RESUMO
INTRODUCTION: Optimal DNA and RNA quantity and purity is essential for downstream molecular biology experimentation and to avoid re-processing of sample. Despite availability of different kits and automated systems for nucleic acid isolation there is limited data on their performance evaluation, more so with pediatric blood samples, that are usually compromised in quantity. Hence, we evaluated the performance of automated QIAcube platform using pediatric blood samples in parallel with manual Qiagen extraction kits. MATERIALS AND METHODS: : A total of 500 samples were analyzed based on groups of PBMC and direct blood input. The isolated DNA and RNA were surveyed for quantity and quality tests by spectrophotometric and downstream analysis. RESULTS: : There was no significant difference in the DNA quantity (ng/ul) between manual and automated method based on similar sample input but quality (260/280) was significantly better with the QIAcube platform when direct blood and or PBMCs were used for extraction respectively (1.82 ± 004 Vs. 1.84.002; P-0.000008 and 1.859 ± 005 Vs. 1.843 ± 0.003; P-0.02). Moreover, the standard error mean was low for both quantity and quality in the QIAcube method suggesting uniformity. Comparison of quality assessment by spectrophotometer and qubit fluorimeter showed that QIAcube sheared DNA less (P- 0.038) as compared to manual method (P-0.013). Also, time taken to process the samples in QIAcube was 23% less than the kit-based method. CONCLUSION: Overall analysis of QIAcube platform suggests that it yields more better, uniform, and less-sheared quality of nucleic acid in a relatively less time as compared to manual extraction kits.
Assuntos
Automação Laboratorial/normas , Células Sanguíneas , DNA/isolamento & purificação , Leucócitos Mononucleares , Biologia Molecular/métodos , RNA/isolamento & purificação , Kit de Reagentes para Diagnóstico/normas , Automação Laboratorial/métodos , Criança , Pré-Escolar , DNA/normas , Humanos , Lactente , Biologia Molecular/instrumentação , Biologia Molecular/normas , RNA/normasRESUMO
Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) are a group of small insects of great concern for Public Health. These dipterous are intensely studied worldwide due to their involvement in the transmission of several pathogens, mainly Leishmania spp. parasites. Nowadays, the molecular tools have been included in Phlebotomine sand flies studies and has shown to be powerful tools in bioecology studies of these dipterous. Thereby, when molecular approaches are employed, there is a great concern regarding the amount and quality of the DNA obtained for analysis. Here, seven methods of DNA extraction, between commercial kits and in house extraction protocols were evaluated. We considered measure of DNA concentration and purity ratios using a spectrophotometer to check the performance of each protocol. In addition, the quality evaluation of the DNA extracted was performed by endogenous gene PCR on samples. The results of the seven evaluated DNA extraction protocols and their implications are discussed.
Assuntos
DNA/isolamento & purificação , Psychodidae/genética , Análise de Variância , Animais , Custos e Análise de Custo , DNA/análise , DNA/normas , Eletroforese em Gel de Ágar , Feminino , Masculino , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Cloreto de Sódio , Espectrofotometria , Fatores de TempoRESUMO
INTRODUCTION: Metagenomics is increasingly considered for clinical diagnostics. In order for this technology to become integrated in the clinical microbiology laboratory, process controls are required. Molecular diagnostic tests typically integrate an internal control (IC) to detect potential sources of variation and technical failure. However, few studies report on the integration of an IC in metagenomics. AIM: We aimed to develop an easy-to-use IC method for the process control of library preparation and sequencing applied to metagenomics in clinical microbiology diagnostics using Thermus thermophilus DNA. METHODOLOGY: DNA was extracted from urine samples and sequenced on the Ion Torrent Proton in the absence and presence of incremental concentrations (0.5-2-5%) of IC. Between aliquots of each sample, we compared the IC relative abundance (RA), and after in silico subtraction of IC reads, analysed microbial composition and the RA of pathogens. The optimal IC concentration was defined as the lowest concentration still detectable in all samples with the smallest impact on the microbial composition. RESULTS: The RA of IC correlated linearly with the spiked IC concentration (r2 = 0.99). IC added in a concentration of 0.5% of the total DNA concentration was detectable in all sample aliquots, regardless of human-bacterial DNA proportion, and after in silico removal gave the smallest difference in RA of pathogens compared to the sample aliquot sequenced in the absence of IC. The microbial composition in the presence and absence of IC was highly similar after in silico removal of IC reads (median BC-dissimilarity per sample: 0.059), provided samples had a mean of >10,000 bacterial reads. CONCLUSION: T. thermophilus DNA at a percentage of 0.5% of the total DNA concentration was successfully applied for the process control of metagenomics of urine samples. We demonstrated negligible alterations in sample microbial composition after in silico subtraction of IC reads. This approach contributes toward implementation of metagenomics in the clinical microbiology laboratory.
Assuntos
Bactérias , Infecções Bacterianas/diagnóstico , DNA/normas , Técnicas de Diagnóstico Molecular/métodos , Thermus thermophilus/genética , Urina/microbiologia , Bactérias/genética , Bactérias/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Metagenoma , Padrões de Referência , Análise de Sequência de DNA/métodosRESUMO
For over 10 years, quantitative PCR (qPCR) for DNA quantitation has been reported in forensics. However, assays have not been described for small qPCR platforms. Thus, technological advancement is not always implemented in small forensic genetics laboratories. A duplex qPCR assay is reported, using a StepOne instrument and targeting a short and a long human DNA region. This study was performed according to international validation guidelines, including sensitivity, repeatability, reproducibility, precision, accuracy, contamination assessment, known and case-type samples, and degradation studies. Characterization of the genetic markers, species specificity, and population studies had already been conducted. Moreover, case-type samples were quantified, amplified using commercial kits and the number of alleles detected was recorded. Sensitivity was shown to be 10 pg/µL. Standard curve replicates demonstrated the assay is accurate, precise, as well as fairly repeatable and reproducible. The NGM Detect kit was shown to yield higher peaks than Identifiler Plus and NGM Select for degraded samples. Moreover, quality sensors were always present and proved useful. The quantification values of the large target showed a correlation with the number of alleles detected in the STR profiles for known and casework samples. The degradation index was shown to be informative, with a value of 10 or higher indicating dropout. It is suggested that after quantitation, samples with low or degraded DNA be amplified using newer amplification kits containing quality sensors to confirm that the low-quality profile was not affected by inhibition.
Assuntos
DNA , Genética Forense/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , DNA/análise , DNA/química , DNA/genética , DNA/normas , Marcadores Genéticos/genética , Humanos , Reprodutibilidade dos Testes , Especificidade da EspécieRESUMO
While genotyping studies are scavenging for suitable samples to analyze, large serum collections are currently left unused as they are assumed to provide insufficient amounts of DNA for array-based genotyping. Long-term stored serum is considered to be difficult to genotype since preanalytical treatments and storage effects on DNA yields are not well understood. Successful genotyping of such samples has the potential to activate large biobanks for future genome-wide association studies (GWAS). We aimed to evaluate genotyping of ultralow amounts of DNA from samples stored up to 45 years in the Janus Serum Bank with two commercially available platforms. 64 samples, with various preanalytical treatments, were genotyped on the Axiom Array from Thermo Fisher Scientific and a subset of 24 samples with slightly higher yield were genotyped on the HumanCoreExome array from Illumina. Our results showed that about 80% of the serum samples produced call rates with the Axiom arrays that would be satisfactory in GWAS. The mean DNA yield was 5.8 ng as measured with PicoGreen, 3-6% of recommended yield. The failed samples had on average lower input amounts of DNA. All serum samples genotyped on the HumanCoreExome with a standard and FFPE protocol produced GWAS satisfactory call rates, with mean 97.57% and 98.35% call rates, respectively. The mean yield was 10.65 ng, 6% of the recommendations. Successful array-based genotyping of ultralow DNA yields from serum samples stored up to 45 years is possible. These results demonstrate the potential to activate large serum biobank collections for future studies.
Assuntos
Preservação de Sangue/efeitos adversos , DNA/química , Sequenciamento do Exoma/métodos , Técnicas de Genotipagem/métodos , Bancos de Sangue , DNA/sangue , DNA/genética , DNA/normas , Testes Genéticos/métodos , Testes Genéticos/normas , Estudo de Associação Genômica Ampla/métodos , Estudo de Associação Genômica Ampla/normas , Técnicas de Genotipagem/normas , Humanos , Sequenciamento do Exoma/normasRESUMO
A specialized DNA extraction method and a SYBR Green quantitative polymerase chain reaction (SyG-qPCR) assay were combined to generate a ready-to-use kit for rapid detection of porcine admixtures in processed meat products. Our qPCR assay utilized repetitive LINE-1 elements specific to the genome of Sus scrofa domesticus (pig) as a target and incorporated internal controls. We improved the genomic DNA extraction method, and reduced extraction times to the minimum. The method was validated for specificity, sensitivity (0.001% w/w) and robustness, and values were compared with those of a commercially available kit. We also tested our method using 121 processed food products and consistently detected amplification only in samples containing pork. Due to its efficiency and cost-effectiveness, our method represents a valuable new method for detecting food adulteration with pork that is superior to existing quality control approaches.
Assuntos
DNA/análise , Contaminação de Alimentos/análise , Compostos Orgânicos/química , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Benzotiazóis , DNA/isolamento & purificação , DNA/normas , Diaminas , Elementos Nucleotídeos Longos e Dispersos/genética , Produtos da Carne/análise , Controle de Qualidade , Quinolinas , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Sus scrofa/genética , SuínosRESUMO
BACKGROUND: Whole Exome Sequencing (WES) utilises overlapping fragments prone to sequencing artefacts. Saliva, a non-invasive source of DNA, has been successfully used in WES studies on various platforms. This study explored the validity and quality of DNA sourced from saliva compared to whole blood on an Ion Platform. METHODS: DNA was extracted from both sample types from four individuals. WES, performed on the Ion Proton platform was assessed for quality metrics (Depth, Genotyping Quality, etc.) and variant identification for the same source sample-pairs. RESULTS: No significant differences in quality metrics were identified between data obtained from whole blood and saliva samples, with several saliva samples demonstrating higher coverage depth. Variants within the same sample, from the two genomic DNA sources, had an average concordance similar to other studies and platforms with different chemistry. CONCLUSION: Saliva-extracted DNA provides comparable sequencing quality to whole blood for WES on Ion Torrent Platforms.
Assuntos
DNA/normas , Sequenciamento do Exoma/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Saliva/química , Adulto , DNA/química , DNA/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Masculino , Sequenciamento do Exoma/normasRESUMO
Germline variants in the DNA mismatch repair (MMR) gene PMS2 cause 1-14% of all Lynch Syndrome cancers. Correct variant analysis of PMS2 is complex due to the presence of multiple pseudogenes and the occurrence of gene conversion. The analysis complexity increases in highly fragmented DNA from formalin-fixed paraffin-embedded (FFPE) tissue. Here we describe a reliable approach to detect true PMS2 variants in fragmented DNA. A custom NGS panel designed for FFPE tissue was used targeting four MMR genes, POLE and POLD1. Amplicon design for PMS2 was based on the position of paralogous sequence variants (PSVs) that distinguish PMS2 from its pseudogenes. PMS2 variants in exons 1-11 can be correctly curated based on this information. For exons 12-15 this is less reliable as these undergo gene conversion. Using this method, we screened PMS2 variants in 125 MMR-deficient tumors. Of the 125 tumors tested, six were unexplained MMR-deficient tumors with solitary PMS2 protein expression loss. In these six tumors two unclassified variants (class 3) and five variants likely affecting function (class 4/5) were detected in PMS2. One microsatellite unstable tumor with positive staining for all MMR proteins was found to carry a frameshift PMS2 variant (class 5). No class 4 or class 5 PMS2 variants were detected in tumors with other patterns of MMR protein expression loss.
Assuntos
Neoplasias Colorretais/genética , DNA/química , Testes Genéticos/métodos , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Inclusão em Parafina/métodos , Análise de Sequência de DNA/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/patologia , DNA/normas , Fragmentação do DNA , DNA Polimerase II/genética , DNA Polimerase III/genética , Feminino , Formaldeído/química , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/genética , Pseudogenes , Sensibilidade e Especificidade , Fixação de Tecidos/métodosRESUMO
Ovarian tissue contains large pools of immature oocytes enclosed in primordial follicles, making it an attractive target for fertility preservation in female cancer patients, livestock and wild species. Compared to cryopreservation, desiccation and long-term storage of samples at supra-zero temperatures (using strategies inspired from small organisms to resist extreme environments) would be more cost-effective and convenient. The objective of the study was to characterize the influence of microwave-assisted dehydration on structural and functional properties of living ovarian tissues. While this method allows preservation of single cells (cat oocytes and sperm cells so far) using trehalose as the xeroprotectant, it has not been developed for multicellular tissues yet. Ovarian cortex biopsies were reversibly permeabilized, exposed to various concentrations of trehalose, and dried for different times using a commercial microwave under thermal control. Effective dehydration of samples along with proper trehalose retention were reached within 30 min of microwave drying. Importantly, the process did not affect morphology and DNA integrity of follicles or stromal cells. Moreover, transcriptional activity and survival of follicles were partially maintained following 10 min of drying, which already was compatible with storage at non-cryogenic temperatures. Present data provide critical foundation to develop dry-preservation techniques for long-term storage of living multicellular tissues.
Assuntos
Dessecação/métodos , Ovário/citologia , Preservação de Tecido/métodos , Animais , Gatos , Sobrevivência Celular , DNA/química , DNA/normas , Feminino , Micro-Ondas , Ovário/metabolismo , RNA Mensageiro/química , RNA Mensageiro/normas , TemperaturaRESUMO
The objective is to identify cofactors of leukocyte telomere length (LTL) in a Latin American population, specifically the association of LTL with 36 socio-demographic, early childhood, and health characteristics, as well as with DNA sample collection and storage procedures. The analysis is based on longitudinal information from a subsample of 1,261 individuals aged 60+ years at baseline from the Costa Rican Study of Longevity and Healthy Aging (CRELES): a nationally representative sample of elderly population. Random effects regression models for panel data were used to estimate the associations with LTL and its longitudinal changes. Sample collection procedures and DNA refrigerator storage time were strongly associated with LTL: telomeres are longer in blood collected in October-December, in DNA extracted from <1-year-old blood cells, and in DNA stored at 4°C for longer periods of time up to five years. The data confirmed that telomeres are shorter at older ages, as well as among males, and diabetic individuals, whereas telomeres are longer in the high-longevity Nicoya region. Most health, biomarkers, and early childhood indicators did not show significant associations with LTL. Longitudinal LTL variation over approximately two years was mainly associated with baseline LTL levels, as found in other studies. Our findings suggest that if there is unavoidable variability in season of sample collection and DNA storage time, these factors should be controlled for in all demographic and epidemiologic studies of LTL. However, due to unobserved components of measurement variation, statistical control may be inadequate as compared to standardization of data collection procedures.
Assuntos
Coleta de Amostras Sanguíneas/métodos , DNA/normas , Leucócitos/química , Longevidade , Telômero/genética , Idoso , Costa Rica , Feminino , Envelhecimento Saudável , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Análise de Regressão , Homeostase do Telômero , Fatores de TempoRESUMO
Incorporation of genome and exome sequencing into fetal and neonatal autopsy investigations has been shown to improve diagnostic yield. This requires deoxyribonucleic acid (DNA) to be extracted from either the placenta or autopsy tissue for molecular testing. However, the sources and quality of DNA obtained are highly variable and there are no adequate published data on what tissue is most ideal to sample for DNA extraction in this setting. Here we compare the quality of DNA extracted from sampling the placenta and various solid organs at fetal and neonatal autopsy, thereby determining the optimal tissue from which to source DNA for ancillary testing as part of the modern perinatal autopsy. A total of 898 tissue samples were obtained at autopsy from 176 fetuses (gestational ages 17-40 weeks) and 44 neonates (age range 0-28 days) at our tertiary institution. Fetal tissue was processed using the QIAsymphony DSP DNA Mini kit and placental tissue was extracted using the New iGENatal Kit. DNA concentration was quantified using the Qubit dsDNA BR Assay Kit. DNA integrity, as stratified by gel electrophoresis was classified as high (≥5 kb) or low quality (<5 kb). Genome sequencing was performed on the extracted DNA, together with respective parental DNA from blood samples, and confirmed absence of maternal contamination in all cases. Analyses used logistic mixed models to test for associations between tissue types, intrauterine retention times, delivery to autopsy and death to autopsy intervals with DNA quality. In the fetal cohort, the placenta had the highest proportion of high quality DNA samples (93.1%), and liver had the lowest proportion (35.3%). Among the neonates, all tissue samples with the exception of liver had over 88% high DNA quality with the placenta also yielding the highest quality (100%). There was statistically significant deterioration in DNA quality with prolonged time interval between demise and autopsy (≥5 days). In the 726 fetal samples, the odds of obtaining higher quality DNA from the placenta, thymus, and spleen were 70.4 [95% confidence interval (CI) 29.2-169.6], 3.6 (95% CI 2.0-6.6) and 3.3 (95% CI 1.8-6.1) times, respectively, more likely than samples from the liver (p values <0.001). DNA yield from other fetal solid organs investigated was not significantly superior to that from the liver. This study shows that, when available, refrigerated unfixed placenta is the most suitable source of high quality DNA during perinatal investigations. Of the solid fetal organs sampled at autopsy, lymphocyte-rich, lytic enzymes-poor organs such as thymus and spleen were significantly more likely to yield good quality DNA than the liver.
Assuntos
DNA/isolamento & purificação , Feto , Genômica , Autopsia , Estudos de Coortes , DNA/normas , Feminino , Humanos , Recém-Nascido , Fígado , Placenta , Gravidez , Refrigeração , Baço , TimoRESUMO
Among the laboratory and bioinformatic processing steps for human microbiome studies, a lack of consistency in DNA extraction methodologies is hindering the ability to compare results between studies and sometimes leading to errant conclusions. The purpose of this article is to highlight the issues related to DNA extraction methods and to suggest minimum standard requirements that should be followed to ensure consistency and reproducibility.
Assuntos
DNA/isolamento & purificação , Genômica/normas , Microbiota , DNA/normas , Fezes/química , HumanosRESUMO
BACKGROUND: Whole blood is currently the most common DNA source for whole-genome sequencing (WGS), but for studies requiring non-invasive collection, self-collection, greater sample stability or additional tissue references, saliva or buccal samples may be preferred. However, the relative quality of sequencing data and accuracy of genetic variant detection from blood-derived, saliva-derived and buccal-derived DNA need to be thoroughly investigated. METHODS: Matched blood, saliva and buccal samples from four unrelated individuals were used to compare sequencing metrics and variant-detection accuracy among these DNA sources. RESULTS: We observed significant differences among DNA sources for sequencing quality metrics such as percentage of reads aligned and mean read depth (p<0.05). Differences were negligible in the accuracy of detecting short insertions and deletions; however, the false positive rate for single nucleotide variation detection was slightly higher in some saliva and buccal samples. The sensitivity of copy number variant (CNV) detection was up to 25% higher in blood samples, depending on CNV size and type, and appeared to be worse in saliva and buccal samples with high bacterial concentration. We also show that methylation-based enrichment for eukaryotic DNA in saliva and buccal samples increased alignment rates but also reduced read-depth uniformity, hampering CNV detection. CONCLUSION: For WGS, we recommend using DNA extracted from blood rather than saliva or buccal swabs; if saliva or buccal samples are used, we recommend against using methylation-based eukaryotic DNA enrichment. All data used in this study are available for further open-science investigation.
Assuntos
Variações do Número de Cópias de DNA/genética , DNA/genética , Sequenciamento Completo do Genoma/normas , Adulto , DNA/sangue , DNA/química , DNA/normas , Metilação de DNA/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa Bucal/química , Polimorfismo de Nucleotídeo Único/genética , Saliva/química , Análise de Sequência de DNA/normasRESUMO
Nucleic acid amplification methods, such as polymerase chain reaction (PCR), are extensively used in many applications to detect target DNA because of their high sensitivity, good reproducibility, and wide dynamic range of quantification. However, analytical quality control when detecting low copy number target DNA is often missing because of a lack of appropriate reference materials. Recent advances in analytical sciences require a method to accurately quantify DNA at the single molecule level. Herein, we have developed a novel method to produce reference material containing a defined copy number of target DNA (referred to as "cell number-based DNA reference material"). In this method, a suspension of cells carrying a single target DNA sequence was ejected by an inkjet head, and the number of cells in each droplet was counted using highly sensitive cameras. The resulting solutions contained a defined copy number of target DNA and could be used as reference materials. The use of the newly developed reference material was compared with that of diluted solutions of target DNA to evaluate the performance of qualitative real-time PCR in terms of the limit of detection (LOD). Our results demonstrated that cell number-based DNA reference material provides more accurate information regarding performance quality. The reference material produced by this method is a promising tool to evaluate assay performance.
Assuntos
Bioimpressão , DNA/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sequência de Bases , DNA/metabolismo , DNA/normas , Variações do Número de Cópias de DNA , Limite de Detecção , Microscopia , Fotometria , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Saccharomyces cerevisiae/genéticaRESUMO
High accuracy, reliability, and reproducibility of genetic analyses in various applications require optimized and validated protocols and standards. Optimal procedures for storing the genetic material extracted from biological samples are equally important. In this study, we investigated the stability of dilute (4000 cp/µL, nominal concentration, equivalent to 0.02 ng/mL) DNA solutions stored at 4, -20, and -80 °C in the presence or absence of nucleic acid carriers. As representative examples, we used different formulations of a linearized plasmid DNA solution considered for characterization as reference materials (RMs) for specific applications. Employing droplet digital PCR, a highly accurate and precise method for quantification of nucleic acid not requiring a calibrant, we demonstrated that inclusion of a carrier nucleic acid in the formulation (at 50 ng/µL) improved the plasmid stability at -20 and -80 °C. For the case of a DNA standard used in real-time PCR assays for human erythropoietin gene, cDNA or transcript, we found that inclusion of yeast RNA in the formulation was preferred over salmon testes DNA as it had no effect on PCR amplification and provided the lowest relative expanded uncertainty for the characterized RM. RNA background may also be preferred as it is applicable to a broader range of DNA RMs. Our findings are important in production of reliable, stable DNA standards, including DNA RMs. These results can be used when selecting protocols for stable storage of DNA either extracted from biological samples or synthesized in a laboratory.
Assuntos
DNA/química , Reação em Cadeia da Polimerase em Tempo Real/normas , Animais , DNA/normas , Eritropoetina/genética , Congelamento , Humanos , Plasmídeos , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos Testes , Salmão/genética , TemperaturaRESUMO
Human biobanks are collections of biological samples and health information that allow the organization of biomedical research for upgrading the knowledge of human disorders from different diseases (cancer, allergies, rare diseases, etc.), and reach real answers for diagnosis and treatment. A wide range of samples can be stored in these biorepositories such as hair, nails, urine, tissue, whole blood, red blood cells, buffy coat, plasma, serum, DNA, and RNA. Among these, buffy coat and whole blood are widely used by researchers because they can obtain DNA and RNA from these matrices. Some preliminary studies have been performed on animals to evaluate the quality and functionality of the nucleic acids obtained from some of these matrices, although more in-depth studies are needed in this area. In this study, blood samples extracted by venipuncture from 30 healthy volunteers were used to obtain DNA from buffy coat and whole blood. The purity and integrity of the nucleic acids obtained were assessed by spectrophotometry, fluorimetry, and agarose electrophoresis, and functionality was assessed by PCR and real-time PCR. Another aspect tested in this study was based on the comparison between short-term and long-term storage at -80°C and fresh samples from both matrices to evaluate the storage conditions at the biobank. Results showed differences in the yield obtained from both matrices as a function of the storage time, although the functionality of all the obtained DNA remained intact.
Assuntos
Coleta de Amostras Sanguíneas/normas , DNA/normas , Buffy Coat/química , DNA/sangue , DNA/genética , Voluntários Saudáveis , Humanos , FlebotomiaRESUMO
In the female reproductive tract, male gametes undergo a natural sperm selection process in order to discriminate spermatozoa on the basis of their quality to maximize the chances of successful reproduction. With the introduction of assisted reproductive technology (ART), scientists and clinicians developed diverse sperm selection strategies focusing on the isolation of competent spermatozoa. With increasing understanding of sperm functions and fertilization mechanism and evolution of available technologies, the initial simple sperm preparation protocols were complemented, and sometimes replaced, by new sperm-sorting techniques. In particular, while in the early years the focus was on obtaining motile spermatozoa, in later years, especially after the introduction of intracytoplasmic sperm injection (ICSI), the focus shifted to the isolation of functional and "healthy" spermatozoa, considering some other important factors, such as sperm DNA integrity. Sperm DNA damage, as well as chromatin structure alterations, in fact, is related to decreased reproductive ability of men, in natural as well as in assisted reproduction.
Assuntos
DNA/normas , Técnicas de Reprodução Assistida , Espermatozoides , Dano ao DNA , Humanos , Masculino , Injeções de Esperma IntracitoplásmicasAssuntos
Animais Selvagens/classificação , Animais Selvagens/genética , Biodiversidade , Classificação/métodos , Conservação dos Recursos Naturais/métodos , DNA/análise , Ecologia/métodos , Animais , DNA/genética , DNA/normas , Contaminação por DNA , Sanguessugas/genética , Especificidade da Espécie , Ursidae/classificação , Ursidae/genéticaRESUMO
The extraction of DNA is a critical step for species identification by PCR analysis in processed food and feed products. In this study, eight DNA extraction procedures were compared-DNeasy Blood and Tissue Kit, DNeasy mericon Food Kit, chemagic DNA Tissue 10 Kit, Food DNA Isolation Kit, UltraPrep Genomic DNA Food Mini Prep Kit, High Pure PCR Template Preparation Kit, phenol-chloroform extraction, and NucleoSpin Food-Using self-prepared samples from both raw and heat-processed and/or mechanically treated muscles and different types of meat products and pet food (pork, beef, and chicken). The yield, purity, and suitability of DNA for PCR amplification was evaluated. Additionally, comparisons between the effectiveness of various extraction methods were made with regard to price, and labor- and time-intensiveness. It was found that the DNeasy mericon Food Kit was the optimal choice for the extraction of DNA from raw muscle, heat-treated muscle, and homemade meat products from multiple and single species.