Guidelines for the Validation of Analytical Methods for Nucleic Acid Sequence‐Based Analysis for the FDA Foods Program, 1St Edition
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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. -
The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments Stephen A
Clinical Chemistry 55:4 611–622 (2009) Special Report The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments Stephen A. Bustin,1* Vladimir Benes,2 Jeremy A. Garson,3,4 Jan Hellemans,5 Jim Huggett,6 Mikael Kubista,7,8 Reinhold Mueller,9 Tania Nolan,10 Michael W. Pfaffl,11 Gregory L. Shipley,12 Jo Vandesompele,5 and Carl T. Wittwer13,14 BACKGROUND: Currently, a lack of consensus exists on SUMMARY: Following these guidelines will encourage how best to perform and interpret quantitative real- better experimental practice, allowing more reliable time PCR (qPCR) experiments. The problem is exac- and unequivocal interpretation of qPCR results. erbated by a lack of sufficient experimental detail in © 2009 American Association for Clinical Chemistry many publications, which impedes a reader’s ability to evaluate critically the quality of the results presented or to repeat the experiments. The fluorescence-based quantitative real-time PCR (qPCR)15 (1–3), with its capacity to detect and mea- CONTENT: The Minimum Information for Publication sure minute amounts of nucleic acids in a wide range of of Quantitative Real-Time PCR Experiments (MIQE) samples from numerous sources, is the enabling tech- guidelines target the reliability of results to help ensure nology par excellence of molecular diagnostics, life sci- the integrity of the scientific literature, promote con- ences, agriculture, and medicine (4, 5). Its conceptual sistency between laboratories, and increase experimen- and practical simplicity, together with its combination tal transparency. MIQE is a set of guidelines that de- of speed, sensitivity, and specificity in a homogeneous scribe the minimum information necessary for assay, have made it the touchstone for nucleic acid evaluating qPCR experiments. -
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. -
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. -
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. -
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. -
Technical Aspects and Recommendations for Single-Cell Qpcr
Molecular Aspects of Medicine xxx (2017) 1e8 Contents lists available at ScienceDirect Molecular Aspects of Medicine journal homepage: www.elsevier.com/locate/mam Technical aspects and recommendations for single-cell qPCR * Anders Ståhlberg a, , Mikael Kubista b, c a Sahlgrenska Cancer Center, Department of Pathology and Genetics, Institute of Biomedicine, The Sahlgrenska Academy, University of Gothenburg, Box 425, 40530 Gothenburg, Sweden b TATAA Biocenter, Odinsgatan 28, 41103 Gothenburg, Sweden c Laboratory of Gene Expression, Institute of Biotechnology, Czech Academy of Sciences, Prumyslova 595, 252 50 Vestec, Prague, Czech Republic article info abstract Article history: Single cells are basic physiological and biological units that can function individually as well as in groups Received 17 May 2017 in tissues and organs. It is central to identify, characterize and profile single cells at molecular level to be Received in revised form able to distinguish different kinds, to understand their functions and determine how they interact with 16 July 2017 each other. During the last decade several technologies for single-cell profiling have been developed and Accepted 24 July 2017 used in various applications, revealing many novel findings. Quantitative PCR (qPCR) is one of the most Available online xxx developed methods for single-cell profiling that can be used to interrogate several analytes, including DNA, RNA and protein. Single-cell qPCR has the potential to become routine methodology but the Keywords: Single-cell analysis technique is still challenging, as it involves several experimental steps and few molecules are handled. qPCR Here, we discuss technical aspects and provide recommendation for single-cell qPCR analysis. The Single-cell qPCR workflow includes experimental design, sample preparation, single-cell collection, direct lysis, reverse Single-cell workflow transcription, preamplification, qPCR and data analysis. -
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. -
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. -
Cov2-ID, a MIQE-Compliant Sub-20-Min 5-Plex RT-PCR Assay
www.nature.com/scientificreports OPEN CoV2‑ID, a MIQE‑compliant sub‑20‑min 5‑plex RT‑PCR assay targeting SARS‑CoV‑2 for the diagnosis of COVID‑19 Stephen Bustin1*, Amy Coward2, Garry Sadler2, Louise Teare2 & Tania Nolan1 Accurate, reliable and rapid detection of SARS‑CoV‑2 is essential not only for correct diagnosis of individual COVID‑19 disease but also for the development of a rational strategy aimed at lifting confnement restrictions and preparing for possible recurrent waves of viral infections. We have used the MIQE guidelines to develop two versions of a unique fve plex RT‑qPCR test, termed CoV2‑ID, that allows the detection of three viral target genes, a human internal control for confrming the presence of human cells in a sample and a control artifcial RNA for quality assessment and potential quantifcation. Viral targets can be detected either individually with separate fuorophores or jointly using the same fuorophore, thus increasing the test’s reliability and sensitivity. It is robust, can consistently detect two copies of viral RNA, with a limit of detection of a single copy and can be completed in around 15 min. It was 100% sensitive and 100% specifc when tested on 23 RNA samples extracted from COVID‑19 positive patients and fve COVID‑19 negative patients. We also propose using multiple cycle fuorescence detection, rather than real‑time PCR to reduce signifcantly the time taken to complete the assay as well as assuage the misunderstandings underlying the use of quantifcation cycles (Cq). Finally, we have designed an assay for the detection of the D614G mutation and show that all of the samples isolated in the Chelmsford, Essex area between mid‑April and June 2020, have the mutant genotype whereas a sample originating in Australia was infected with the wild type genotype. -
BMC Molecular Biology
BMC Molecular Biology This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. MIQE precis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments BMC Molecular Biology 2010, 11:74 doi:10.1186/1471-2199-11-74 Stephen A Bustin ([email protected]) Jean-Francois Beaulieu ([email protected]) Jim Huggett ([email protected]) Rolf Jaggi ([email protected]) Frederick SB Kibenge ([email protected]) Pal A Olsvik ([email protected]) Louis C Penning ([email protected]) Stefan Toegel ([email protected]) ISSN 1471-2199 Article type Editorial Submission date 13 September 2010 Acceptance date 21 September 2010 Publication date 21 September 2010 Article URL http://www.biomedcentral.com/1471-2199/11/74 Like all articles in BMC journals, this peer-reviewed article was published immediately upon acceptance. It can be downloaded, printed and distributed freely for any purposes (see copyright notice below). Articles in BMC journals are listed in PubMed and archived at PubMed Central. For information about publishing your research in BMC journals or any BioMed Central journal, go to http://www.biomedcentral.com/info/authors/ © 2010 Bustin et al. , licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. -
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 ...............................