Mircury lna universal rt microrna pcr cdna synthesis kit

Manufactured by Qiagen

Sourced in Denmark

The MiRCURY LNA™ Universal RT microRNA PCR cDNA synthesis kit is a laboratory equipment product designed for the reverse transcription and amplification of microRNA samples. It provides a streamlined workflow for the detection and quantification of microRNA expression levels.

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Lab products found in correlation

Sybr green reagents, by Quanta Biosciences (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Superscript 3, by Thermo Fisher Scientific (1 mentions) Rneasy mini, by Qiagen (1 mentions) Cfx connect real time pcr detection system, by Bio-Rad (1 mentions) Sybr green based assay, by Thermo Fisher Scientific (1 mentions) Rnase inhibitor, by Thermo Fisher Scientific (1 mentions) Dnase treatment, by Qiagen (1 mentions) Mircury lna universal rt microrna pcr, by Qiagen (1 mentions) Mircury lna universal rt microrna pcr protocol, by Qiagen (1 mentions) Abi 7900 real time pcr detection system, by Thermo Fisher Scientific (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

CDNA synthesis kit (89 citations) RT-PCR kit (45 citations) MiRCURY LNA Universal RT microRNA cDNA synthesis kit (9 citations) MiRCURY LNA™ Universal RT microRNA PCR/Universal cDNA Synthesis Kit II (9 citations) MiRCURY RT Kit (7 citations) LNA miRCuRY (2 citations)

4 protocols using mircury lna universal rt microrna pcr cdna synthesis kit

Quantitative RT-PCR Methodology

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For quantitative RT-PCR, total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA) or RNeasy Mini (Qiagen, Valencia, CA) and reverse transcribed by Superscript III (Invitrogen) or miRCURY LNA™ Universal RT microRNA PCR cDNA synthesis kit (EXIQON, Woburn, MA). cDNAs were used for PCR with SYBR Green reagents (Quanta Biosciences, Gaithersburg, MD) on MX3000 bioanalyzer (Stratagene, La Jolla, CA) and CFX Connect real-time PCR detection system (Bio Rad, Hercules, CA). The data was normalized to β-actin expression, or 5S rRNA expression for miR-203 expression. Product sizes were confirmed by gel electrophoresis. Primer sets for miR-203 and 5S rRNA were purchased from EXIQON. Additional primer sequences are provided in Table S1.

Dainichi T., Hayden M.S., Park S.G., Oh H., Seeley J.J., Grinberg-Bleyer Y., Beck K.M., Miyachi Y., Kabashima K., Hashimoto T, & Ghosh S. (2016). PDK1 is a Regulator of Epidermal Differentiation that Activates and Organizes Asymmetric Cell Division. Cell reports, 15(8), 1615-1623.

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Quantification of miR200 Expression in Clinical Samples

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For miR200 expression analyses, total RNA was extracted using either fibroblasts, iPSCs, or serum samples obtained from three clinical groups (Control, Medalist −C, and Medalist +C) using standard TRIzol-based RNA extraction protocols in the presence of RNase inhibitors (Life Technologies) and DNase treatment (QIAGEN). RNA measurements used ND-1000 spectrophotometer V3.5 (NanoDrop Technologies, Inc.). Samples with a 260/280 optical densitometry ratios of 2 were used for further analyses that included cDNA conversion using miRCURY LNA Universal RT microRNA PCR cDNA synthesis kit (Exiqon). A total of 5 ng/µl cDNA was thereafter subjected to a nested quantitative real-time PCR analysis using primers for miR200a, miR200b, and miR200c in equimolar ratios (500 nM each) in a standard 10-µl reaction volume and 384-well format, SYBR Green-based assay (Life Technologies, Roche). Primer sequences can be found in Table S7.

Bhatt S., Gupta M.K., Khamaisi M., Martinez R., Gritsenko M.A., Wagner B.K., Guye P., Busskamp V., Shirakawa J., Wu G., Liew C.W., Clauss T.R., Valdez I., Ouaamari A.E., Dirice E., Takatani T., Keenan H.A., Smith R.D., Church G., Weiss R., Wagers A.J., Qian W.J., King G.L, & Kulkarni R.N. (2015). Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes. Cell metabolism, 22(2), 239-252.

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Validation of Serum MicroRNA Panel

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For the validation study, the miRCURY LNA Universal RT MicroRNA PCR protocol (Exiqon, QIAGEN Ltd, Manchester, UK) was used to conduct the first-strand cDNA synthesis and qRT-PCR, using the individual assays for human miR-874 and Pick-&-Mix microRNA PCR Panel. The custom serum focus microRNA panel was used for the validation set, which focuses on miRNAs of interest, includes controls, reference genes, RNA spike-in, and interplate calibrators.
Fourteen selected and two endogenous control miRNAs were evaluated from serum in a 96-well plate with all the relevant controls (see Supplementary Table S3 for the panel assay list). In addition, the RNA spike-ins technology ensured the quality of RT and qRT-PCR reactions.²⁶ (link) A 2 µL RNA was reverse transcribed in 10 µL reactions using the miRCURY LNA Universal RT MicroRNA PCR cDNA synthesis kit (Exiqon). cDNA was diluted 50 times and assayed in 10 µL PCR reactions according to the protocol for miRCURY LNA Universal RT MicroRNA PCR (Exiqon) to determine the C_T value. All PCR reactions for each sample were in triplicate. The qRT-PCR was performed to validate detection of selected miRNAs.

ElShelmani H., Wride M.A., Saad T., Rani S., Kelly D.J, & Keegan D. (2020). Identification of Novel Serum MicroRNAs in Age-Related Macular Degeneration. Translational Vision Science & Technology, 9(4), 28.

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Quantifying Nestin and miR-940 Expression

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Total RNA from patient tissue samples and cell lines was extracted using TRIzol reagent (Invitrogen) following the manufacturer's protocol. RNA concentrations and quality were determined by spectrophotometry (Eppendorf, Hamburg, Germany) and gel analysis. Forward and reverse primer pairs were designed for Nestin and 18S rRNA as follows: Nestin-Q-PCR (forward: 5′-GGAAGAGTCTGACCCTGT-3′ reverse: 5′-AGACTAGCGGCATTCCTT-3′) and 18S rRNA Q-PCR (forward: 5′-CCTGGATACCGCAGCTAGGA-3′ reverse: 5′-GCGGCGCAATACGAATGCCCC-3′). Reverse transcription was performed using the PrimeScriptTM RT-PCR Kit (Takara Bio lnc, Otsu, Shiga, Japan). The expression of mature miRNAs was assayed using the miRCURY LNA Universal RT microRNA PCR cDNA Synthesis Kit (Exiqon, Vedbæk, Denmark) with specific primers for hsa-miR-940 or U6 snRNA as a control (Exiqon). Reverse transcription was performed using 1 μg of total RNA according to the mercury LNA SYBR Green Master Mix Kit (Exiqon) protocol and measured with an ABI7900 Real-Time PCR Detection System (Applied Biosystems, Foster city, CA, USA). The ΔΔCt method for relative quantization was used to determine gene expression.

Ma J., Sun F., Li C., Zhang Y., Xiao W., Li Z., Pan Q., Zeng H., Xiao G., Yao K., Hong A, & An J. (2014). Depletion of intermediate filament protein Nestin, a target of microRNA-940, suppresses tumorigenesis by inducing spontaneous DNA damage accumulation in human nasopharyngeal carcinoma. Cell Death & Disease, 5(8), e1377-.

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