Magna pure 96 cellular rna large volume kit

Manufactured by Roche

Sourced in Germany, Denmark

The MagNA Pure 96 Cellular RNA Large Volume Kit is a laboratory equipment product designed for the automated extraction and purification of cellular RNA. It facilitates the isolation of high-quality RNA from a wide range of sample types, enabling downstream applications such as gene expression analysis.

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6 protocols using magna pure 96 cellular rna large volume kit

Quantitative RNA Extraction and Analysis

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Cell pellets (550 μL) were added 1.53 mL PaxGene Blood RNA tubes solution (PreAnalytiX (Hombrechtikon, Switzerland) and stored at −80 ºC until RNA extraction. Total RNA was extracted using the MagNA Pure 96 Cellular RNA large volume kit (Roche Diagnostics, Mannheim, Germany) by the manufacturer’s instructions, and the amount analysed using the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). cDNA was synthesized using High Capacity cDNA reverse transcription kit and 2720 Thermal cycler (Applied Biosystems, Foster City, CA) and stored at −80 ºC. TF mRNA levels were measured using the StepOne Plus Real-Time PCR systems (Applied Biosystems). Gene expression was detected by TaqMan Fast Universal PCR Master Mix reagents and pre-developed TaqMan® gene expression assays using the target gene TF (TF, Hs 0017225) with reference gene human beta-2-microglobulin (TaqMan B2M Probe Dye Fam, 4333766 – 0804015) analyzed using qPCR with cycle conditions according to the manual.

Gravastrand C.S., Steinkjer B., Halvorsen B., Landsem A., Skjelland M., Jacobsen E.A., Woodruff T.M., Lambris J.D., Mollnes T.E., Brekke O.L., Espevik T, & Rokstad A.M. (2019). Cholesterol Crystals Induce Coagulation Activation through Complement-Dependent Expression of Monocytic Tissue Factor. The Journal of Immunology Author Choice, 203(4), 853-863.

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Quantitative RT-PCR Analysis of miRNA and mRNA

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Total RNA was isolated using a MagNA Pure 96 system (MagNA Pure 96 Cellular RNA Large Volume Kit, Roche Diagnostics). cDNAs were synthesized using TaqMan miRNA assays (Applied Biosystems) for miRNA experiments and detection for antimiR, or using Transcriptor Universal cDNA Master (Roche) for mRNA experiments. Subsequent qRT-PCR analyses were normalized to U6 small RNA for miRNA experiments or to Actb (beta-actin) mRNA for mRNA experiments and were performed using TaqMan primers (Applied Biosystems, Supplementary Table S2) in a LightCycler 480 System with a LightCycler 480 Probes Master kit (Roche). All the studies were performed in accordance with Minimum Information for publication of quantitative real-time PCR Experiments (MIQE) guidelines (33 (link)).

Yoshioka K., Kunieda T., Asami Y., Guo H., Miyata H., Yoshida-Tanaka K., Sujino Y., Piao W., Kuwahara H., Nishina K., Hara R.I., Nagata T., Wada T., Obika S, & Yokota T. (2019). Highly efficient silencing of microRNA by heteroduplex oligonucleotides. Nucleic Acids Research, 47(14), 7321-7332.

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Quantitative Analysis of CD8 Expression

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Total RNA were isolated from livers using the Magna Pure 96 cellular RNA large volume kit (Roche Diagnostics, Indianapolis IN) according to the manufacturer’s instructions. RNA samples were then pretreated with DNAse (Thermo Fischer Scientific, Waltham, MA) and reverse transcribed using RevertAid H minus first strand cDNA synthesis kit (Thermo Fischer Scientific). RT-qPCR was performed using SybrGreen (Thermo Fisher Scientific) with primers specific for CD8 (Supplementary Table 4). Gene expression was calculated using the ΔΔC_t method relative to GAPDH housekeeping gene (Supplementary Table 4) and by normalizing to the mean expression of untreated mice.

Meliani A., Boisgerault F., Hardet R., Marmier S., Collaud F., Ronzitti G., Leborgne C., Costa Verdera H., Simon Sola M., Charles S., Vignaud A., van Wittenberghe L., Manni G., Christophe O., Fallarino F., Roy C., Michaud A., Ilyinskii P., Kishimoto T.K, & Mingozzi F. (2018). Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration. Nature Communications, 9, 4098.

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Quantitative Cytokine Expression Analysis

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The expression of cellular IFN-γ, IL-2, TNF-α and IL-4 mRNAs were measured using one-step RT-qPCR protocol. Total RNA of the stimulated PBMCs was isolated using RNeasy Mini kit (Qiagen) or MagNA Pure 96 Cellular RNA Large Volume Kit in MagnaPure 96 System (Roche) according to manufacturer’s protocols. For amplification and quantitation, 5 µl of purified RNA was used in One Step PrimeScript III RT-qPCR Kit (Takara Bio Inc) with predesigned TaqMan FAM-MGB IFN-γ (Hs00989291_m1), IL-2 (Hs00174114_m1), IL-4 (Hs00174122_m1), TNF-α (Hs00174128_m1) and β-actin (Hs01060665_g1) primer/probe sets (Thermo Fisher Scientific) in Rotor-Gene Q (Qiagen). The conditions for the qRT-PCR thermal cycling were the following: One reverse transcription cycle at 55°C for 10 min and 95°C for 10 sec followed by 45 amplification cycles at 95°C for 5 sec and 58°C for 30 sec. The relative fold changes of the target genes were obtained with the 2^−ΔΔCt method by using β-actin Ct-values for normalization and for each participant their corresponding DMSO treated sample as the calibrator.

Hurme A., Jalkanen P., Heroum J., Liedes O., Vara S., Melin M., Teräsjärvi J., He Q., Pöysti S., Hänninen A., Oksi J., Vuorinen T., Kantele A., Tähtinen P.A., Ivaska L., Kakkola L., Lempainen J, & Julkunen I. (2022). Long-Lasting T Cell Responses in BNT162b2 COVID-19 mRNA Vaccinees and COVID-19 Convalescent Patients. Frontiers in Immunology, 13, 869990.

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Whole Blood RNA Extraction and qPCR

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PBS was added to whole blood pellets (equal amounts to retracted plasma), before adding PaxGene solution (1 ml blood sample/2.76 ml PaxGene solution) for RNA stabilization. Total RNA was extracted by the MagNa Pure 96 Cellular RNA large volume kit (Roche Diagnostics GmbH, Roche Applied BioScience, Mannheim, Germany) according to the manufacturer’s instructions. The extractions of the RNA were completed through MagNa Pure 96 instrument from Roche. The RNA concentration was estimated by the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). The cDNA was synthesized using High Capacity cDNA reverse transcription kit and 2720 Thermal cycler (Applied Biosystems) and stored at −80°C. The TF mRNA levels were measured using the 7500 Fast Real-Time PCR system (Applied Biosystems), TaqMan Fast Universal PCR Master Mix reagents and predeveloped TaqMan® gene expression assays. The target gene TF (TF, Hs 0017225, Applied Biosystems), and the reference gene human beta-2-microglobulin (TaqMan B2M Probe Dye Fam, 4333766 – 0804015, Applied Biosystems), were analyzed using qPCR with cycle conditions according to the manual. This reference gene was chosen because the expression of this gene was stable through the whole blood assay.

Gravastrand C., Hamad S., Fure H., Steinkjer B., Ryan L., Oberholzer J., Lambris J.D., Lacík I., Mollnes T.E., Espevik T., Brekke O.L, & Rokstad A.M. (2017). Alginate microbeads are coagulation compatible, while alginate microcapsules activate coagulation secondary to complement or directly through FXII. Acta biomaterialia, 58, 158-167.

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Mononuclear Cell RNA Isolation and Reverse Transcription

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Mononuclear cells from PB and BM were isolated by Lymphoprep density centrifugation (AXIS-SHIELD PoC AS, Oslo, Norway), resuspended in MagNA Pure LC mRNA lysis buffer (Roche Diagnostics, Mannheim, Germany), and stored at -80°C. Total RNA was prepared from 300,000 to 1,000,000 mononuclear cells using either a MagNA Pure LC robotic instrument (Roche Diagnostics, Hvidovre, Denmark) and the MagNA Pure LC RNA Isolation Kit-High Performance or a MagNA Pure 96 robotic instrument and the MagNA Pure 96 Cellular RNA Large Volume Kit (Roche Diagnostics) as described by the manufacturer. RNA from 100,000 to 400,000 mononuclear cells in 19 μL of MagNA Pure RNA elution buffer was reversed transcribed using either 4 μL of cDNA primer N9 and 17.6 μL of cDNA mix consisting of 8 μL of First Strand Buffer, 4 μL of DDT, 0.8 μL of M-MLV Reverse Transcriptase (Invitrogen, Taastrup, Denmark), 0.8 μL of Protector RNase inhibitor (Roche), and 4 μL of dNTP or 5.4 μL of 5× VILO Reaction Mix and 2.7 μL of 10× Superscript Enzyme (Thermo Fisher Scientific, Waltham, MA).

Skou A.S., Juul-Dam K.L., Hansen M., Lausen B., Stratmann S., Holmfeldt L., Aggerholm A., Nyvold C.G., Ommen H.B, & Hasle H. (2021). Measurable Residual Disease Monitoring of SPAG6, ST18, PRAME, and XAGE1A Expression in Peripheral Blood May Detect Imminent Relapse in Childhood Acute Myeloid Leukemia. The Journal of molecular diagnostics : JMD, 23(12).

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