Ez1 robot

Manufactured by Qiagen

Sourced in Germany, Spain, United States

The EZ1 robot is an automated sample preparation system designed for nucleic acid extraction and purification. It performs a range of essential laboratory tasks, including DNA, RNA, and viral nucleic acid extraction, with consistent and reliable results. The EZ1 robot's core function is to simplify and streamline the sample preparation process, enabling efficient and reproducible sample processing.

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11 protocols using ez1 robot

Maternal Cell-Free DNA Extraction

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Maternal blood samples were processed as previously described [5 (link)]. Total DNA was extracted from 1 ml of plasma using the MagNaPure Compact Nucleic Acid Isolation Kit I-Large Volume kit and the Total NA Plasma 1000 v3 protocol of the MagNaPure Compact (Roche, Mannheim, Germany). Final elution volume was 50μl.
Control DNAs included paternal genomic DNA (pDNA) and maternal genomic DNA (mDNA) obtained from peripheral blood and fetal DNA (fDNA) obtained from CVS or AF. Genomic pDNA and mDNA were extracted from 1 ml of peripheral blood using the MagNaPure Compact (Roche, Mannheim, Germany) and eluted in 200 μl. Fetal genomic DNA samples were extracted with the EZ1 Robot (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol.

Perlado S., Bustamante-Aragonés A., Donas M., Lorda-Sánchez I., Plaza J, & Rodríguez de Alba M. (2016). Fetal Genotyping in Maternal Blood by Digital PCR: Towards NIPD of Monogenic Disorders Independently of Parental Origin. PLoS ONE, 11(4), e0153258.

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Comprehensive Respiratory Pathogen Evaluation

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All recipients who developed signs and symptoms of a URTD and/or LRTD underwent a detailed virologic, bacterial, and fungal evaluation. When bronchoscopy was performed, a detailed microbiologic evaluation including respiratory viruses; bacterial, fungal, and acid-fast bacilli cultures; Aspergillus galactomannan assay; and detection of cytomegalovirus was performed. Patients with URTD symptoms underwent nasopharyngeal aspiration, nasopharyngeal swabs, or an induced sputum test, whereas BAL was performed in patients with an LRTD whenever possible.
All clinical samples were tested by RT-PCR using the Luminex xTAG RVP Fast v1 assay (Luminex, Austin, TX), as described in detail elsewhere [25] (link). Briefly, all specimens were received at the laboratory within 30 minutes of collection and were conserved at 4°C until processed (within 18 hours of receipt). Nucleic acid extraction was performed using the Qiagen EZ-1 viral extraction kit with a EZ1 Robot (Qiagen, Valencia, CA). The Luminex xTAG RVP Fast v1 assay can detect adenoviruses; HBoV; CoV 229E, HKU1, NL63, and OC43; influenza A virus/H₁N₁, influenza A virus/H₃N₂, and other influenza A viruses (non-subtypifiable); influenza B virus; HMPV A and B; HPiV 1, 2, 3, and 4A-4B; RSV A-B; and enterovirus/rhinovirus (EvRh).

Piñana J.L., Madrid S., Pérez A., Hernández-Boluda J.C., Giménez E., Terol M.J., Calabuig M., Navarro D, & Solano C. (2017). Epidemiologic and Clinical Characteristics of Coronavirus and Bocavirus Respiratory Infections after Allogeneic Stem Cell Transplantation: A Prospective Single-Center Study. Biology of Blood and Marrow Transplantation, 24(3), 563-570.

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Multiplex PCR for MSI Analysis

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A multiplex PCR protocol was applied for MSI analysis. Samples for PCR were set up using the 2× Qiagen Multiplex PCR Kit to amplify five mononucleotide markers which were fluorescently labeled: BAT25(c-kit), BAT26(MSH2), NR21(SLC7A8), NR24(ZNF-2) and MONO27(MAP4K3) [6 (link)]. One microlitre of the template DNA extracted from formalin fixed tissue using the EZ1 Robot (Qiagen) was added to 25 ul PCR reaction. The following PCR programme was used to amplify selected markers: (Heated lid at 110 °C) 95 °C for 10 min, then 34 cycles of 94 °C for 1 min, 58 °C for 1 min and 72 °C 1 min, followed by 72 °C for 10 min. Following amplification, 1 ul of the PCR products were added to 0.5 ul of GeneScan™ 500 ROX™ Size Standard (an internal lane size standard for the Applied Biosystems fluorescence-based DNA electrophoresis systems) and 8.5 ul of formamide. Following denaturation (95 °C for 2 min and snap cooled on ice), the products were run on the 3130 genetic analyser (Applied Biosystems) and data processed using Genemarker software. Data supporting the conclusion of this article are included within the article.

Kerr L., Rewhorn M.J., Longmuir M., Fraser S., Walsh S., Andrew N, & Leung H.Y. (2016). A cohort analysis of men with a family history of BRCA1/2 and Lynch mutations for prostate cancer. BMC Cancer, 16, 529.

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Automated DNA extraction and array CGH analysis

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DNA extraction was done with the EZ1® robot and EZ1® DNA tissue kit (Qiagen, Hilden, Germany). The automatic protocol was done following the manufacturer’s instructions. For the purpose, 1 ml of cell suspension from the culture was used. The microarray-based comparative genomic hybridation (array CGH) technique was done using the PerkinElmer/Signature Genomics 8x60K platform following the manufacturer’s protocol (CGX™ Array training guide, version 1.2, 8x60K). Data analysis and interpretation were done with the Genoglyphix® program (Genoglyphix user guide 3.0) and following the European guidelines for constitutional cytogenomic analysis.

Hervás-Salcedo R., Fernández-García M., Hernando-Rodríguez M., Quintana-Bustamante O., Segovia J.C., Alvarez-Silva M., García-Arranz M., Minguez P., del Pozo V., de Alba M.R., García-Olmo D., Ayuso C., Lamana M.L., Bueren J.A, & Yañez R.M. (2021). Enhanced anti-inflammatory effects of mesenchymal stromal cells mediated by the transient ectopic expression of CXCR4 and IL10. Stem Cell Research & Therapy, 12, 124.

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Viral RNA Extraction and Reverse Transcription from Stool

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Viral RNA was extracted from 200 µl of 1:10 stool suspensions using High Pure Viral RNA Kit (Roche Diagnostics) following the manufacturer's instructions. A total of 50 µl of RNA was collected and stored at −20 °C until reverse transcription. Reverse transcription (RT) was carried out as described previously and cDNA was stored at −20 °C until used [Bucardo et al., 2008 (link)]. RNA from household and asymptomatic samples from 2010 and 2011 were purified by using automated RNA purification (either EZ1 robot (QIAGEN) or KingFisher magnetic particle processor (Thermo Fisher Scientific).

Bucardo F., Reyes Y., Becker-Dreps S., Bowman N., Gruber J.F., Vinjé J., Espinoza F., Paniagua M., Balmaseda A., Svensson L, & Nordgren J. (2017). Pediatric norovirus GII.4 infections in Nicaragua, 1999–2015. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 55, 305-312.

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BRAF Codon 600 Mutation Detection

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The region of BRAF codon 600 (exon 15) was sequenced using the Pyrosequencer (PSQ 96MA) and associated software (Qiagen, UK). Tumour DNA was extracted from the FFPE tissue using the Qiagen EZ1 robot in conjuction with the Tissue Extraction kit (Qiagen, UK) as per the manufacturer’s guidelines. Codon 600 was amplified using a non-nested 25µl polymerase chain reaction. Each sample was tested in triplicate. The amplicons were sequenced using the Pyrosequencer (PSQ 96MA) and associated software. Sequencing primer 5’-TGATTTTGGTCTAGCTACA-3’. Pyrograms were genotyped by two independent blinded scientists.

Newton K., Jorgensen N., Wallace A., Buchanan D., Lalloo F., McMahon R., Hill J, & Evans D. (2014). Tumour MLH1 promoter region methylation testing is an effective pre-screen for Lynch Syndrome (HNPCC). Journal of medical genetics, 51(12), 789-796.

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Filovirus RNA Extraction Protocol

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Viruses used in this study include the ebolaviruses SUDV, EBOV, RESTV, TAFV, and BDBV. MARV variants included Musoke, Ci67, and Angola. All viruses are maintained at USAMRIID, and IRB approval was not required for use. For each of the filoviruses, total RNA was purified from the supernatant of virus-infected cells using TRIzol (Life Technologies, Grand Isle, NY). Total nucleic acid from 100 µl cell culture supernatant was isolated directly from the TRIzol mixture using the EZ1 Virus 2.0 kit (Qiagen, Valencia, CA) and the EZ1 robot (Qiagen) according to the manufacturer's directions. Nucleic acid was eluted in a final volume of 60 µl elution buffer.

Koehler J.W., Hall A.T., Rolfe P.A., Honko A.N., Palacios G.F., Fair J.N., Muyembe J.J., Mulembekani P., Schoepp R.J., Adesokan A, & Minogue T.D. (2014). Development and Evaluation of a Panel of Filovirus Sequence Capture Probes for Pathogen Detection by Next-Generation Sequencing. PLoS ONE, 9(9), e107007.

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Bat Tissue DNA Extraction and Sequencing

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A pool of approximately 1 mm³ of frozen kidney, lung, and spleen tissue from individual bat specimens was crushed in DMEM medium using two 3-mm tungsten beads in a TissueLyser II (Qiagen, Valencia, CA, USA) [13 (link)]. Subsequently, the mixture was centrifuged at 10,000 rpm for 5 min and the supernatant used for DNA extraction. Genomic DNA was extracted using an EZ1 robot with the viral mini kit v2.0 according to manufacturer’s protocol (Qiagen, Valencia, CA, USA). A 648-bp fragment of the mitochondrial cytochrome C oxidase subunit I (COI) gene was PCR amplified with primers COIintF and COIintR as described elsewhere [22 (link), 39 (link), 40 (link)]. All PCRs were conducted in 25-μl reactions containing 12.5 μl of GoTaq Hot Start Green Master Mix (Promega, Madison, WI, USA), 1 μl of each primer, 1 μl of DNA template, and 9.5 μl of nuclease-free water. The amplification profile was 94°C for 5 min followed by 40 cycles of 45 s at 94°C, 45 s at 52° and 90 s at 72°C. Five μl of each PCR product was loaded on a 1.7% agarose gel stained with 1X GelRed dye (Biotium Inc., CA, USA) and visualized after electrophoresis under UV light. Amplicons were sequenced at Genoscreen (Lille, France) using both forward and reverse primers.

Ramasindrazana B., Dellagi K., Lagadec E., Randrianarivelojosia M., Goodman S.M, & Tortosa P. (2016). Diversity, Host Specialization, and Geographic Structure of Filarial Nematodes Infecting Malagasy Bats. PLoS ONE, 11(1), e0145709.

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Nucleic Acid Extraction and Quantification Protocol

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All organisms used in this study (listed in S1 File) are maintained at United States Army Medical Research Institute of Infectious Diseases (USAMRIID) or were provided by the Unified Culture Collection (UCC) or the American Type Culture Collection (ATCC, Manassas, VA). Samples included bacterial, parasite DNA, cell culture supernatant from virus-infected cells treated with TRIzol LS (ThermoFisher Scientific, Waltham, MA) or gamma irradiation. Total nucleic acid from each unpurified sample was extracted using the EZ1 Virus Mini Kit v2.0 (Qiagen, Valencia, CA) with the EZ1 robot (Qiagen) according to the manufacturer’s instructions. Total nucleic acid was eluted in 90 μl elution buffer.
Due to a limited supply, Coxiella burnetii DNA was amplified using the REPLI-g Whole Genome Amplification Kit (Qiagen) according to the manufacturer’s instructions. The number of C. burnetii genome equivalents (GE) was approximated using the genome of C. burnetii RSA493 (GenBank# NC_002971) and the C+G (42.7%) and A+T (57.3%) genome percentages. Based on these calculations, 1 GE is approximately 2.05 fg. The approximate number of GE for Plasmodium falciparum 3D7 DNA (ATCC) was similarly determined to be approximately 23.89 fg.

Koehler J.W., Douglas C.E, & Minogue T.D. (2018). A highly multiplexed broad pathogen detection assay for infectious disease diagnostics. PLoS Neglected Tropical Diseases, 12(11), e0006889.

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SARS-CoV-2 RNA Extraction and Sensitivity Testing

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RNA from the cell culture supernatant was extracted using TRIzol LS (ThermoFisher) and the Qiagen EZ1 robot with the EZ1 Virus Mini Kit (Qiagen, Valencia, Ca) according the manufacturer’s directions. Sensitivity testing was conducted for each assay using total nucleic acid isolated from cell culture supernatants from PICV and PTV infected Vero E6 cells. These supernatants were previously titered by plaque assay, so the limit of detection (LOD) was determined based on the number of PFU/ml before sample extraction with the EZ-1 kits. For analytical experiments, extracted RNA was serially diluted 10-fold into water, and the diluted RNA was run with each assay in triplicate. The preliminary LOD was determined based on 3/3 replicates being positive (<40 Cq), and 60 replicates at this preliminary LOD was conducted for LOD confirmation. For mock clinical samples, cell culture supernatant was 10-fold serially diluted in water, serum, and whole blood (BioreclamationIVT, Baltimore, MD), and each dilution was extracted using TRIzol LS and the EZ1 (Qiagen) according to the manufacturer’s instructions.

Stefan C.P., Chase K., Coyne S., Kulesh D.A., Minogue T.D, & Koehler J.W. (2016). Development of real-time reverse transcriptase qPCR assays for the detection of Punta Toro virus and Pichinde virus. Virology Journal, 13, 54.

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