scholarly journals Sample collection/stabilization and DNA/RNA extraction from swab samples for microbiome or metagenome analyses

BioTechniques ◽  
2021 ◽  
Author(s):  
David Navarro ◽  
A González ◽  
A Saiz O y Odriozola
Keyword(s):  
Microbial Communities ◽  
Rna Extraction ◽  
Genetic Material ◽  
New Product ◽  
Storage Medium ◽  
Sample Collection ◽  
Bacterial Composition ◽  
Good Preservation ◽  
Dna And Rna ◽  
And Storage

Good preservation and storage are essential to preserving microorganisms’ genetic material in microbial communities from wide array of sample inputs and accurately represent the bacterial composition for further analysis and applications. The objective is to develop a proper preservation and storage medium to preserve DNA and RNA from those microorganisms. DANAGEN-BIOTED has developed a new product to deal with this problem. Click on the To read the full Application forum, click on the View Article button above and download the PDF.

Variability in, variability out: best practice recommendations to standardize pre-analytical variables in the detection of circulating and tissue microRNAs

2017 ◽  
Vol 55 (5) ◽  
Author(s):  
Jenna Khan ◽  
Joshua A. Lieberman ◽  
Christina M. Lockwood
Keyword(s):  
Best Practice ◽  
Rna Extraction ◽  
Storage Conditions ◽  
Sample Collection ◽  
Ischemic Time ◽  
Cold Ischemic Time ◽  
C Storage ◽  
Tissue Specimens ◽  
And Storage

Abstract:microRNAs (miRNAs) hold promise as biomarkers for a variety of disease processes and for determining cell differentiation. These short RNA species are robust, survive harsh treatment and storage conditions and may be extracted from blood and tissue. Pre-analytical variables are critical confounders in the analysis of miRNAs: we elucidate these and identify best practices for minimizing sample variation in blood and tissue specimens. Pre-analytical variables addressed include patient-intrinsic variation, time and temperature from sample collection to storage or processing, processing methods, contamination by cells and blood components, RNA extraction method, normalization, and storage time/conditions. For circulating miRNAs, hemolysis and blood cell contamination significantly affect profiles; samples should be processed within 2 h of collection; ethylene diamine tetraacetic acid (EDTA) is preferred while heparin should be avoided; samples should be “double spun” or filtered; room temperature or 4 °C storage for up to 24 h is preferred; miRNAs are stable for at least 1 year at –20 °C or –80 °C. For tissue-based analysis, warm ischemic time should be <1 h; cold ischemic time (4 °C) <24 h; common fixative used for all specimens; formalin fix up to 72 h prior to processing; enrich for cells of interest; validate candidate biomarkers with in situ visualization. Most importantly, all specimen types should have standard and common workflows with careful documentation of relevant pre-analytical variables.

scholarly journals A Fast and Accessible Method for the Isolation of RNA, DNA, and Protein to Facilitate the Detection of SARS-CoV-2

2020 ◽  
Author(s):  
Jose Carlos Ponce-Rojas ◽  
Michael S. Costello ◽  
Duncan A. Proctor ◽  
Kenneth S. Kosik ◽  
Maxwell Z. Wilson ◽  
...  
Keyword(s):  
Low Cost ◽  
Rna Extraction ◽  
Genetic Material ◽  
Simple Method ◽  
Dna And Rna ◽  
Alternative Method ◽  
Accessible Method ◽  
Comparable Performance ◽  
Viral Envelopes ◽  
Common Laboratory

AbstractManagement of the COVID-19 pandemic requires widespread SARS-CoV-2 testing. A main limitation for widespread SARS-CoV-2 testing is the global shortage of essential supplies, among these, RNA extraction kits. The need for commercial RNA extraction kits places a bottleneck on tests that detect SARS-CoV-2 genetic material, including PCR-based reference tests. Here we propose an alternative method we call PEARL (Precipitation Enhanced Analyte RetrievaL) that addresses this limitation. PEARL uses a lysis solution that disrupts cell membranes and viral envelopes while simultaneously providing conditions suitable for alcohol-based precipitation of RNA, DNA, and proteins. PEARL is a fast, low-cost, and simple method that uses common laboratory reagents and offers comparable performance to commercial RNA extraction kits. PEARL offers an alternative method to isolate host and pathogen nucleic acids and proteins to streamline the detection of DNA and RNA viruses, including SARS-CoV-2.

scholarly journals A Fast and Accessible Method for the Isolation of RNA, DNA, and Protein to Facilitate the Detection of SARS-CoV-2

2020 ◽  
Author(s):  
Jose Carlos Ponce-Rojas ◽  
Michael S. Costello ◽  
Duncan A. Proctor ◽  
Kenneth S. Kosik ◽  
Maxwell Z. Wilson ◽  
...  
Keyword(s):  
Low Cost ◽  
Rna Extraction ◽  
Genetic Material ◽  
Simple Method ◽  
Dna And Rna ◽  
Alternative Method ◽  
Accessible Method ◽  
Comparable Performance ◽  
Viral Envelopes ◽  
Common Laboratory

Management of the COVID-19 pandemic requires widespread SARS-CoV-2 testing. A main limitation for widespread SARS-CoV-2 testing is the global shortage of essential supplies, among these, RNA extraction kits. The need for commercial RNA extraction kits places a bottleneck on tests that detect SARS-CoV-2 genetic material, including PCR-based reference tests. Here we propose an alternative method we call PEARL (Precipitation Enhanced Analyte RetrievaL) that addresses this limitation. PEARL uses a lysis solution that disrupts cell membranes and viral envelopes while simultaneously providing conditions suitable for alcohol-based precipitation of RNA, DNA, and proteins. PEARL is a fast, low-cost, and simple method that uses common laboratory reagents and offers comparable performance to commercial RNA extraction kits. PEARL offers an alternative method to isolate host and pathogen nucleic acids and proteins to streamline the detection of DNA and RNA viruses, including SARS-CoV-2.

Effective optimization of SARS-CoV-2 laboratory testing variables in an era of supply chain constraints

2020 ◽  
Vol 15 (15) ◽  
pp. 1483-1487
Author(s):  
Nikhil S Sahajpal ◽  
Ashis K Mondal ◽  
Allan Njau ◽  
Sudha Ananth ◽  
Kimya Jones ◽  
...  
Keyword(s):  
Supply Chain ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Sample Collection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Short Supply ◽  
Viral Transport ◽  
3D Printed ◽  
Bridging Studies

RT-PCR-based assays for the detection of SARS-CoV-2 have played an essential role in the current COVID-19 pandemic. However, the sample collection and test reagents are in short supply, primarily due to supply chain issues. Thus, to eliminate testing constraints, we have optimized three key process variables: RNA extraction and RT-PCR reactions, different sample types and media to facilitate SARS-CoV-2 testing. By performing various validation and bridging studies, we have shown that various sample types such as nasopharyngeal swab, bronchioalveolar lavage and saliva, collected using conventional nasopharyngeal swabs, ESwab or 3D-printed swabs and, preserved in viral transport media, universal transport media, 0.9% sodium chloride or Amies media are compatible with RT-PCR assay for COVID-19. Besides, the reduction of PCR reagents by up to fourfold also produces reliable results.

scholarly journals Vaginal Microbiota of Adolescent Girls Prior to the Onset of Menarche Resemble Those of Reproductive-Age Women

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Roxana J. Hickey ◽  
Xia Zhou ◽  
Matthew L. Settles ◽  
Julie Erb ◽  
Kristin Malone ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Adolescent Girls ◽  
Vaginal Microbiota ◽  
Reproductive Age ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gardnerella Vaginalis ◽  
Bacterial Composition ◽  
Prospective Longitudinal Study ◽  
Prospective Longitudinal

ABSTRACTPuberty is an important developmental stage wherein hormonal shifts mediate the physical and physiological changes that lead to menarche, but until now, the bacterial composition of vaginal microbiota during this period has been poorly characterized. We performed a prospective longitudinal study of perimenarcheal girls to gain insight into the timing and sequence of changes that occur in the vaginal and vulvar microbiota during puberty. The study enrolled 31 healthy, premenarcheal girls between the ages of 10 and 12 years and collected vaginal and vulvar swabs quarterly for up to 3 years. Bacterial composition was characterized by Roche 454 pyrosequencing and classification of regions V1 to V3 of 16S rRNA genes. Contrary to expectations, lactic acid bacteria, primarily Lactobacillus spp., were dominant in the microbiota of most girls well before the onset of menarche in the early to middle stages of puberty.Gardnerella vaginaliswas detected at appreciable levels in approximately one-third of subjects, a notable finding considering that this organism is commonly associated with bacterial vaginosis in adults. Vulvar microbiota closely resembled vaginal microbiota but often exhibited additional taxa typically associated with skin microbiota. Our findings suggest that the vaginal microbiota of girls begin to resemble those of adults well before the onset of menarche.IMPORTANCEThis study addresses longitudinal changes in vaginal and vulvar microbial communities prior to and immediately following menarche. The research is significant because microbial ecology of the vagina is an integral aspect of health, including resistance to infections. The physiologic changes of puberty and initiation of cyclic menstruation are likely to have profound effects on vaginal microbiota, but almost nothing is known about changes that normally occur during this time. Our understanding has been especially hampered by the lack of thorough characterization of microbial communities using techniques that do not rely on the cultivation of fastidious bacteria, as well as a dearth of studies on girls in the early to middle stages of puberty. This study improves our understanding of the normal development of vaginal microbiota during puberty and onset of menarche and may better inform clinical approaches to vulvovaginal care of adolescent girls.

Numerical Investigation of Nitrate Leakage and Migration in the Soil after Rainfall

2014 ◽  
Vol 1010-1012 ◽  
pp. 429-436
Author(s):  
Jin Hua Shan ◽  
Jing Ding ◽  
Jian Feng Lu
Keyword(s):  
Heat Transfer ◽  
Thermal Power ◽  
Nitrate Salt ◽  
Storage Medium ◽  
Migration Process ◽  
Solar Thermal Power ◽  
Salt Layer ◽  
Thermal Power System ◽  
And Migration ◽  
And Storage

Nitrate salt is important heat transfer and storage medium in solar thermal power system, but nitrate salt leakage and pollution in groundwater is seldom investigated. In this paper, the nitrate salt leakage and migration in the soil after rainfall are simulated and analyzed. During the nitrate leakage process, the liquid nitrate will solidify, and then a thin solidification layer of nitrate forms. According to the simulation result, the radius of the leakage opening mainly affects the radius of nitrate solidification layer, while the leakage velocity will influence the radius and thickness of salt layer. During the nitrate migration process after rainfall, the nitrate will gradually migrate to the groundwater, and the final migration domain of nitrate in the soil will be mainly determined by the radius of nitrate solidification layer.

Optimizing Protocols for High-Quality RNA Extraction from Blood and Liver Tissues of the Broad-Snouted Caiman

2021 ◽  
Vol 28 (4) ◽  
pp. 197-204
Author(s):  
Evelyn Cecilia López González ◽  
Lucía Magdalena Odetti ◽  
Gisela Laura Poletta ◽  
Nancy Denslow ◽  
Kevin J. Kroll ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Biological Processes ◽  
High Quality ◽  
Efficient Procedure ◽  
Physiological Conditions ◽  
Environmental Perturbation ◽  
Preservation Methods ◽  
Cell Tissue ◽  
And Storage ◽  
Liver Tissues

Transcriptomic information provides fundamental insights into biological processes and can be used to determine gene expression in cell, tissue, or organism under specific physiological conditions, or in response to any environmental perturbation. Extraction of high quality RNA is a challenging step mainly in non-traditional organisms, and protocols for preservation and isolation need to be adjusted in many cases. In the present work, we aimed to develop a protocol for preservation and isolation of high-quality and quantity of RNA from blood and liver tissues of Caiman latirostris. Three preservation methods were tested: 1) flash freezing (LN2) and storage at –80°C; 2) RNAlater® conservation with progressive cooling up to –80°C); 3) preservation in TRIzol® reagent, flash freezing in LN2 and storage at –80°C. Methods 1 and 2 were tested for liver, while 2 and 3 for blood. Our results showed that both preservation methods resulted in excellent outcomes for liver samples. For blood samples however, TRIzol® preservation was an efficient procedure for adequate RNA quality, quantity, and integrity, while conservation in RNAlater® solution was inadequate in both quality and quantity for an optimal RNA extraction. Appropriate protocols were established for each tissue and are being used now for transcriptomic studies in this sentinel organism.

scholarly journals Recent developments in nucleic acid based techniques for use in rumen manipulation

2009 ◽  
Vol 38 (spe) ◽  
pp. 341-351 ◽  
Author(s):  
Christopher McSweeney ◽  
Seungha Kang ◽  
Emma Gagen ◽  
Carl Davis ◽  
Mark Morrison ◽  
...  
Keyword(s):  
Microbial Community ◽  
Nucleic Acid ◽  
Microbial Communities ◽  
Marker Gene ◽  
Molecular Ecology ◽  
Functional Gene ◽  
Rumen Microorganisms ◽  
Dna And Rna ◽  
Conventional Culture ◽  
Reference Strains

Nucleic acid-based techniques which can be used to characterise complex microbial communities without incubation are now being employed regularly in ruminant nutrition studies. Conventional culture-based methods for enumerating rumen microorganisms (bacteria, archaea, protozoa, and fungi) have been superseded and are now used mainly to obtain pure isolates of novel organisms and reference strains that are required for the development and validation of the nucleic acid approaches. These reference strains are also essential for physiological studies of the lifestyle of the organisms as well as sources of genomic DNA and RNA that can be analysed for functional gene activity. The foundation of the molecular ecology techniques is 16S/18S rDNA sequence analysis which has provided a phylogenetically based classification scheme for enumeration and identification of microbial community members. The use of this marker gene in assays involving the use of single nucleic acid probes or primer sets is rapidly evolving to high throughput approaches such as microarray analysis and new generation sequencing technologies. While these analyses are very informative for determining the composition of the microbial community and monitoring changes in population size, they can only infer function based on these observations. The focus of nucleic acid research is now shifting to the functional analysis of the ecosystem which involves the measurement of functional genes and their expression in the predominant or specific members of the rumen microbial community. Functional gene studies are less developed than 16S rDNA-based analysis of community structure. Also for gene expression studies there are inherent problems involved in extracting high quality RNA from digesta, and priming cDNA synthesis from bacterial mRNA. This paper reviews nucleic acid based molecular methods which have recently been developed for studying the structure and function of rumen microbial communities.

scholarly journals Guidance for laboratories performing molecular pathology for cancer patients

2014 ◽  
Vol 67 (11) ◽  
pp. 923-931 ◽  
Author(s):  
Ian A Cree ◽  
Zandra Deans ◽  
Marjolijn J L Ligtenberg ◽  
Nicola Normanno ◽  
Anders Edsjö ◽  
...  
Keyword(s):  
Molecular Pathology ◽  
Rna Extraction ◽  
Internal Audit ◽  
Turnaround Time ◽  
Careful Consideration ◽  
Internal Quality ◽  
Molecular Testing ◽  
Pathology Report ◽  
Sufficient Information ◽  
Dna And Rna

Molecular testing is becoming an important part of the diagnosis of any patient with cancer. The challenge to laboratories is to meet this need, using reliable methods and processes to ensure that patients receive a timely and accurate report on which their treatment will be based. The aim of this paper is to provide minimum requirements for the management of molecular pathology laboratories. This general guidance should be augmented by the specific guidance available for different tumour types and tests. Preanalytical considerations are important, and careful consideration of the way in which specimens are obtained and reach the laboratory is necessary. Sample receipt and handling follow standard operating procedures, but some alterations may be necessary if molecular testing is to be performed, for instance to control tissue fixation. DNA and RNA extraction can be standardised and should be checked for quality and quantity of output on a regular basis. The choice of analytical method(s) depends on clinical requirements, desired turnaround time, and expertise available. Internal quality control, regular internal audit of the whole testing process, laboratory accreditation, and continual participation in external quality assessment schemes are prerequisites for delivery of a reliable service. A molecular pathology report should accurately convey the information the clinician needs to treat the patient with sufficient information to allow for correct interpretation of the result. Molecular pathology is developing rapidly, and further detailed evidence-based recommendations are required for many of the topics covered here.

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