Killing two birds with one stone: simultaneous extraction of DNA and RNA from activated sludge biomass

1999 ◽  
Vol 45 (3) ◽  
pp. 269-272 ◽  
Author(s):  
Zhongtang Yu ◽  
William W Mohn
Keyword(s):  
Activated Sludge ◽  
Ammonium Acetate ◽  
Rna Extraction ◽  
Cell Lysis ◽  
Biomass Density ◽  
Simple Method ◽  
Dna And Rna ◽  
Acid Recovery ◽  
Bacterial Aggregate ◽  
Soil And Sediment

DNA and RNA are usually extracted from activated sludge samples using two separate methods developed for soil and sediment samples. However, activated sludge differs from soil and sediment in at least three aspects: high biomass density, low humic acid content, and the presence of bacterial aggregate flocs. Taking these characteristics into consideration, we developed a simple and rapid method allowing simultaneous DNA and RNA extraction from activated sludge samples. This method combines (i) mini-bead beating, which is most efficient in breaking bacterial aggregate flocs and cells, (ii) protection of RNA with diethyl pyrocarbonate, and (iii) precipitation of impurities with ammonium acetate. Phenol/chloroform extraction and column purification are not necessary. The resulting DNA and RNA are suitable for PCR and reverse transcriptase - PCR, respectively. The efficiencies of cell lysis and nucleic acid recovery were high enough to permit detection by PCR of 102cells/mL of mixed liquor. By simultaneously extracting both DNA and RNA from a single sample, this method eliminates variability in cell lysis between extraction of DNA and RNA using two different methods. This extraction method is rapid, and within 1 h, one person can process four or more samples. This simple method makes it easier to analyze a large number of activated sludge samples.Key words: activated sludge, DNA, extraction, PCR, RNA.

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.

Nitrification preservation in activated sludge during curative bulking chlorination

2003 ◽  
Vol 47 (11) ◽  
pp. 85-92 ◽  
Author(s):  
E. Cotteux ◽  
P. Duchene
Keyword(s):  
Activated Sludge ◽  
Pilot Plant ◽  
Wastewater Treatment Plants ◽  
Oxygen Uptake Rate ◽  
Ammonia Concentration ◽  
Synthetic Wastewater ◽  
Biological Wastewater Treatment ◽  
Simple Method ◽  
Method Of Measurement ◽  
The Impact

The bulking that occurs in biological wastewater treatment plants using activated sludge is very often controlled by the injection of sodium hypochlorite into the return activated sludge (RAS) stream. In the present study undertaken at two pilot plants fed with synthetic wastewater, the impact of the pass frequency of the sludge at the chlorine dosing point on the nitrifying flora is analysed. The pass frequency is one for the pilot plant 1 and two for the pilot plant 2. A dose of chlorine of 4.85 ± 0.05 g/kg/MLVSS per day was applied at both pilots. The preservative effect on nitrifying activity of the lowest concentration of chlorine at the dosing point and therefore of the highest pass frequency was evidenced. Among other tools, a simple method of measurement of the oxygen uptake rate enabled us to monitor the effect of chlorination on nitrification before recording an increase in the ammonia concentration in the bulking.

Estimation of Kinetic Parameters of Heterotrophic Biomass under Aerobic Conditions and Characterization of Wastewater for Activated Sludge Modelling

1992 ◽  
Vol 25 (6) ◽  
pp. 125-139 ◽  
Author(s):  
J. Kappeler ◽  
W. Gujer
Keyword(s):  
Wastewater Treatment ◽  
Activated Sludge ◽  
Kinetic Parameters ◽  
Specific Growth ◽  
Wastewater Treatment Plants ◽  
Simple Method ◽  
Batch Tests ◽  
Different Types ◽  
Heterotrophic Biomass

To predict the behaviour of biological wastewater treatment plants, the Activated Sludge Model No. 1 is often used. For the application of this model kinetic parameters and wastewater composition must be known. A simple method to estimate kinetic parameters of heterotrophic biomass and COD wastewater fractions is presented. With three different types of batch-tests these parameters and fractions can be determined by measuring oxygen respiration. Our measurements showed that the maximum specific growth rate µmax of heterotrophic biomass depends on temperature, reactor configuration and SRT. In typical wastewater treatment plants of Switzerland the amount of readily biodegradable substrate was generally small (about 9 % of the COD in primary effluent). The same method can also be used to determine kinetic parameters of nitrifying biomass.

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.

scholarly journals Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0240769
Author(s):  
Prasanna Channathodiyil ◽  
Jonathan Houseley
Keyword(s):  
Flow Cytometry ◽  
Transcriptome Analysis ◽  
Mammalian Cells ◽  
Single Cells ◽  
Rna Extraction ◽  
Cyclin B1 ◽  
Immunofluorescent Staining ◽  
Rna Seq ◽  
Simple Method ◽  
Staining Methods

A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal—a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells.

scholarly journals Optimisation of DNA and RNA extraction from archival formalin-fixed tissue

1999 ◽  
Vol 27 (16) ◽  
pp. i-iii ◽  
Author(s):  
N. J. Coombs ◽  
A. C. Gough ◽  
J. N. Primrose
Keyword(s):  
Rna Extraction ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Dna And Rna ◽  
Formalin Fixed

scholarly journals Determination of Total Interleukin-8 in Whole Blood after Cell Lysis

2000 ◽  
Vol 46 (9) ◽  
pp. 1387-1394 ◽  
Author(s):  
Jochen Reinsberg ◽  
Jörg Dembinski ◽  
Christoph Dorn ◽  
Daniela Behrendt ◽  
Peter Bartmann ◽  
...  
Keyword(s):  
Whole Blood ◽  
Healthy Subjects ◽  
Room Temperature ◽  
Sample Pretreatment ◽  
Cell Lysis ◽  
Interleukin 8 ◽  
Simple Method ◽  
Sample Handling ◽  
A Cell

Abstract Background: It has been shown that a high percentage of interleukin-8 (IL-8) in blood is cell associated. Recently, a simple method for determination of cell-associated IL-8 in whole blood after cell lysis has been described. The purpose of this study was to evaluate this method, to examine the influence of preanalytic sample handling, and to establish the concentration range of total IL-8 and its relation to age and sex in healthy subjects. Methods: Total IL-8 content of whole blood was determined after lysing blood cells with Milenia® cell lysis solution. IL-8 in the resulting blood lysate was measured with the IMMULITE® IL-8 immunoassay. Results: When freshly drawn blood was stored up to 48 h on ice, no significant changes in total IL-8 were measured in the subsequently prepared lysate, whereas with storage at room temperature, total IL-8 increased after 3 h from 94 ± 13 ng/L to 114 ± 16 ng/L (n = 10). In lysate stored for 48 h at 4 °C, marginal changes of the IL-8 concentration were noted, with storage at room temperature, only 76% ± 5% (n = 12) of initial concentration was recovered. From lysate frozen at −20 and −80 °C, respectively, 84% ± 4% and 93% ± 2% of initial IL-8 was recovered after 70 days (n = 10). IL-8 was measured with comparable precision in plasma (CV, 3.2–4.2%) and blood lysate (CV, 3.7–4.1%). When plasma was diluted with cell lysis solution, a slightly overestimated recovery (125% ± 3%) was observed; for lysate specimens with a cell lysis solution content ≥75%, the recovery after dilution was 98% ± 2%. In lysate prepared from 12 blood samples with exogenous IL-8 added, IL-8 recovery was 104% ± 2% (recovery from plasma <35%). The median total IL-8 in blood lysates from 103 healthy subjects (22–61 years) was 83 ng/L of blood (2.5–97.5 percentile range, 49–202 ng/L of blood). In females but not in males, total IL-8 increased significantly with advancing age (P <0.002). We found grossly increased total IL-8 in six pregnant women with amniotic infection syndrome. Conclusions: The evaluated method allows the assessment of total IL-8 in blood with good performance when appropriate conditions of sample pretreatment are considered. The values in healthy volunteers all were above the detection limit of the IL-8 assay; therefore, slight changes of total IL-8 could be noted. Thus, the present method is a suitable tool to study the diagnostic relevance of total IL-8 in blood.

scholarly journals Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride

BioTechniques ◽  
2020 ◽  
Vol 69 (3) ◽  
pp. 178-185 ◽  
Author(s):  
Yinhua Zhang ◽  
Guoping Ren ◽  
Jackson Buss ◽  
Andrew J Barry ◽  
Gregory C Patton ◽  
...  
Keyword(s):  
Guanidine Hydrochloride ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Simple Method ◽  
Diagnostic Assays ◽  
Loop Mediated Isothermal Amplification ◽  
Dna And Rna ◽  
Versatile Technique ◽  
Primer Sets

Loop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 pandemic has presented an urgent need for new and better diagnostic methods, with colorimetric LAMP utilized in numerous studies for SARS-CoV-2 detection. However, the sensitivity of colorimetric LAMP in early reports has been below that of the standard RT-qPCR tests, and we sought to improve performance. Here we report the use of guanidine hydrochloride and combined primer sets to increase speed and sensitivity in colorimetric LAMP, bringing this simple method up to the standards of sophisticated techniques and enabling accurate, high-throughput diagnostics.

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