Purelink micro to midi total rna purification kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PureLink Micro-to-Midi total RNA purification kit is a laboratory equipment product designed for the extraction and purification of total RNA from various sample types. It provides a standardized protocol for efficient RNA isolation, suitable for a wide range of sample sizes, from microscale to mid-scale applications.

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9 protocols using purelink micro to midi total rna purification kit

Hantavirus Detection in Tissue Samples

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Total RNA was extracted from 20–50 mg of tissue, using the PureLink Micro-to-Midi total RNA purification kit (Invitrogen, San Diego, CA). cDNA, synthesized using the SuperScript III First-Strand Synthesis Systems (Invitrogen), were analyzed for hantavirus RNA by RT-PCR, using oligonucleotide primers designed from highly conserved regions of hantavirus genomes (Table 2) (Song et al., 2007b (link), 2007c (link), 2009 (link); Kang et al., 2009a (link), 2009b (link); Gu et al., 2013 (link), 2014a (link), 2014b (link)).
Nested or hemi-nested PCR was performed in 20-μL reaction mixtures, containing 250 μM dNTP, 2.5 mM MgCl₂, 1 U of Takara LA Taq polymerase (Takara, Shiga, Japan) and 0.25 μM of each primer (Table 2). Initial denaturation at 94°C for 2 min was followed by two cycles each of denaturation at 94°C for 30 sec, two-degree step-down annealing from 46°C to 38°C for 40 sec, and elongation at 72°C for 1 min, then 30 cycles of denaturation at 94°C for 30 sec, annealing at 42°C for 40 sec, and elongation at 72°C for 1 min, in a GeneAmp PCR 9700 thermal cycler (Perkin-Elmer, Waltham, MA) (Arai et al., 2008b (link)). PCR products were separated, using MobiSpin S-400 spin columns (MoBiTec, Goettingen, Germany), and amplicons were sequenced directly using an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA).

Gu S.H., Hejduk J., Markowski J., Kang H.J., Markowski M., Połatyńska M., Sikorska B., Liberski P.P, & Yanagihara R. (2014). Co-circulation of Soricid- and Talpid-borne Hantaviruses in Poland. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 28, 296-303.

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Cardiac Gene Expression Profiling

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RNA was extracted from frozen cardiac tissues (n = 3/group) using the TRIzol® reagent in conjunction with the PureLink™ Micro-to-Midi Total RNA Purification kit (Invitrogen, CA, USA) with on-column DNase treatment (PureLink DNase, Invitrogen, CA, USA). The RNA was reverse transcribed using the RT² First Strand Kit (Qiagen, CA, USA) as per the manufacturer’s instructions. Real-time PCR was used to determine the relative gene expression levels using a customized RT² PCR Array plate (Qiagen, CA, USA) on a Bio-Rad CFX thermocycler (Bio-Rad, CA, USA). The comparative ΔΔCt method was used to analyze the genes of interest relative to the β-actin reference gene as described^{59 (link)}.

Chandramouli C., Reichelt M.E., Curl C.L., Varma U., Bienvenu L.A., Koutsifeli P., Raaijmakers A.J., De Blasio M.J., Qin C.X., Jenkins A.J., Ritchie R.H., Mellor K.M, & Delbridge L.M. (2018). Diastolic dysfunction is more apparent in STZ-induced diabetic female mice, despite less pronounced hyperglycemia. Scientific Reports, 8, 2346.

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Soricomorph-borne Hantavirus Detection

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Total RNA was extracted from mole lung tissues, using the PureLink Micro-to-Midi total RNA purification kit (Invitrogen, San Diego, CA), and cDNA was prepared using the SuperScript III First-Strand Synthesis System (Invitrogen) and random hexamers. PCR was performed as described previously using oligonucleotide primers designed from NVAV and other soricomorph-borne hantaviruses (Song et al., 2007 (link); Arai et al., 2008 (link)) and DNA sequencing was performed using an ABI Prism 377XL Genetic Analyzer (Applied Biosystems, Foster City, CA).

Hugot J.P., Gu S.H., Feliu C., Ventur J., Ribas A., Dormion J., Yanagihara R, & Nicolas V. (2014). Genetic Diversity of Talpa Europaea and Nova Hanta Virus (NVAV) in France. Bulletin de l'Academie veterinaire de France, 167(3), 10.4267/2042/54201.

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Knockdown of Dux in mESCs

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Chaf1a (s77588) and negative control Silencer Select siRNAs were purchased from LifeTechnologies. Dux siRNA pools were generated using Giardia Dicer. Briefly, primers were designed to amplify two ~400bp fragments of the endogenous Dux locus from genomic mouse DNA and add T7 handles (see Supplementary Table 14). Purified PCR products were then used as template for in vitro transcription using the MEGAscript® T7 Transcription Kit (ThermoFischer, AM1334). Template DNA was then degraded and the ssRNA allowed to anneal before dicing. Diced siRNAs were purified using the PureLink™ Micro-to-Midi Total RNA purification Kit (Invitrogen, 12183-018) with modifications. siRNA concentration was measured with the Qubit® RNA HS Assay Kit (ThermoFisher, Q32852). mESCs containing the MERVL:GFP reporter were transfected with 20pmol (10pmol of each) of total siRNA using RNAiMax (Life Technologies). All siRNA transfections were performed twice (on back to back days) to ensure knockdown.

Hendrickson P.G., Doráis J.A., Grow E.J., Whiddon J.L., Lim J.W., Wike C.L., Weaver B.D., Pflueger C., Emery B.R., Wilcox A.L., Nix D.A., Peterson C.M., Tapscott S.J., Carrell D.T, & Cairns B.R. (2017). Conserved roles for murine DUX and human DUX4 in activating cleavage stage genes and MERVL/HERVL retrotransposons. Nature genetics, 49(6), 925-934.

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Orthohantavirus Detection from Tissue Samples

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Total RNA was extracted from 20–50 mg of each tissue using the PureLink Micro-to-Midi total RNA purification kit (Invitrogen, San Diego, CA, USA). cDNA was prepared using the SuperScript III First-Strand Synthesis System (Invitrogen) and oligonucleotide primer (5’-TAGTAGTAGACTCC-3’) designed from the conserved 3’-end of the S, M, and L segments of orthohantaviruses.
Gene amplification was carried out in 20-μL reaction mixtures containing 250 μM dNTP, 2 mM MgCl₂, 1 U of AmpliTaq polymerase (Roche, Basel, Switzerland), and 0.25 μM of oligonucleotide primers, designed from highly conserved regions of previously identified soricid-borne orthohantaviruses. A listing of the oligonucleotide primers used to amplify the S-, M-, and L-genomic segments is provided in Table S1. Initial denaturation was followed by touchdown cycling (two-degree step-down annealing from 48 °C to 38 °C for 40 sec) and elongation at 72 °C for 1 min, then 32 cycles of denaturation at 94 °C for 40 sec, annealing at 42 °C for 40 sec, and elongation at 72 °C for 1 min, in a GeneAmp PCR 9700 thermal cycler (Perkin-Elmer, Waltham, MA, USA). Amplified products were separated by electrophoresis on 1.5% agarose gels and purified using the QIAQuick Gel Extraction Kit (Qiagen, Hilden, Germany). DNA was sequenced directly using an ABI Prism 377XL Genetic Analyzer (Applied Biosystems Inc., Foster City, CA, USA).

Liphardt S.W., Kang H.J., Dizney L.J., Ruedas L.A., Cook J.A, & Yanagihara R. (2019). Complex History of Codiversification and Host Switching of a Newfound Soricid-Borne Orthohantavirus in North America. Viruses, 11(7), 637.

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Giardia Dicer-generated siRNA Pools

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siRNA pools were generated with Giardia Dicer. Briefly, primers were designed to amplify two ~100–300-bp fragments of the indicated ORF from genomic mouse DNA and to add T7 handles (Supplementary Table 1). Purified PCR products were then used as a template for in vitro transcription with a MEGAscript T7 Transcription kit (Thermo Fisher, AM1334). Template DNA was then degraded, and the dsRNA was allowed to anneal before dicing. Diced siRNAs were purified with a PureLink Micro-to-Midi Total RNA purification kit (Invitrogen, 12183–018) with modifications. siRNA concentration was measured with a Qubit RNA HS Assay kit (Thermo Fisher, Q32852). mESCs containing the MERVL:GFP reporter were transfected with 20 pmol (10 pmol of each) of total siRNA with RNAiMAX (Life Technologies). 24 hours after siRNA transfection, mESCs were treated with doxorubicin or vehicle control for 6 hours, then washed and medium was replaced with 2iLIF for 18 hours. MERVL-GFP⁺ was measured as per below.

Grow E.J., Weaver B.D., Smith C.M., Guo J., Stein P., Shadle S.C., Hendrickson P.G., Johnson N.E., Butterfield R.J., Menafra R., Kloet S.L., van der Maarel S.M., Williams C.J, & Cairns B.R. (2021). p53 convergently activates Dux/DUX4 in embryonic stem cells and in facioscapulohumeral muscular dystrophy cell models. Nature genetics, 53(8), 1207-1220.

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Knockdown of Dux in mESCs

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Screening for NVAV in Mole Lung Tissues

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As an initial screening for NVAV infection, total RNA was extracted from 20–50 mg of mole lung tissues, using the PureLink Micro-to-Midi total RNA purification kit (Invitrogen, San Diego, CA), and cDNA was synthesized using the SuperScript III First-Strand Synthesis Systems (Invitrogen), then analyzed for NVAV RNA by RT-PCR, using NVAV-specific oligonucleotide primers33 (link)34 (link).

Gu S.H., Kumar M., Sikorska B., Hejduk J., Markowski J., Markowski M., Liberski P.P, & Yanagihara R. (2016). Isolation and partial characterization of a highly divergent lineage of hantavirus from the European mole (Talpa europaea). Scientific Reports, 6, 21119.

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Mammarenavirus Detection from Tissue Samples

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The total RNA was extracted from liver and kidney tissue fragments using the PureLink Micro-To-Midi total RNA Purification Kit (Invitrogen, San Diego, CA, USA) according to the manufacturer’s protocol. Mammarenavirus detection was performed according to previously described protocols targeting fragments of GPC and NP genes from the S segment of mammarenaviruses^{8 (link),44 (link)}.

Fernandes J., Guterres A., de Oliveira R.C., Chamberlain J., Lewandowski K., Teixeira B.R., Coelho T.A., Crisóstomo C.F., Bonvicino C.R., D’Andrea P.S., Hewson R, & de Lemos E.R. (2018). Xapuri virus, a novel mammarenavirus: natural reassortment and increased diversity between New World viruses. Emerging Microbes & Infections, 7, 120.

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