproducibility) and robustness of PCR fragment concentration determination. RESULTS: The trueness and precision of the size determination was below 3% and 2% respectively. The repeatability of the concentration determination was below 15%. The difference in concentration determination increases when Multiplex-PCR/storage step is added between the two measurements of one sample. CONCLUSIONS: The characteristics established in our study are in concordance with the manufacturer's specifications established for a ladder as a sample. However, the concentration determination may vary depending on chip preparation, sample storage and concentration. The 15% variation of concentration determination repeatability was shown to be partly proportional and can be suppressed by proper normalization.
- MeSH
- aktiny genetika MeSH
- antigeny povrchové genetika MeSH
- DNA nádorová genetika MeSH
- erbB receptory genetika MeSH
- glutamátkarboxypeptidasa II genetika MeSH
- laboratoř na čipu * MeSH
- lidé MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- nádorové biomarkery genetika MeSH
- nádorové cirkulující buňky metabolismus MeSH
- nádory prostaty rezistentní na kastraci krev genetika MeSH
- prostatický specifický antigen genetika MeSH
- reagenční diagnostické soupravy MeSH
- regulace genové exprese u nádorů * MeSH
- reprodukovatelnost výsledků MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
MicroRNAs (miRNAs) are small RNA molecules, which act as post-transcriptional regulators of a gene expression, with important functions within the cell physiology. Whilst many authors have focused on the study of miRNA expression in physiological and pathological processes, various technical variables related to miRNA isolation have simultaneously emerged and the stability of the stored miRNA samples has been questioned. A robust method for RNA isolation is essential for reproducible results and miRNAs instability in the stored samples would make for an alarming situation for most expression studies. Here these issues are discussed and we investigate the stability of miRNAs isolated from clinical samples of B lymphocytes (chronic lymphocytic leukemia) by the most commonly utilized method based on a Trizol/TRI-Reagent solution (RNAs stored at -80 degrees C). To assess the stability of miRNAs, a Real Time-PCR analysis was performed for a panel of 29 miRNAs from a freshly isolated RNA sample and after 14 days storage at -80 degrees C. Furthermore, a Real Time-PCR analysis was repeatedly performed for a stored RNA sample over a period of approximately 10 months. We observed high stability of isolated miRNAs and respective cDNAs. The reproducibility and efficiency of the Trizol/TRI-Reagent isolation method was also tested and compared to the mirVana Isolation kit (Ambion) and RNeasy kit (Qiagen). In conclusion, Trizol/TRI-Reagent based isolation is a robust reproducible method, and obtained miRNA samples do not show any tendency to degradation when properly stored and handled.
- MeSH
- B-lymfocyty chemie MeSH
- chronická lymfatická leukemie diagnóza MeSH
- fenoly chemie MeSH
- guanidiny chemie MeSH
- lidé MeSH
- mikro RNA chemie izolace a purifikace MeSH
- odběr biologického vzorku MeSH
- polymerázová řetězová reakce MeSH
- reprodukovatelnost výsledků MeSH
- roztoky MeSH
- stabilita RNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
... SAFETY, 13 -- Standard Versus Universal Precautions, 13 -- PART II FOUNDATIONS: BASIC SCIENCES AND REAGENTS ... ... : Overview and Applications, 47 -- SECTION 1: INTRODUCTION TO BLOOD BANKING REAGENTS, 48 -- Regulation ... ... of Reagent Manufacture, 48 Reagent Quality Control, 49 -- SECTION 2: COMMERCIAL ANTIBODY REAGENTS, 50 ... ... -- Polyclonal Versus Monoclonal Antibody Products, 50 -- Reagents for ABO Antigen Typing, 52 Reagents ... ... for D Antigen Typing, 53 Low-Protein Reagent Control, 54 -- SECTION 3: REAGENT RED CELLS, 55 -- Aj and ...
Fourth edition x, 406 stran : ilustrace ; 28 cm
- MeSH
- antigeny krevních skupin imunologie MeSH
- hematologické testy metody MeSH
- krevní banky MeSH
- krevní nemoci imunologie MeSH
- krevní transfuze * metody MeSH
- určování krevní skupiny a křížové zkoušky metody MeSH
- Publikační typ
- programovaná výuka MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- hematologie a transfuzní lékařství
