DOCSLIB.ORG

DNA / RNA Extraction DNA / RNA Extraction – Key to Successful Analysis

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
DNA / RNA Extraction DNA / RNA Extraction – Key to Successful Analysis BIOTECON Diagnostics DNA / RNA Extraction DNA / RNA Extraction – Key to Successful Analysis StarPrep Kits ShortPrep Kits Sample Preparation Kits Magnetic Preparation Kits Fast and easy lysis by mixing samples with buffer High purity DNA/RNA clean-up, manual column based or automated magnetic bead based Reliable analysis of food and feed samples with real-time extraction kits have been optimized in combination with PCR require appropriate sample preparation. Optimized our foodproof® real-time PCR Detection, Identification and and validated methods for the extraction of nucleic acids Quantification Kits. The isolated DNA/RNA can be used from any kind of biological material are of high importance. directly in all our real-time PCR assays. The processed sample must be free of contaminants, especially inhibitors, while retaining small amounts of Direct Lysis, Column Based or Automated the target’s genetic information. The extraction method The foodproof® ShortPrep, StarPrep, Sample Preparation should be as fast and convenient as possible without and Magnetic Preparation Kits assist you in efficiently compromising safety or reproducibility. working with all types of sample matrices, including difficult complex samples like e.g. cheeses, spices or cacao. Optimized Combination of Extraction Kit and PCR Assay BIOTECON Diagnostics has developed a broad range of With fast, straight-forward and easy-to-use solutions, you fast, safe and easy-to-use DNA and RNA extraction kits for can overcome the challenges of complex matrices, PCR all kinds of food, beverage or environmental samples. Our inhibitors and hidden targets. 2 DNA / RNA Extraction – Key to Successful Analysis Table 1: Comparison of foodproof® extraction kits for food and beverage-relevant parameters Rapid Highly purified Target Bead based/ Bulk Prefilled Column based Automated Sample Magnetic Gram-negative bacteria StarPrep One* ShortPrep I Preparation I Preparation I, V Sample Magnetic Gram-positive bacteria StarPrep Two* ShortPrep II Preparation II Preparation II Enterobacteriaceae, Magnetic StarPrep One - - Cronobacter Preparation IV Yeast & Mold, Dekkera, S. StarPrep Two - - - cerevisiae var. diastaticus Vibrio, STEC, StarPrep Three* - - - Beer spoilage bacteria Sample Magnetic GMOs, Animal ID, Allergens StarPrep Five** - Preparation III Preparation III Sample Magnetic Viruses - - Preparation IV Preparation Kit VI * High-throughput protocol available. See 8-strip DNA extraction protocols. **Only for Animal ID 3 StarPrep Kits: Versatile WORKFLOW e.g. StarPrep One Enjoy convenient and flexible DNA StarPrep can be used for rapid DNA extraction: the foodproof® StarPrep extraction from enrichment cultures, Kits are designed for quick and direct and rinse samples. The reagents ADD SAMPLE easy extraction of DNA from food contain no hazardous chemicals or CENTRIFUGE 5 min DISCARD SUPERNATANT and beverage samples by heat- compounds, unlike other sample induced lysis. StarPrep is a cost- preparation methods. The entire DNA effective, versatile solution for nearly preparation can be performed in a ADD LYSIS BUFFER all kind of foods, with optimized lysis single tube, minimizing handling steps, reagents and protocols for different reducing risk of cross-contamination applications. and exposure to biohazardous material. StarPrep One Kit Bulk solution for fast and flexible DNA extraction MIX Table 2: Overview of foodproof® StarPrep Kits foodproof® StarPrep One foodproof® StarPrep Two foodproof® StarPrep Three foodproof® StarPrep Five Gram-positive and other Vibrio, INCUBATE bacteria, e.g. Listeria, 10 min STEC, beer spoilage Gram-negative bacteria, Campylobacter, E. coli Targets bacteria, Salmonella Animal ID e.g. Salmonella O157, Alicyclobacillus; spp. plus Enteritidis & Yeast and Mold, Typhimurium Legionella MIX Suitable for a wide range of food, animal feed and Enrichment cultures, Matrices Meat, processed food environmental samples beverage samples Mechanical disruption and Principle Heat-induced lysis Heat-induced lysis Heat-induced lysis CENTRIFUGE heat-induced lysis 1 min Isolations 100 / 500 96 96 96 Additional - Resuspension Reagent - - SUPERNATANT Products FOR DETECTION MicroVal, AOAC, Approvals AOAC, NordVal and DAFF AOAC-RI - NordVal and DAFF 4 ShortPrep Kits: Ready-to-Use WORKFLOW The fast and easy DNA extraction method for your enriched e.g. ShortPrep I samples: the foodproof® ShortPrep Kits are designed for quick and easy DNA extraction of bacterial DNA to be used directly in PCR. Simply add enriched sample to pre-aliquoted buffer, and in less than 30 minutes, samples from bacterial enrichment cultures are ready for PCR. The entire extraction ADD SAMPLE is performed in a single tube, minimizing handling steps and to prefilled lysis buffer reducing risk of cross-contamination. The lysis buffers are free of dangerous reagents. The kits are validated and certified by internationally MIX recognized institutions such as AOAC, NordVal and DAFF, and can be used for a variety of foods, like raw materials or processed food, environmental samples and pet foods. ShortPrep I Kit Prefilled tubes for fast DNA extraction INCUBATE in less than 30 minutes 10 min Table 3: Overview of foodproof® ShortPrep Kits foodproof® ShortPrep I foodproof® ShortPrep II MIX Gram-positive and other bacteria, e.g. Listeria, Targets Gram-negative bacteria, e.g. Salmonella Campylobacter and Alicyclobacillus Matrices* Suitable for a wide range of food, animal feed and environmental samples CENTRIFUGE Principle Heat-induced lysis Mechanical disruption and heat-induced lysis 1 min Convenience 96 individual ready-to-use reaction mixes for maximum efficiency and easy DNA extraction Approvals AOAC, NordVal and DAFF SUPERNATANT FOR DETECTION * DNA extracts from some raw materials of plant origin (e.g. spices) and from some beverage samples may still contain substances that interfere with PCR amplification or with fluorescent signal detection. For such materials, preparation and purification with the BIOTECON Diagnostics’ foodproof Sample Preparation Kit I and II are recommended. 5 Sample Preparation Kits: High Quality Purification WORKFLOW e.g. Sample Preparation III Kit* Get rid of unwanted PCR contaminants and inhibitors: the foodproof® Sample Preparation Kits are designed to isolate and purify DNA or RNA even from challenging ADD EXTRACTION BUFFER matrices containing high amounts of PCR inhibitors. Kit to sample reagents and consumables are optimized to obtain the best results for each parameter. Protocols are easy to Sample Preparation III Kit MIX follow and implement in daily routine use. The quality of the INCUBATE 30 min Column-based DNA obtained with the kits is suitable for any application extraction for CENTRIFUGE 10 min using real-time PCR. highly purified DNA TRANSFER SUPERNATANT ADD BINDING BUFFER & PROTEINASE K Table 4: Overview of foodproof® Sample Preparation Kits INCUBATE 10 min ADD ISOPROPANOL foodproof® Sample foodproof® Sample foodproof® Sample foodproof® Sample Preparation I Kit Preparation II Kit Preparation III Kit Preparation IV Kit TRANSFER SUPERNATANT to filter column Gram-positive and other Genetically modified CENTRIFUGE 1 min Gram-negative bacteria, bacteria, e.g. Listeria, organisms (GMOs), Viruses Targets DISCARD FLOW THROUGH e.g. Salmonella Campylobacter and animal identification (e.g. Norovirus) REPEAT ONCE Alicyclobacillus allergens ADD WASH BUFFER Matrices Suitable for a wide range of food, beverages, animal feed and environmental samples CENTRIFUGE 1 min The RNA is bound to a DISCARD FLOW THROUGH The DNA is bound to a glass fiber filter, washed thoroughly and the pure DNA is eluted Principle glass fiber filter, washed REPEAT ONCE from the columns and eluted ADD ELUTION BUFFER Isolation of highly purified DNA, virtually free of other cellular components and PCR inhibitors CENTRIFUGE 1 min Isolation of viral RNA, Features Suitable for matrices containing high amount of PCR inhibitors FLOW THROUGH process control included FOR DETECTION Sample Preparation Kit II and Sample Preparation Kit III include a lysozym and Proteinase K digestion step, respectively, for maximum performance * Workflow for GMOs & allergens; animal ID slightly different protocol 6 Sample Preparation Kits: High Quality Purification Magnetic Preparation Kits: Automated Purification foodproof RoboPrep® BIOTECON Diagnostics offers a variety of solutions The foodproof RoboPrep® 32 is perfect for automated high-throughput molecular testing for for labs with low to medium sample pathogens, GMOs, animal identification and allergens. throughput allowing the automated The foodproof® Magnetic Preparation Kits are based on extraction of up to 32 samples magnetic bead technology, providing ultra-pure, ready- simultaneously. Despite being small and to-use genomic DNA of bacterial, plant or animal origin economic, the RoboPrep® 32 offers the for enhanced performance in sensitive real-time PCR Magnetic Preparation Kit I full spectrum in terms of efficiency and applications. The process proceeds with DNA being bound Bead-based convenience. Nucleic acid extraction with to magnetic beads and washed thoroughly through multiple DNA extraction magnetic beads is combined with heating steps. In the final step, highly purified DNA is eluted from for automation blocks enhancing sample lysis and the beads and is ready for use. elution. The result is highly purified DNA / Table 5: Overview of foodproof® Magnetic Preparation Kits RNA, free of inhibitory components, which can be directly used for PCR assays. MPK I MPK II MPK III MPK IV
Load more

Recommended publications
  • RNA Isolation and Purification for Every Sample, RNA Type, and Application Isolate and Purify RNA with Confidence
    RNA isolation and purification For every sample, RNA type, and application Isolate and purify RNA with confidence RNA isolation is a crucial step in your quest to understand gene expression levels. With all the solutions that Thermo Fisher Scientific has to offer, you can be confident that you’re getting started on the right foot. Go ahead and push the limits of your research. We’ll be there to support you with robust RNA purification kits, trusted RNA tools, and experienced technical support, all backed by nearly 30 years of leadership and innovation in RNA technologies. • Isolate from any sample type, for any application • Obtain high-purity, intact RNA • Achieve high yields, even from small sample quantities Contents Methods of RNA isolation 4 RNA and sample types 5 RNA applications 6 Organic RNA extraction 8 Spin column RNA extraction 10 Lysate-based RNA extraction 11 Automated RNA purification 12 Transcriptome purification 14 mRNA purification 15 MicroRNA and small RNA purification 16 Viral RNA purification 18 RNA from FFPE samples 20 RNA isolation technology guide 22 Avoiding RNA degradation 26 Tips for handling RNA 27 RNA quantitation 28 Gene expression solutions 30 Gene expression research considerations 31 Superior cDNA synthesis for any application 32 Complete kit with flexible priming options 33 Which instrument fits your needs? 34 Which thermal cycler or PCR instrument fits your needs? 35 RNA technical resources 36 Services and support 37 Ordering information 38 Methods of RNA isolation For every application, sample, and RNA type For over three decades, our scientists have developed and professional support. Our RNA isolation products innovative and robust RNA isolation products designed include organic reagents, columns, sample lysate, and to make your job as a scientist easier.
    [Show full text]
  • Virus Purification, Rna Extraction, and Targeted
    VIRUS PURIFICATION, RNA EXTRACTION, AND TARGETED GENOME CAPTURE IN ONE CHIP Miyako Niimi1*, Taisuke Masuda1, Kunihiro Kaihatsu2, Nobuo Kato2, and Fumihito Arai1 1Nagoya University, JAPAN and 2Osaka University, JAPAN ABSTRACT In this research, we demonstrated a microfluidic chip to pretreat the samples for viral genome assay. The microfluidic chip has the following three functions; (1) Virus purification and enrichment, (2) Viral RNA extraction, and (3) Capture of the targeted virus genome. (1) Hydroxyapatite chromatography, Boom method, and PNA (2) (Peptide Nucleic Acid) were used for the above three (3) functions, respectively. These three functions were integrated in one chip. Furthermore PNA immobilized on the glass can detect the targeted virus genome so that in situ virus detection would be possible by anybody, anywhere, anytime. KEYWORDS: Virus purification, RNA extraction and detection, Infectious disease diagnosis INTRODUCTION For the purpose of diagnosing the infectious diseases Figure 1. Concept of the microfluidic chip. The microflu- quickly and accurately, DNA sequencers for gene analysis idic chip consists of the three parts: (1) hydroxyapatite- of infectious viruses have been developed rapidly. The packed microcolumn for virus purification, (2) silica- latest DNA sequencers can treat the massive numbers of packed microcolumn for viral RNA extraction, and (3) samples such as saliva and nasal at one time. However, it PNA immobilized glass for capture of the targeted virus is necessary to purify and enrich the virus and extract the genome. viral RNA in the sample as the pretreatments before gene (1) (2) analysis. Hydroxyapatite chromatography[1] have been Sample Elution Buffer used extensively for purification and fractionation of Hydroxy- various biochemical substances such as protein and virus.
    [Show full text]
  • A Suitable RNA Preparation Methodology for Whole Transcriptome Shotgun Sequencing Harvested from Plasmodium Vivax‑Infected Patients Catarina Bourgard1, Stefanie C
    www.nature.com/scientificreports OPEN A suitable RNA preparation methodology for whole transcriptome shotgun sequencing harvested from Plasmodium vivax‑infected patients Catarina Bourgard1, Stefanie C. P. Lopes2,3, Marcus V. G. Lacerda2,3, Letusa Albrecht1,4* & Fabio T. M. Costa1* Plasmodium vivax is a world‑threatening human malaria parasite, whose biology remains elusive. The unavailability of in vitro culture, and the difculties in getting a high number of pure parasites makes RNA isolation in quantity and quality a challenge. Here, a methodological outline for RNA‑ seq from P. vivax isolates with low parasitemia is presented, combining parasite maturation and enrichment with efcient RNA extraction, yielding ~ 100 pg.µL−1 of RNA, suitable for SMART‑Seq Ultra‑Low Input RNA library and Illumina sequencing. Unbiased coding transcriptome of ~ 4 M reads was achieved for four patient isolates with ~ 51% of transcripts mapped to the P. vivax P01 reference genome, presenting heterogeneous profles of expression among individual isolates. Amongst the most transcribed genes in all isolates, a parasite‑staged mixed repertoire of conserved parasite metabolic, membrane and exported proteins was observed. Still, a quarter of transcribed genes remain functionally uncharacterized. In parallel, a P. falciparum Brazilian isolate was also analyzed and 57% of its transcripts mapped against IT genome. Comparison of transcriptomes of the two species revealed a common trophozoite‑staged expression profle, with several homologous genes being expressed. Collectively, these results will positively impact vivax research improving knowledge of P. vivax biology. Plasmodium vivax is the most prevalent malaria parasite outside Sub-Saharan Africa, causing the most geographi- cally widespread type of malaria, placing millions of people at risk of infection 1.
    [Show full text]
  • RNA Extraction and Quantification, Reverse Transcription, and Real
    RNA Extraction and Quantification, Reverse Transcription, and Real-time PCR (q-PCR) General Surgery Labs SOP 9-10-2014 Sladjana Stratomirovic RNA Extraction and Quantification, Reverse Transcription, and Real-time PCR (q-PCR) Preparation of Samples Cells: o Remove media and wash cells 2X with cold PBS. (2 ml for 6 well plate or 3 ml for 6cm plate) . Keep on ice at all times! o Add 200-500 ul cold PBS o Scrape cells and collect a 1.5 eppendorf tube o Spin down to collect pellet at 12000 rpm for 10 min (4 degree) o Remove PBS completely . store the pellet at -80 freezer if you are not extracting RNA immediately Snap frozen tissue: o Homogenize tissue by placing it in liquid nitrogen-cooled aluminum foil and crushing it into fine pieces (using a hammer) for use in Western blots, RNA extractions etc. Only a small amount of tissue is needed for RNA extraction (20-30 mg) o The rest of the homogenized tissue can be aliquoted and stored at -80. RNA Extraction RNA extraction is done using different RNA extraction kits specific for certain cells, tissue, blood, etc. You can learn more about different kits for different tissues on the Qiagen website: http://www.qiagen.com. The RNeasy Mini Kit from Qiagen is the kit used most often in our lab. The kit is supplied with a Quick–Start protocol for RNA extraction. You can find more detailed explanation in the Qiagen handbook on their website:www.qiagen.com/handbooks. Notes before start using Qiagen kit: Add 10ul β –mercacptoethanol (β-ME) to 1ml RLT buffer provided in the kit.
    [Show full text]
  • Development of an RNA Extraction Protocol for Norovirus from Raw Oysters and Detection by Qrt-PCR and Droplet-Digital RT-PCR
    foods Article Development of an RNA Extraction Protocol for Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR Daniel Plante 1 , Julio Alexander Bran Barrera 1, Maude Lord 1, Irène Iugovaz 1 and Neda Nasheri 2,3,* 1 Microbiology Laboratory, Regulatory Operations and Enforcement Branch, Health Canada, 1001 St-Laurent Street West, Longueil, QC J4K 1C7, Canada; [email protected] (D.P.); [email protected] (J.A.B.B.); [email protected] (M.L.); [email protected] (I.I.) 2 National Food Virology Reference Centre, Bureau of Microbial Hazards, Food Directorate, Health Canada, 251 Sir Frederick Banting Driveway, Ottawa, ON K1A 0K9, Canada 3 Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada * Correspondence: [email protected] Abstract: Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load Citation: Plante, D.; Bran Barrera, J.A.; Lord, M.; Iugovaz, I.; Nasheri, N. quantification was performed by both qRT-PCR and droplet-digital RT-PCR.
    [Show full text]
  • Bacterial RNA Extraction and Purification from Whole Human
    Technical Note pubs.acs.org/ac Bacterial RNA Extraction and Purification from Whole Human Blood Using Isotachophoresis Anita Rogacs, Yatian Qu, and Juan G. Santiago* Department of Mechanical Engineering, Stanford University, Stanford, California, 94305 *S Supporting Information ABSTRACT: We demonstrate a novel assay for physico- chemical extraction and isotachophoresis-based purification of 16S rRNA from whole human blood infected with Pseudomonas putida. This on-chip assay is unique in that the extraction can be automated using isotachophoresis in a simple device with no moving parts, it protects RNA from degradation when isolating from ribonuclease-rich matrices (such as blood), and produces a purified total nucleic acid sample that is compatible with enzymatic amplification assays. We show that the purified RNA is compatible with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and demonstrate a clinically relevant sensitivity of 0.03 bacteria per nanoliter using RT-qPCR. ucleic acid amplification methods, including reverse magnetic beads. These approaches are well-established in N transcription-quantitative polymerase chain reaction traditional settings but require multiple reagent wash steps and (RT-qPCR), targeting the 16S rRNA (16S rRNA) enable fast specialized fabrication or manipulation (e.g., pumping liquids and specific detection of bacteria in complex samples such as for wash steps or moving magnets for beads). Further, SPE whole blood.1,2 16S rRNA is a universal constituent of bacterial binding capacity can be low due to competitive protein ribosomes present at high copy numbers (103−104 per actively absorptions (e.g., to silica) and the presence of PCR inhibiting 3,4 growing cell). Targeting these biomarkers can potentially chemistry such as guanidinium thiocyanate (GuCN), guanidi- increase assay sensitivity compared with the assays targeting the nium chloride (GuHCl), isopropyl alcohol, or ethanol.
    [Show full text]
  • Rapid Plant DNA and RNA Extraction Protocol Using a Bench Drill Brief Note
    Rapid plant DNA and RNA extraction protocol using a bench drill Brief Note C.F. Ferreira1*, D.L. Gutierrez2*, J.F. Kreuze3, M.L. Iskra-Caruana4,5, M. Chabannes4,5, A.C.O. Barbosa6, T.A. Santos6, A.G.S. Silva6, R.M.F. Santos7, E.P. Amorim1, S.A.S. de Oliveira1 and O.N. Jesus1 1 Embrapa Mandioca e Fruticultura, Cruz das Almas, BA, Brasil 2 INIA - Instituto Nacional de Innovación Agraria, La Molina, Lima, Peru 3CIP - Centro Internacional de la Papa, Lima, Peru 4 CIRAD, UMR BGPI, F-34398, Montpellier, France 5 BGPI, CIRAD, Univ. Montpellier, INRA, Montpellier SupAgro, Montpellier, France 6 Universidade Federal do Recôncavo da Bahia, Cruz das Almas, BA, Brasil 7 Universidade Estadual de Feira de Santana, Feira de Santana, BA, Brasil * These authors contributed equally to this study. Corresponding author: C.F. Ferreira E-mail: [email protected] Genet. Mol. Res. 18 (3): gmr18394 Received June 12, 2019 Accepted July 18, 2019 Published August 14, 2019 DOI http://dx.doi.org/10.4238/gmr18394 ABSTRACT. Plant DNA and RNA extraction methods are well established, with a wide range of protocols, depending on the purposes of each laboratory/research. Nowadays, quick, inexpensive and easy plant DNA and RNA extraction methods are highly sought after. We developed an optimized protocol for plant DNA and RNA extraction that uses an inexpensive bench drill and plastic bags and does not require liquid nitrogen. DNA from leaves and RNA from leaves and roots of banana, pineapple, citrus, papaya, passion fruit and cassava, were extracted using a basic cetyltrimethylammonium bromide method.
    [Show full text]
  • Aquapreserve™ DNA/RNA/Protein Order # Preservation and Extraction Kit 8001MT, 8060MT
    AquaPreserve™ DNA/RNA/Protein Order # Preservation and Extraction Kit 8001MT, 8060MT © MoBiTec GmbH 2014 Page 2 Contents 1. Description .............................................................................................. 3 2. Kit Contents ............................................................................................. 3 3. Terms & Conditions ................................................................................ 3 4. AquaPreserve Blood DNA/RNA Extraction Protocol ............................ 4 4.1 Lyse the blood cells............................................................................ 4 4.2 Pellet the proteins. ............................................................................. 4 4.3 Pellet the DNA/RNA ........................................................................... 4 4.4 Rinse the DNA/RNA pellet ................................................................. 4 4.5 Solubilize the DNA/RNA pellet ........................................................... 4 5. AquaPreserve for Total Blood DNA/RNA Biobanking .......................... 5 5.1 Stabilize the blood sample. ................................................................ 5 5.2 Transport the blood sample................................................................ 5 5.3 Store the blood sample. ..................................................................... 5 5.4 Extract the DNA and RNA. ................................................................. 5 6. AquaPreserve Blood Protein Extraction Protocol ...............................
    [Show full text]
  • A Simple RNA Preparation Method for SARS-Cov-2 Detection by RT-Qpcr
    www.nature.com/scientificreports OPEN A simple RNA preparation method for SARS‑CoV‑2 detection by RT‑qPCR Aniela Wozniak1,5, Ariel Cerda2,5, Catalina Ibarra‑Henríquez2,5, Valentina Sebastian3, Grace Armijo2, Liliana Lamig2, Carolina Miranda3, Marcela Lagos1, Sandra Solari1, Ana María Guzmán1, Teresa Quiroga1, Susan Hitschfeld2, Eleodoro Riveras2, Marcela Ferrés1,4, Rodrigo A. Gutiérrez2* & Patricia García1* The technique RT‑qPCR for viral RNA detection is the current worldwide strategy used for early detection of the novel coronavirus SARS‑CoV‑2. RNA extraction is a key pre‑analytical step in RT‑qPCR, often achieved using commercial kits. However, the magnitude of the COVID‑19 pandemic is causing disruptions to the global supply chains used by many diagnostic laboratories to procure the commercial kits required for RNA extraction. Shortage in these essential reagents is even more acute in developing countries with no means to produce kits locally. We sought to fnd an alternative procedure to replace commercial kits using common reagents found in molecular biology laboratories. Here we report a method for RNA extraction that takes about 40 min to complete ten samples, and is not more laborious than current commercial RNA extraction kits. We demonstrate that this method can be used to process nasopharyngeal swab samples and yields RT‑qPCR results comparable to those obtained with commercial kits. Most importantly, this procedure can be easily implemented in any molecular diagnostic laboratory. Frequent testing is crucial for individual patient management as well as for public health decision making in this pandemic. Implementation of this method could maintain crucial testing going despite commercial kit shortages.
    [Show full text]
  • Establishment of the Total RNA Extraction System for Lily Bulbs with Abundant Polysaccharides
    African Journal of Biotechnology Vol. 10(78), pp. 17907-17915, 7 December, 2011 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB10.2523 ISSN 1684–5315 © 2011 Academic Journals Full Length Research Paper Establishment of the total RNA extraction system for lily bulbs with abundant polysaccharides Xueyan Li, Chunxia Wang, Hongmei Sun* and Tianlai Li Shenyang Agricultural University, College of Horticulture, Key Laboratory of Protected Horticulture of Liaoning Province, Shenyang, Liaoning 110161, China. Accepted 4 November, 2011 Isolating high quality RNA from lily bulbs is made difficult by the presence of plentiful polysaccharides. To solve this problem, an efficient and steady RNA extraction technique from different parts of scales in three hybrids lily bulbs has been developed through modifications of three common RNA extraction methods, cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and Trizol method. NaCl concentration of both CTAB and SDS protocols was increased in order to get rid of the polysaccharides in scales. The resulting RNA is of high-quality, as judged by agarose gel electrophoresis and UV-spectroscopic analysis. Furthermore, the middle scales obtain the highest RNA yield followed by the exterior and the interior scales. However, Trizol method was also improved to remove polysaccharides but failed. The results of reverse-transcription-polymerase chain reaction (RT- PCR) indicated that only the improved CTAB method is suitable for subsequent purposes, such as reverse transcription PCR and cDNA library construction. Key words: Lilium bulbs, polysaccharides-rich tissues, RNA isolation, improved cetyltrimethylammonium bromide (CTAB) method. INTRODUCTION Total RNA extraction from plant tissues is a crucial for example, seeds and abnormal organs, where problems prerequisite for plant molecular biology studies.
    [Show full text]
  • Standard Operating Procedures for Interlaboratory and Methods Assessment of the SARS‐Cov‐2 Genetic Signal in Wastewater
    Standard Operating Procedures for Interlaboratory and Methods Assessment of the SARS‐CoV‐2 Genetic Signal in Wastewater 2020 Introduction Interlaboratory and Methods Assessment of the SARS‐CoV‐2 Genetic Signal in Wastewater provides an assessment of the reliability and repeatability of laboratory methods currently being used to test for the genetic signal of SARS‐CoV‐2 in untreated wastewater. A number of the study’s participating labs agreed to publicly share their Standard Operating Procedures, and the ones provided here cover each of the eight major method groups described in the Environmental Science: Water Research & Technology manuscript about this project. Contents SOP 1.1 .......................................................................................................................................................... 1 SOP 1.2(H) ..................................................................................................................................................... 6 SOP 1S.1(H) ................................................................................................................................................. 20 SOP 1S.2H .................................................................................................................................................... 26 SOP 1S.3(H) ................................................................................................................................................. 31 SOP 2.1 .......................................................................................................................................................
    [Show full text]
  • Eukaryotic Operon-Like Transcription of Functionally Related Genes in Drosophila
    Eukaryotic operon-like transcription of functionally related genes in Drosophila Yehuda Ben-Shahar*†, Kishore Nannapaneni‡§, Thomas L. Casavant‡§¶ʈ, Todd E. Scheetz‡§¶, and Michael J. Welsh*†**†† *Howard Hughes Medical Institute, and Departments of †Internal Medicine, **Physiology and Biophysics, and ¶Ophthalmology and Visual Sciences, Roy J. and Lucille A. Carver College of Medicine Center for Bioinformatics and Computational Biology, Departments of ʈElectrical and Computer Engineering and §Biomedical Engineering, University of Iowa, Iowa City, IA 52242 Contributed by Michael J. Welsh, November 2, 2006 (sent for review October 2, 2006) Complex biological processes require coordinated function of CheB42a llz many genes. One evolutionary solution to the problem of coordi- % identity (100bp intervals) nately expressing functionally related genes in bacteria and nem- D. melanogaster atodes is organization of genes in operons. Surprisingly, eukaryotic D. simulans 100 operons are considered rare outside the nematode lineage. In 50 Drosophila melanogaster, we found lounge lizard (llz), which D. yakuba 100 encodes a degenerin/ENaC cation channel, cotranscribed with 50 CheB42a, a nonhomologous gene of unknown function residing 100 D. erecta <100 bp upstream. These two genes were transcribed from a single 50 promoter as one primary transcript and were processed posttran- 100 scriptionally to generate individual mRNAs. The mechanism did not D. pseudoobscura 50 involve alternative splicing, and it differed from the trans splicing used in nematode operons. Both genes were expressed in the same 0.5 Kb Exon tissues, and previous work suggested that both may be involved in UTR courtship behavior. A bioinformatic approach identified numerous additional loci as potential Drosophila operons. These data reveal Fig.
    [Show full text]