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Mircury lna mirna pcr system

Manufactured by Qiagen
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Sourced in Germany

The MiRCURY LNA miRNA PCR System is a laboratory equipment designed for the detection and quantification of microRNA (miRNA) expression levels. It utilizes Locked Nucleic Acid (LNA) technology to provide sensitive and specific amplification of miRNA targets.

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Lab products found in correlation

Mirneasy mini kit, by Qiagen (3 mentions) Bravo liquid handling station, by Agilent Technologies (3 mentions) Mircury lna sybr green pcr kit, by Qiagen (2 mentions) Mircury lna rt kit, by Qiagen (2 mentions) Lightcycler 480, by Roche (2 mentions) 7900ht pcr system, by Thermo Fisher Scientific (2 mentions) Sybr green master mix, by Qiagen (2 mentions) Paxgene blood rna tube, by Qiagen (1 mentions) Mircury lna universal rt kit, by Qiagen (1 mentions) Quantstudio 7 pro real time pcr system, by Thermo Fisher Scientific (1 mentions) Mircury lna mirna pcr assay, by Qiagen (1 mentions) Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions) Rna spike in kit, by Qiagen (1 mentions) Geneglobe web tool, by Qiagen (1 mentions) Cfx96 real time pcr detection system, by Bio-Rad (1 mentions) Primescript rt master mix, by Takara Bio (1 mentions) Rnaiso reagent, by Takara Bio (1 mentions) Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Rotor gene q, by Qiagen (1 mentions)

16 protocols using mircury lna mirna pcr system

1

Profiling Blood and Brain miRNAs

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In total, 46 miRNAs were analysed, including 38 prioritised candidate miRNAs (32 in the blood and six in the brain) (5 (link)) selected based on our recent systematic review and meta-analysis of over 100 studies and another eight endogenous miRNAs with reportedly stable expression in the blood (23 (link)). Two spike-in controls, UniSp3 and UniSp6, were used as serial quality controls throughout the qPCR process. Blood samples were collected in PAXgene Blood RNA tubes (Qiagen, Venlo, The Netherlands). Subsequent qPCR analyses were performed on the miRCURY LNA miRNA PCR System at Qiagen laboratories. Further details can be found in the supplementary material.
During quality control of the qPCR data, qPCR cycle threshold (Ct) values > 35 (i.e., the number of amplification cycles needed for the target to be detected above the background signal) were considered not available, according to the manufacturer’s recommendations. MiRNAs with Ct values > 35 in more than 50% of the samples were removed (total miRNAs removed: n=18 (39.13%), which is comparable to other studies (24 (link))) (eTable 1 in the Supplement). Finally, normalization was undertaken using the geNorm algorithm, preferred over other normalization approaches according to a recent comparative study (24 (link)). In a final step, Ct values were converted to fold changes.
Sadlon A., Takousis P., Evangelou E., Prokopenko I., Alexopoulos P., Udeh-Momoh C.M., Price G., Middleton L, & Perneczky R. (2023). Association of Blood MicroRNA Expression and Polymorphisms with Cognitive and Biomarker Changes in Older Adults. The Journal of Prevention of Alzheimer's Disease, 11(1), 230-240.
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2

Quantification of miRNA Expression in Tdrd7-/- Lens

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Selected Tdrd7-/- lens misexpressed candidate miRNAs were analyzed by custom-designed PCR primers using miRCURY LNA miRNA PCR system (Qiagen, Germantown, MD). Total RNA was isolated from P4 Tdrd7-/- and control lenses in three biological replicates using miRNeasy mini kit (Qiagen Catalog: 217004). First-strand cDNA synthesis was performed using miRCURY LNA Universal RT kit (Qiagen Catalog: 339340) and miRNA expression was quantified using miRCURY LNA SYBR Green PCR kit (Qiagen Catalog: 339345) according to the manufacturer’s instructions. qPCR was run on BioRad CFX RT-PCR thermal cycler. MiRNA expression was normalized to miR-17, which exhibits robust expression in the lens and is not altered in Tdrd7-/- lens, as well as the housekeeping genes Gapdh and Actb. Relative expression was estimated using the 2–ΔΔCT followed by statistical two-level nested analysis of variance test to calculate p-values.
Anand D., Al Saai S., Shrestha S.K., Barnum C.E., Chuma S, & Lachke S.A. (2021). Genome-Wide Analysis of Differentially Expressed miRNAs and Their Associated Regulatory Networks in Lenses Deficient for the Congenital Cataract-Linked Tudor Domain Containing Protein TDRD7. Frontiers in Cell and Developmental Biology, 9, 615761.
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3

miRNA Expression Profiling via qPCR

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The miRNAs miR‐141, miR‐200a, miR‐200b, miR‐200c, and miR‐429 were analysed using the miRCURY LNA miRNA PCR system (Qiagen) based on SYBR Green detection. As reference genes (RGs), miR‐27a‐3p, miR‐193a‐5p, and let‐7g‐5p were used according to published literature [33 (link)]. All reagents were from Qiagen unless otherwise indicated. For reverse transcription, an miRCURY LNA RT Kit was used according to the manufacturer's instructions in a 10 μl final volume containing the reaction master mix and 10 ng of total RNA including the UniSp6 spike‐in.
The resulting cDNA was diluted ten‐fold and 3 μl was used in a 10 μl final qPCR reaction volume according to the manufacturer's instructions. qPCR was carried out using the QuantStudio 7 Pro Real‐Time PCR System (Thermo Fisher Scientific). All qPCR reactions were run in duplicates. To test the qPCR efficiency, cDNA samples were first pooled and then qPCR was performed as described above. qPCR reaction efficiency was tested in triplicates for each analysed miRNA using three‐fold dilutions. The signal was collected after each cycle. Following amplification, melting curve analysis of PCR products was performed to verify the specificity and identity. Melting curves were acquired on the SYBR channel using a ramping rate of 0.7 °C/60 s for 60–95 °C.
Pavlič A., Boštjančič E., Kavalar R., Ilijevec B., Bonin S., Zanconati F, & Zidar N. (2022). Tumour budding and poorly differentiated clusters in colon cancer – different manifestations of partial epithelial–mesenchymal transition. The Journal of Pathology, 258(3), 278-288.
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4

Quantifying Differential miRNA Expression

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Differentially expressed miRNAs were analyzed by qRT-PCR using the miRCURY LNA™ miRNA PCR System (Qiagen). Mature miRNAs were retrotranscribed using the miRCURY LNA RT Kit (Ref. 339340, Qiagen), which allows for universal cDNA synthesis through the addition of a poly(A) tail to miRNA templates. For the miRNA measurements from cell lysates, 25 ng of RNA was used as the starting material, whereas 7 µL of the eluates were used for cDNA synthesis in the case of conditioned media and extracellular vesicles. In the study involving miRNA measurements in patients, 1.12 µL of RNA eluates were used for cDNA synthesis. qRT-PCR was carried out with miRCURY LNA™ miRNA PCR Assays (Qiagen) in a ViiA™ 7 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific). The Ct values were analyzed using the ΔΔCt method and the fold change values were calculated as 2−ΔΔCrt with the Thermo Fisher Cloud Relative Quantification software [21 (link)]. U6 and spike-in controls were used as the endogenous and exogenous controls, respectively.
de la Cruz-Ojeda P., Schmid T., Boix L., Moreno M., Sapena V., Praena-Fernández J.M., Castell F.J., Falcón-Pérez J.M., Reig M., Brüne B., Gómez-Bravo M.A., Giráldez Á., Bruix J., Ferrer M.T, & Muntané J. (2022). miR-200c-3p, miR-222-5p, and miR-512-3p Constitute a Biomarker Signature of Sorafenib Effectiveness in Advanced Hepatocellular Carcinoma. Cells, 11(17), 2673.
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5

Quantitative Analysis of Wound Healing

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Flash-frozen skin tissues collected after wound closure were sent to Qiagen Inc. (Germantown, MD, USA) for quantitative RT-PCR analysis (RT2 PCR) and microRNA (miR) qPCR analysis. RNA was isolated from flash-frozen tissues using the miRNeasy mini kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s standard procedure (Qiagen, MD). RT2 PCR rat wound healing array was performed by Qiagen according to their standard protocol for Cat# PARN-121Z. For miR analysis, Qiagen used the extremely specific and sensitive LNA technology and the miRCURY LNA miRNA PCR system to run panel I+II for 752 miRs (Cat# YAMR-312Y).
Dhall S., Park M.S., Li C, & Sathyamoorthy M. (2021). Regenerative Effects of Hypoxia Primed Flowable Placental Formulation in Muscle and Dermal Injury. International Journal of Molecular Sciences, 22(13), 7151.
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6

Profiling Plasma miRNA Biomarkers

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Based on the literature review, specific miRNAs were selected with potentially stable plasma expression (reference miRNAs, e.g., miR-16-5p, miR-103a-3p, miR-191-5p, miR-1228-3p, miR-520d-5p) and target miRNAs (miR-10b-5p, miR-99a-5p, miR-130a-3p, miR-342-3p, miR-484, miR-486-5p, miR-1260a) [10 (link),17 (link),52 (link),60 (link)] with elevated expression in the plasma of cancer patients. The expression level of selected target and reference miRNAs was determined by the miRCURY LNA miRNA PCR system (Qiagen), and the quality of samples was tested by synthetic spike-in oligonucleotides (RNA Spike-in kit, Qiagen). All protocols for miRNA quantification were performed using a Bravo liquid handling station (Agilent, Santa Clara, CA, USA) and run on a LightCycler 480 instrument (Roche Diagnostics GmbH, Mannheim, Germany) with rapid analysis on LC480 instrument software by the second derivative method for Cq calculation. Secondary analysis of Cq values was performed in the GeneGlobe web tool (Qiagen), where isolation and transcription quality controls were evaluated, a stabilisation factor for reference miRNAs was calculated and data were normalised. These factors were taken into account when calculating the change in expression between the control group and the group of breast cancer patients with the corresponding p-value.
Holubekova V., Kolkova Z., Grendar M., Brany D., Dvorska D., Stastny I., Jagelkova M., Zelinova K., Samec M., Liskova A., Laucekova Z., Kudela E., Bobrovska M., Kalman M., Zubor P, & Dankova Z. (2020). Pathway Analysis of Selected Circulating miRNAs in Plasma of Breast Cancer Patients: A Preliminary Study. International Journal of Molecular Sciences, 21(19), 7288.
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7

Quantifying miRNA Expression Levels

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Measuring the expression levels of selected mature miRNAs was done with MiRCURY™ LNA™ miRNA PCR System (Qiagen). Total RNA (50 ng) was subjected to reverse transcription using miRCURY LNA™ Universal RT miRNA PCR and cDNA synthesis kit (Qiagen) and subsequently diluted ×25. A total of 5 µL of PCR reaction contained 2 µL of diluted cDNA, 1 × SYBR Green Master Mix (Qiagen) and MiRNA LNA PCR primerset (Qiagen) and was run in 384-well format using 7900HT PCR system (Applied Biosystems, Foster City, CA, USA). qRT-PCR assays are listed in Supplementary Table S5. All qPCR measurements were performed in technical triplicates.
Boresowicz J., Kober P., Rusetska N., Maksymowicz M., Paziewska A., Dąbrowska M., Zeber-Lubecka N., Kunicki J., Bonicki W., Ostrowski J., Siedlecki J.A, & Bujko M. (2020). DNA Methylation Influences miRNA Expression in Gonadotroph Pituitary Tumors. Life, 10(5), 59.
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8

MicroRNA Quantification via LNA-PCR

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microRNA transfection levels were assessed using the miRCURY LNA miRNA PCR System (Qiagen), per manufacturer's directions. The real-time PCR amplification was performed, using the PCR primer sets (Supplementary File 1) for hsa-miR-125a-5p and U6 snRNA, as an internal control. Real-time PCR amplification was performed on the Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad). The data was analyzed using the 2-ΔΔCt method [16] , using the U6 snRNA as an endogenous control.
Vo D.T., Karanam N.K., Ding L., Saha D., Yordy J.S., Giri U., Heymach J.V, & Story M.D. (2019). miR-125a-5p Functions as Tumor Suppressor microRNA And Is a Marker of Locoregional Recurrence And Poor prognosis in Head And Neck Cancer. Neoplasia (New York, N.Y.), 21(9), 849-862.
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9

RNA Extraction and Quantification of Chondrocytes and BMSCs

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For RNA extraction of chondrocytes or micromass-cultured BMSCs, the cultured cells or cell micromass were homogenized with RNAiso reagent (Takara, Japan). After homogenization, RNA was extracted following the manufacturer’s guidance. The extracted RNA was then quantified and reverse transcribed into cDNA using PrimeScript RT Master Mix (TaKaRa, Japan). Quantitative real-time PCR was performed using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, USA). The relative gene expression was calculated, normalized, and compared using the 2−ΔΔCT method following the previous protocol.16 (link) The miRNA reverse transcription and real-time PCR was performed with miRCURY LNA miRNA PCR System (Qiagen, German). The relative miRNA expression was normalized to hsa-miR-103a-3p or mmu-miR-103a-3p. The primers used for cloning construction are listed in Table S1.
Feng L., Yang Z., Li Y., Pan Q., Zhang X., Wu X., Lo J.H., Wang H., Bai S., Lu X., Wang M., Lin S., Pan X, & Li G. (2022). MicroRNA-378 contributes to osteoarthritis by regulating chondrocyte autophagy and bone marrow mesenchymal stem cell chondrogenesis. Molecular Therapy. Nucleic Acids, 28, 328-341.
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10

Plasma miR-126 Quantification Using qRT-PCR

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Total RNA was extracted from 200 µL of plasma by using TRIzol (Invitrogen, Life Technologies, Carlsbad, CA, USA), according to the manufacturer instructions. For plasma miR-126 determination, the Qiagen miRCURY® LNA®miRNA PCR System was used: miRCURY LNA RT Kit for miRNA-specific cDNA synthesis and the miRCURY LNA SYBR Green PCR Kit and miRCURY LNA miRNA PCR Assay for quantitative real-time PCR amplifications. The analyses were performed using the Rotor-Gene Q thermal cycler (Qiagen, Hilden, Germany) and 40 cycles at 9°C for 15 s, 55°C for 30 s and 70°C for 30 s. RNU6B was used as endogenous control and relative expressions were calculated as 2−ΔCt values.
Luceri C., D’Ambrosio M., Bigagli E., Cinci L., Russo E., Staderini F., Cricchio M., Giudici F, & Scaringi S. (2022). Involvement of MIR-126 and MMP9 in the Pathogenesis of Intra-Abdominal Fistulizing Crohn’s Disease: A Brief Research Report. Frontiers in Surgery, 9, 822407.
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