Mircury rna isolation kit biofluid

Manufactured by Qiagen

Sourced in Denmark, United States

The MiRCURY RNA Isolation Kit-Biofluids is a laboratory equipment product designed to isolate and purify RNA from various biofluids, including plasma, serum, urine, and cerebrospinal fluid. The kit utilizes a spin column-based method to efficiently extract and concentrate RNA for downstream applications.

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Lab products found in correlation

Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions) Mirneasy, by Qiagen (3 mentions) Universal cdna synthesis kit 2, by Qiagen (3 mentions) Rna spike in kit, by Qiagen (3 mentions) Steponeplus, by Thermo Fisher Scientific (2 mentions) Mircury lna universal rt microrna pcr, by Qiagen (2 mentions) Magmax mirvana total rna isolation kit, by Thermo Fisher Scientific (2 mentions) Glycogen, by Roche (2 mentions) Mircury exosome isolation kit serum and plasma, by Qiagen (2 mentions) 2100 bioanalyzer, by Agilent Technologies (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Mircury rna isolation kit cell and plant, by Qiagen (2 mentions) Exilent sybr green master mix, by Qiagen (2 mentions) Mircury lna universal rt microrna pcr system, by Qiagen (1 mentions) Steponeplus system, by Thermo Fisher Scientific (1 mentions) Mircury lna universal rt cdna synthesis kit, by Qiagen (1 mentions) Tri reagent, by Merck Group (1 mentions) Universal cdna synthesis kit, by Qiagen (1 mentions) 2720 thermal cycler, by Thermo Fisher Scientific (1 mentions) Taqman microrna assay, by Thermo Fisher Scientific (1 mentions)

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RNA isolation kit (344 citations)

48 protocols using mircury rna isolation kit biofluid

Plasma microRNA Isolation and Quantification

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RNA from previously collected plasma samples was isolated using miRCURY RNA Isolation Kit Biofluids (Exiqon) according to the manufacturer’s instructions. The RNA spike-in controls (Exiqon) were used according to the manufacturer’s instruction for validation of RNA isolation (UNiSp2, UNiSp4 and UNiSp5), cDNA synthesis and PCR amplification (UniSp6, cel-miR-39-3p). Moreover, an additional template-free control was purified by the samples and profiled. Briefly, EDTA-plasma samples were thawed on melting ice and centrifuged to avoid presence of cell debris. Next, debris-free samples were lysed using lysing solution and RNA isolation was performed. Isolated RNA was reverse-transcribed using a miRCURY LNA Universal RT cDNA Synthesis kit (Exiqon). The cDNA was diluted 50× and assayed according to the protocol for the miRCURY LNA Universal RT microRNA PCR system (Exiqon). All LNA primers were obtained from Exiqon. The Step-One Plus system was used for amplification (Applied Biosystems). All data were normalized to the cel-miR-39-3p control.
Results were derived by the (2^–^Δ^CT) × 10⁴ (miR-1) or (2^–^Δ^CT) × 10² (miR-126) and normalized to the volume and to the expression of cel-miR-39-p3.

Kazimierczyk E., Eljaszewicz A., Kazimierczyk R., Tynecka M., Zembko P., Tarasiuk E., Kaminski K., Sobkowicz B., Moniuszko M, & Tycinska A. (2020). Altered microRNA dynamics in acute coronary syndrome. Postępy w Kardiologii Interwencyjnej = Advances in Interventional Cardiology, 16(3), 287-293.

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Exosomal RNA Isolation Protocol

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RNA isolation of cell pellets and exosomes was carried out using the Tri-Reagent (Sigma) and miRcury RNA isolation kit-Biofluids (Exiqon), according to the manufacturer's instructions. Samples were dissolved in DEPC-treated water and stored at −80°C. Exosomes from supernatants were isolated as described earlier.

Khurana R., Ranches G., Schafferer S., Lukasser M., Rudnicki M., Mayer G, & Hüttenhofer A. (2017). Identification of urinary exosomal noncoding RNAs as novel biomarkers in chronic kidney disease. RNA, 23(2), 142-152.

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Serum miRNA Profiling in Asthma

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Three miRNAs, viz. miRNA-106a-5p, miRNA-146a-5p and miRNA-19b-3p, were selected for the study based on miRNA profiling in PBMCs isolated from elderly and non-elderly asthma patients and healthy subjects. The profiling results and the outline of the study are briefly presented in the Additional file 1. From the miRNAs whose expression significantly differed in asthmatics and controls in the profiling study, we chose those that had been previously tested in serum and were related to the presence of asthma or its clinical features [15 (link), 20 (link)]. Additionally, although not shown as significant in the profiling results, miRNA-126a-5p was also included in the analysis, as many studies have reported it to have an important role in anti-inflammatory responses [20 (link)].
The miRNA was isolated from the serum using a miRCURY RNA Isolation Kit-Biofluids (Exiqon, Vedbaek, Denmark) according to the manufacturer's protocol. Equal volumes of RNA were used to prepare complementary DNA (cDNA) with the universal cDNA Synthesis Kit (Exiqon). They were used to perform quantitative PCR (StepOne Plus; Applied Biosystems, Foster City, CA, US) with miR-93 as a reference gene. Serum expression levels of miRNA-106a-5p, miRNA-146a-5p, miRNA-19b-3p and miRNA-126a-5p were evaluated with real-time PCR. miRNA expression is presented as 2^−ΔCT.

Wardzyńska A., Pawełczyk M., Rywaniak J., Makowska J., Jamroz-Brzeska J, & Kowalski M.L. (2021). Circulating miRNA expression in asthmatics is age-related and associated with clinical asthma parameters, respiratory function and systemic inflammation. Respiratory Research, 22, 177.

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Serum RNA Extraction Using miRCURY Kit

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Total RNA was extracted from serum using the miRCURY RNA Isolation Kit—Biofluids (Exiqon, Vedbaek, Denmark) as per the manufacturer's instructions (See Appendix A). The extracted RNA was stored at −80°C prior to transfer to Exiqon, Denmark, 2 months later, on dry ice.

Mackie F.L., Baker B.C., Beggs A.D., Stodolna A., Morris R.K, & Kilby M.D. (2019). MicroRNA changes in maternal serum from pregnancies complicated by twin‐twin transfusion syndrome: A discovery study. Prenatal Diagnosis, 39(8), 616-634.

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Cardiac miRNA Profiling: Isolation, Quantification, and Normalization

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Left ventricle portion of heart was homogenized in a proprietary buffer and miRNA was isolated using commercially available kits (PureLink^™ miRNA Isolation kit, Cat# K1570-01, Invitrogen, CA, USA for heart and miRCURY RNA Isolation Kit-Biofluids, Cat#300112, Exiqon, MA, USA for plasma), cDNA was synthesized using miRCURY LNA^™ Universal RT microRNA PCR, Cat#203301, Exiqon, MA, USA in 2720 Thermal Cycler (Applied Biosystems, CA, USA), purified using PureLink^™ Quick PCR Purification Kit, (Cat# K310001,Invitrogen, Poststraβe, Germany) and PCR was run in LightCycler 2.0 (Roche, Basel, Switzerland) using readymade specific forward and reverse primer mix (TaqMan^® MicroRNA Assays, Applied Biosystems, CA, USA; microRNA LNA^™ PCR primer sets, Exiqon, MA, USA) and the amplification curves were analyzed using 2^-ΔΔCt method. Micro RNAs quantified were hsa-miR-25-3p (Cat# 204361), mmu-miR-155-5p (Cat# 205930), hsa-miR-99b-3p (Cat# 204064) and mmu-miR-451(Cat# 204734) and normalized with RNU5G (a small nuclear RNA) in heart and hsa-miR-30e-5p (Cat# 204714) in plasma.

Amara V.R., Surapaneni S.K, & Tikoo K. (2017). Dysregulation of microRNAs and renin-angiotensin system in high salt diet-induced cardiac dysfunction in uninephrectomized rats. PLoS ONE, 12(7), e0180490.

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Plasma and Macrophage MicroRNA Extraction

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MicroRNA-enriched total RNA from plasma and macrophages was isolated with miRCURY™ RNA Isolation Kit-Biofluids (Exiqon, Madrid, Spain) and miRCURY™ RNA Isolation Kit-Cell & Plant (Exiqon, Madrid, Spain), respectively. The extracted RNA was quantified using NanoDrop 2000 (Isogen LifeSciences, Utrecht, Netherlands).

Daimiel L., Micó V., Díez-Ricote L., Ruiz-Valderrey P., Istas G., Rodríguez-Mateos A, & Ordovás J.M. (2020). Alcoholic and Non-Alcoholic Beer Modulate Plasma and Macrophage microRNAs Differently in a Pilot Intervention in Humans with Cardiovascular Risk. Nutrients, 13(1), 69.

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Optimizing RNA Isolation from Cerebrospinal Fluid

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For the study of RNA isolation technical parameters, ten lumbar
puncture-harvested CSF samples were mixed and spun at 3000 × g for 5 min.
Aliquots of 200 μl supernatant from the mixed sample were used for each
RNA isolation procedure. Four replicates were evaluated for each method. Total
RNA was extracted and compared using commercially available kits: MagMAX™
mirVana™ Total RNA Isolation Kit (Thermo Fisher Scientific),
miRCURY™ RNA Isolation Kit–Biofluids (Exiqon), miRNeasy
Serum/Plasma Kit (QIAGEN), NucleoSpin miRNA Plasma (MACHEREYNAGEL),
PureLink™ RNA Mini Kit (Thermo Fisher Scientific), and TRIzol LS reagent
(Thermo Fisher Scientific). RNA isolations were carried out following
manufacturers’ instructions except for TRIzol LS reagent which was done
exactly as described previously [3 (link), 22 (link), 23 (link)]. RNA isolation experiments were performed with or without
nucleic acid carriers, glycogen, and bacteriophage MS2 RNA (both from Roche).
RNA was eluted or dissolved with 30 μl of nuclease-free water containing
RNAsin (0.5 U/μl, Promega). After a preliminary comparison by single-tube
TaqMan® miRNA assays (data not shown), three kits including
miRCURY™ RNA Isolation Kit–Biofluids, miRNeasy Serum/Plasma Kit,
and TRIzol LS reagent were selected for further assessments by miR-15/107
TaqMan® Low-Density Array (TLDA) analysis [24 (link)].

Wang W.X., Fardo D.W., Jicha G.A, & Nelson P.T. (2016). A Customized Quantitative PCR MicroRNA Panel Provides a Technically Robust Context for Studying Neurodegenerative Disease Biomarkers and Indicates a High Correlation Between Cerebrospinal Fluid and Choroid Plexus MicroRNA Expression. Molecular neurobiology, 54(10), 8191-8202.

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Comprehensive Gene Expression Analysis

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Total RNA was isolated from treated cells using TRIzol Reagent (Life Technologies), and then RervertAid First Strand cDNA Synthesis Kit (Ferments Life Science) was used to synthesize cDNA. Real-time RT-PCR for DKK1 detection was performed using SYBR Select Master Mix (Life Technologies) following the manufactory instruction. GAPDH was used as an internal control. The primers for GAPDH were: forward primer 5′-ATTCCATGGCACCGTCAAGGCTGA-3′, reverse primer 5′-TTCTCCATGGTGGTGAAGACGCCA-3′; for DKK1 were: forward primer 5′-CCTTGAACTC GGTTCTCAATTCC-3′, reverse primer 5′-CAATGGTCT GGTACTTATTCCCG-3′. The gene expression threshold cycle (CT) values of DKK1 were calculated by normalizing with internal control GAPDH and relative quantization values were calculated.
miRNAs were purified from serum specimens by miRCURY^™ RNA Isolation Kit-Biofluids (EXIQON), cDNA was generated with the miScript II RT Kit (QIAGEN) and real-time RT-PCR was done by miScript SYBR Green PCR Kit (QIAGEN) following the manufacturer's instructions. Primers for miR-493 and endogenous control RNU6 were purchased from QIAGEN. The gene expression threshold cycle (CT) values of miRNAs were calculated by normalizing with internal control RNU6 and relative quantization values were calculated.

Jia X., Li N., Peng C., Deng Y., Wang J., Deng M., Lu M., Yin J., Zheng G., Liu H, & He Z. (2016). miR-493 mediated DKK1 down-regulation confers proliferation, invasion and chemo-resistance in gastric cancer cells. Oncotarget, 7(6), 7044-7054.

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Isolation of Exosomal RNA from Plasma

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RNA from plasma exosomes was isolated using miRCURY™ RNA Isolation Kit – Biofluids (Exiqon) according to the manufacturer’s protocol. In detail, 300 µL supernatant from exosome isolation were mixed with 90 µL Lysis solution BF, vortex for 5 s and incubated for 10 min at RT. 1 µL RNA Spike-in template mixture (miRCURY LNA™ Universal RT microRNA PCR, RNA Spike-in kit) was added to each sample for downstream PCR analysis. Thereafter, 30 µL Protein Precipitation Solution BF was added to samples and then vortexed, incubated for 1 min at RT and centrifuged for 3 min at 11,000 g. After transferring the supernatants 400 µL isopropanol was added, vortexed for 5 s and loaded in microRNA Mini Spin Column BF. Columns were incubated for 2 min at RT, centrifuged for 30 s at 10,000 g, washed with Wash Solution 1 BF and twice with Wash Solution BF 2. After that, columns were centrifuged for 2 min at 11,000 g to dry membranes and RNA was eluted adding 80 µL RNase-free H₂O directly onto the membrane of the spin column BF. Columns were incubated for 1 min at RT and then centrifuged for 1 min at 11,000 g. The purified RNA samples were stored at −80°C.

Colletti M., Tomao L., Galardi A., Paolini A., Di Paolo V., De Stefanis C., Mascio P., Nazio F., Petrini S., Castellano A., Russo I., Caruso R., Piga S., De Vito R., Pascucci L., Peinado H., Masotti A., Locatelli F, & Di Giannatale A. (2020). Neuroblastoma-secreted exosomes carrying miR‐375 promote osteogenic differentiation of bone-marrow mesenchymal stromal cells. Journal of Extracellular Vesicles, 9(1), 1774144.

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Isolation and Purification of Extracellular Vesicles from Plasma

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MiRCURY Exosome Isolation Kit-Serum and Plasma (Exiqon, Denmark) was used for isolation of plasmatic EVs. Briefly, 600 μl of plasma was treated with thrombin for 5 minutes at room temperature to remove clotting factors. The supernatant was mixed with 200 μl of precipitation buffer and incubated at 4°C for overnight and centrifuged (10,000 g for 30 min at room temperature). The pellet was vortexed for 5–15 minutes in 270 μl resuspension buffer until completely re-suspended. The EV miRNA was extracted using miRCURY RNA isolation kit-biofluids (Exiqon, Denmark) according to the manufacturer´s instruction. RNA spike-in template mixture (Exiqon, Denmark) was added as a quality control of the downstream PCR analysis. dx.doi.org/10.17504/protocols.io.tuienue [PROTOCOL DOI]

Svedman F.C., Lohcharoenkal W., Bottai M., Brage S.E., Sonkoly E., Hansson J., Pivarcsi A, & Eriksson H. (2018). Extracellular microvesicle microRNAs as predictive biomarkers for targeted therapy in metastastic cutaneous malignant melanoma. PLoS ONE, 13(11), e0206942.

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