Mircury rt kit

Manufactured by Qiagen

Sourced in Germany

The MiRCURY RT Kit is a laboratory equipment product designed for the reverse transcription of microRNA (miRNA) samples. It provides a reliable and efficient method for the conversion of miRNA molecules into complementary DNA (cDNA) for subsequent analysis.

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Lab products found in correlation

Mirneasy mini kit, by Qiagen (2 mentions) Mircury sybr green mix, by Qiagen (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Mircury spike in kit, by Qiagen (1 mentions) Lc480 2 instrument, by Roche (1 mentions) Lightcycler lc480 2, by Roche (1 mentions) Qiazol lysis reagent, by Qiagen (1 mentions) Mirneasy kit, by Qiagen (1 mentions) Quantitect rt kit, by Qiagen (1 mentions) Lc 480ii instrument, by Roche (1 mentions)

7 protocols using mircury rt kit

Serum microRNA extraction and quantification

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Using a different RNA isolation kit, the miRNeasy Mini Kit (Qiagen, Germany), total RNA was extracted from the same serum samples (n = 39) following the manufacturer’s protocol. A synthetic RNA oligonucleotide mix obtained from the miRCURY Spike-In kit (Qiagen, Germany) was added to each sample at equimolar amounts prior to RNA extraction. These spike-ins were subsequently used to monitor RNA extraction efficiency. The extracted total RNA and miR were stored at –80°C until further analysis.
Further, from total RNA samples, cDNA was synthesized using the miRCURY RT Kit (Qiagen, Germany). Reaction conditions were set according to recommendations by the manufacturer and 2μL of total RNA was used as input in a 10μL reaction. Quantitative RT-PCR reactions were set up using miRCURY SYBR^® green master mix with a 1:50 diluted cDNA and commercial LNA-enhanced primer assay. Reactions were performed in a 384-well primer spotted plates in a Roche LC480 II instrument (Roche, Germany), with the following temperature settings: 95°C for 10 min, 45 cycles of 95°C for 10 s, and 60°C for 60 s. Using the obtained Cq values, the relative miR expression was calculated, as aforementioned, normalizing against the geometric mean of an exogenous spike-in control (cel-miR-39-3p) and an endogenous control (hsa-miR-24-3p), as followed: ΔCq = Averaged Cq_- geomean –Average Cq_- miR.

Tsamou M., Kalligerou F., Ntanasi E., Scarmeas N., Skalicky S., Hackl M, & Roggen E.L. (2023). A Candidate microRNA Profile for Early Diagnosis of Sporadic Alzheimer’s Disease. Journal of Alzheimer's Disease Reports, 7(1), 235-248.

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Quantifying miRNA Expression in Mice

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Total RNA was purified using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol with the exception of an overnight RNA precipitation at −80°. cDNA synthesis was performed using 5 ng RNA and a miRCURY RT kit (QIAGEN cat. #339320) according to manufacturer’s protocol. miRCURY LNA miRNA PCR SYBER Green (QIAGEN cat. #339320) was used for qPCR amplification using Mm-miR-124-3P (YP00206026) and mm-miR-24-3P (YP00204260) primer mixes. miR-103-3p served as control.

Fekry B., Ribas-Latre A., Baumgartner C., Mohamed A.M., Kolonin M.G., Sladek F.M., Younes M, & Eckel-Mahan K. (2019). HNF4α-deficient Fatty Liver Provides a Permissive Environment for Sex-independent Hepatocellular Carcinoma. Cancer research, 79(22), 5860-5873.

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Plasma RNA Isolation and qPCR Analysis

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Ribonucleic acid (RNA) isolation was performed from 200 μl of rat plasma as described previously (41 (link)) using the miRNeasy Mini Kit (Qiagen, Germany) together with glycogen to enhance precipitation. The miRCURY RNA Spike-Ins (Qiagen, Germany) were added to the lysis buffer Qiazol prior to RNA isolation. Total RNA was eluted in 30 μl nuclease-free water and frozen at−80°C until further analysis. Reverse transcription was performed using the miRCURY RT Kit (Qiagen, Germany) with 2 μl total RNA input. RT was performed at 42°C for 60 min followed by heat inactivation at 95°C for 2 min. Quantitative polymerase chain reaction (qPCR) was performed on a Roche LightCycler LC480 II with 45 amplification cycles (95°C for 10 s, 60°C for 60 s) followed by melting curve analysis. Cq-Values were calculated using the 2nd derivative maximum method. Data normalization: Equal biofluid volumes were used throughout the analysis. Homogeneous efficiency of all steps in the workflow was confirmed using spike-in controls. RNA spike-in control was used for normalization to adjust for analytical noise using the equation: Cq = Cq^(UniSp4) – Cq^(miRNA).

Luís A., Hackl M., Jafarmadar M., Keibl C., Jilge J.M., Grillari J., Bahrami S, & Kozlov A.V. (2020). Circulating miRNAs Associated With ER Stress and Organ Damage in a Preclinical Model of Trauma Hemorrhagic Shock. Frontiers in Medicine, 7, 568096.

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Monocyte TF Gene Expression Quantification

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Monocytes resuspended in PBS were spun down, resuspended in QIAzol lysis reagent (Qiagen, USA) and froze at -80° C to preserve the RNA before further isolation. Total RNA was then isolated using a miRNeasy kit according to manufacturer’s protocol (Qiagen, USA). An aliquot of the RNA was reverse transcribed using a miRCURY RT kit (Qiagen, USA) for qPCR analyses of Tissue Factor (F3). The F3 gene expression was normalized to two housekeeping genes: actin B (ACTB) and TATA-box binding protein (TBP) using the 2−ΔΔCT method (17 (link)) and presented as relative fold change in comparison to normoxia control.

Wahlund C.J., Çaglayan S., Czarnewski P., Hansen J.B, & Snir O. (2023). Sustained and intermittent hypoxia differentially modulate primary monocyte immunothrombotic responses to IL-1β stimulation. Frontiers in Immunology, 14, 1240597.

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Quantitative Real-Time PCR Protocol

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Extracted RNA was reverse transcribed using the QuantiTect RT kit (Qiagen, for total RNA) or miRCURY RT kit (Qiagen, for small RNAs) according to manufacturer's instructions. The resulting cDNA was diluted as per kit instructions and quantified with specific primers (Supplementary Table S9) using either QuantiNova SYBR green assays (Qiagen, for cDNA from total RNA) or miRCURY SYBR green assays (Qiagen, for cDNA from small RNAs). Small RNA miRCURY qPCR primer assays were purchased from Qiagen. Primers for the PSG lncRNA were designed to match the PSG lncRNA perfectly but have a mismatch at the 3′ end to the parental ZFAND5 gene. Mismatches located in the 3′ end were shown to impair PCR amplification (21–23 (link)) and allow for differentiation between PSG lncRNA and parental gene. ZFAND5 parental gene primers are located in exon 6 (forward primer) and exon 7 (reverse primer), which according to our data are not expressed at the PSG locus and should only amplify mature mRNA. Data were analysed using the 2^−ΔΔCt method (24 (link)).

Ressel S., Kumar S., Bermúdez-Barrientos J.R., Gordon K., Lane J., Wu J., Abreu-Goodger C., Schwarze J, & Buck A.H. (2024). RNA–RNA interactions between respiratory syncytial virus and miR-26 and miR-27 are associated with regulation of cell cycle and antiviral immunity. Nucleic Acids Research, 52(9), 4872-4888.

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RT-qPCR Validation of NGS Data

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The identical RNA samples that were used for NGS analysis, were used for results verification by RT-qPCR. Starting from total RNA samples, cDNA was synthesized using the miRCURY RT Kit (Qiagen, Germany). Reaction conditions were set according to recommendations by the manufacturer. In total, 2 µl of total RNA were used per 10 µl reverse transcription (RT) reaction. To monitor RT efficiency and presence of impurities with inhibitory activity, a synthetic RNA spike-in (cel-miR-39-3p) was added to the RT reaction. Validated LNA-enhanced forward and reverse miRCURY primer assays for all targets, including spike-in controls, were obtained from Qiagen. PCR amplification was performed in a 96-well plate format in a Roche LC480 II instrument (Roche, Germany) using miRCURY SYBR® Green mix (Qiagen, Germany) with the following settings: 95 °C for 10 min, 45 cycles of 95 °C for 10 s, and 60 °C for 60 s, followed by melting curve analysis. To calculate the cycle of quantification values (Cq-values), the second derivative method was used. Cq-values were normalized to the RNA spike-in control level, by subtracting the individual miRNA Cq-value from the RNA Spike-In Cq, thus obtaining delta-Cq (ΔCq) values that were used for the analysis.

Jirak P., Wernly B., Lichtenauer M., Franz M., Knost T., Abusamrah T., Kelm M., Bimpong-Buta N.Y, & Jung C. (2020). Next-generation sequencing analysis of circulating micro-RNA expression in response to parabolic flight as a spaceflight analogue. NPJ Microgravity, 6, 31.

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Robust RT-qPCR Validation of NGS Data

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The identical RNA samples that were used for NGS analysis were used for results verification by RT-qPCR. cDNAwas synthesized using the miRCURY RT Kit (Qiagen, Germany). Reaction conditions were set according to recommendations by the manufacturer. In total, 2 µL of total RNA were used per 10 µL reverse transcription (RT) reaction. To monitor RT efficiency and presence of impurities with inhibitory activity, a synthetic RNA spike-in (UniSp6) was added to the RT reaction. Validated LNA-enhanced forward and reverse miRCURY primer assays for all targets, including spike-in controls, were obtained from Qiagen. PCR amplification was performed in a 96-well plate format in a Applied Biosystems 7500 instrument (Applied Biosystems, Waltham, MA, USA) using miRCURY SYBR^® Green mix (Qiagen, Germany) with the following settings: 95 °C for 10 min, 45 cycles of 95 °C for 10 s, and 60 °C for 60 s, followed by melting curve analysis. To calculate the cycle of quantification values (Cq-values), the second derivative method was used. Cq-values were normalized to the RNA spike-in control level, by subtracting the individual miRNA Cq-value from the RNA Spike-In Cq, thus obtaining delta-Cq (ΔCq) values that were used for the analysis.

Vashukova E.S., Kozyulina P.Y., Illarionov R.A., Yurkina N.O., Pachuliia O.V., Butenko M.G., Postnikova T.B., Ivanova L.A., Eremeeva D.R., Zainulina M.S., Bespalova O.N, & Glotov A.S. (2021). High-Throughput Sequencing of Circulating MicroRNAs in Plasma and Serum during Pregnancy Progression. Life, 11(10), 1055.

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