Taqman mirna abc purification kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The TaqMan miRNA ABC Purification kit is a laboratory equipment product designed for the isolation and purification of microRNA (miRNA) from various biological samples. The kit utilizes an advanced technology to efficiently extract and purify miRNA molecules, which are small non-coding RNA molecules that play a crucial role in gene expression regulation.

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TaqMan kits (18 citations) TaqMan miRNA kit (14 citations) ABC kit (4 citations) TaqMan miRNA ABC Purification Bead kit Human panel A (4 citations) TaqMan miRNA ABC Purification Buffer Kit (3 citations) TaqMan® miRNA ABC purification kit–Human Panel A (2 citations)

12 protocols using taqman mirna abc purification kit

Quantification of miRNA-29a-3p in HCC Plasma

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Pre-TACE plasma samples were collected and stored at −70°C until processing for miRNA analyses. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to identify and quantify the miRNA levels in plasma. Magnetic beads were utilized for miRNA extraction, using the TaqMan^® miRNA ABC Purification Kit (Applied Biosystems, Carlsbad, CA, USA), according to the manufacturer's instructions. TaqMan^® miRNA hsa-miRNA-specific primers (miRNA-29a-3p #002112; Applied Biosystems) and TaqMan^® MicroRNA Reverse Transcription Kit (Applied Biosystems) were used to identify complementary DNA strands from the isolated miRNAs. An ABI PRISM 7300 instrument (Life Technologies, Foster City, CA, USA) and TaqMan^® Universal Master Mix (Applied Biosystems) were used for polymerase chain reaction (PCR) amplification. The expression levels of miRNA were detected as Ct values, and the relative level of miRNA expression (fold change) was calculated by the 2^(−ΔΔCt) method. miRNA-16 was selected as the internal control to normalize miRNA data. Samples from patients with HCC were compared with 5 reference samples obtained from patients with liver cirrhosis. The calculations used were as follows:
The mean was calculated after the qRT-PCR assays were performed in duplicate, using the same ABI PRISM 7300 instrument.

Kim S.S., Cho H.J., Nam J.S., Kim H.J., Kang D.R., Won J.H., Kim J., Kim J.K., Lee J.H., Kim B.H., Lee M.Y., Cho S.W, & Cheong J.Y. (2017). Plasma MicroRNA-21, 26a, and 29a-3p as Predictive Markers for Treatment Response Following Transarterial Chemoembolization in Patients with Hepatocellular Carcinoma. Journal of Korean Medical Science, 33(1), e6.

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Quantitative Profiling of Circulating miRNAs

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Using TaqMan miRNA ABC Purification kit (Applied Biosystems), free circulating microRNAs were separated from 50μl of serum samples by magnetic beads, and miRNA expression was determined using the Megaplex Pool A Protocol and Megaplex preAmp protocol on microfluidic card type A (Applied Biosystems). Experiments were performed on Viia7 Thermalcycler (Applied Biosystems) and for each microfluidic card, the Ct of every miRNA was determined using Viia7 software (Applied Biosystems).

Lamberti M., Capasso R., Lombardi A., Di Domenico M., Fiorelli A., Feola A., Perna A.F., Santini M., Caraglia M, & Ingrosso D. (2015). Two Different Serum MiRNA Signatures Correlate with the Clinical Outcome and Histological Subtype in Pleural Malignant Mesothelioma Patients. PLoS ONE, 10(8), e0135331.

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Magnetic Bead-Based miR-16 Purification

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Purification of miR-16 using magnetic beads was performed using the TaqMan miRNA ABC Purification kit – human panel A (Thermo Fisher). Manufacturer’s instructions were exactly followed.

Zhou L., Hayden A., Chandrasekaran A.R., Vilcapoma J., Cavaliere C., Dey P., Mao S., Sheng J., Dey B.K., Rangan P, & Halvorsen K. (2021). Sequence-selective purification of biological RNAs using DNA nanoswitches. Cell Reports Methods, 1(8), 100126.

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miRNA Isolation and Purification

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Sections were sectioned and deparaffinized as described above. Taqman miRNA ABC Purification Kit (Thermo Fisher Scientific) was used to isolate and purify total miRNA, following the manufacturer’s instructions.

Wang E., Chen S., Wang H., Chen T, & Chakrabarti S. (2023). Non-coding RNA-mediated endothelial-to-mesenchymal transition in human diabetic cardiomyopathy, potential regulation by DNA methylation. Cardiovascular Diabetology, 22, 303.

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Magnetic Bead-Based miR-16 Purification

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Purification of miR-16 using magnetic beads was performed using the TaqMan miRNA ABC Purification kit – human panel A (Thermo Fisher). Manufacturer’s instructions were exactly followed.

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Plasma miRNA Analysis in COPD

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Blood samples were collected from 20 COPD patients and 20 healthy volunteers at the Binzhou Medical University Hospital. The miRNAs in plasma were isolated using the TaqMan^® miRNA ABC Purification Kit (Thermo Fisher, OR, USA). According to the manufacturer’s instruction, 50 μL plasma was mixed with 100 μL ABC buffer and vortexed for 30 s. The sample was placed on ice and added 80 μL Human Panel Beads. The beads were then hybridized with miRNA at 20°C for 40 min. Subsequently, 100 μL wash buffer was used to wash the sample, and miRNA was eluted by 100 μL elution buffer.
The miRNA was then converted to cDNA and downstream analysis by quantitative real-time PCR (qRT-PCR). The microRNA levels of miR-361-5p and miR-196-5p in plasma were evaluated by qRT-PCR. Small nuclear RNA (snRNA) U6 was used as an endogenous control. Levels of miRNAs were normalized to a reference U6. This study was approved by the Ethics Committee of the Binzhou Medical University Hospital and was carried out in accordance with the Declaration of Helsinki. All the patients and volunteers obtained informed consent for experimentation.

Shen H.F., Liu Y., Qu P.P., Tang Y., Li B.B, & Cheng G.L. (2021). MiR-361-5p/abca1 and MiR-196-5p/arhgef12 Axis Involved in γ-Sitosterol Inducing Dual Anti-Proliferative Effects on Bronchial Epithelial Cells of Chronic Obstructive Pulmonary Disease. International Journal of Chronic Obstructive Pulmonary Disease, 16, 2741-2753.

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Profiling Tumor miRNA Expression

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Four patients in T1, T2, T3 or T4 stage (one in each stage) were enrolled for miRNA microarray analysis. The tumor and the paired adjacent tissues from patients were collected for total RNA extraction using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was concentrated in isopropanol, and then the RNA purity was detected using NanoDrop 2000C (Thermo Fisher Scientific Inc., Waltham, MA, USA) and the RNA quality was determined by formaldehyde denatured agarose gel electrophoresis. Then, 50 μg total RNA was purified using a Taqman miRNA ABC purification kit (Thermo Fisher) and hybridized with the beads. The beads were then washed with magnet, and the miRNAs were eluted 3 times to collect the miRNAs. Thereafter, the collected miRNAs were labeled using a miRNA Complete Labeling and Hyb Kit (Agilent, USA). In brief, 100 μg total RNA was incubated in 10 μg Labeling Spike-In at 37 °C for 30 min, treated with DMSO at 16 °C for 2 h, dried in a vacuum concentrator at 45 °C for 3 h, and then treated with Hybridization Mixture and Hyb labeling at 55 °C for 20 h of hybridization. Then, the miRNAs were further hybridized with Human miRNA Microarray Release 14.0,8 (Agilent, USA) and scanned using a SureScan Dx Microarray Scanner (Agilent, USA). The obtained data were subjected to Quality Center analysis and normalization to produce the heatmap for differentially expressed miRNAs.

Chen D., Cheng L., Cao H, & Liu W. (2021). Role of microRNA-381 in bladder cancer growth and metastasis with the involvement of BMI1 and the Rho/ROCK axis. BMC Urology, 21, 5.

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Comprehensive RNA Extraction and Analysis

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Total RNA was extracted using an RNAiso Plus kit from Takara (Dalian, China). Total miRNAs were extracted and purified using a TaqMan® miRNA ABC Purification Kit from ThermoFisher Scientific (Carlsbad, CA, USA) (cel-miR-39-3p was added as spike-in as previously reported [20 (link)]). After cDNA of lncRNA-MEG3 was amplified with M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA), qRT-PCR was carried out using SYBR Green Real-Time PCR Master Mixes (Takara, Dalian, China) (using GAPDH as internal control); to detect the levels of miR-223-3p in serum, the cDNAs were amplified and applied for qRT-PCR analysis using a mirVana™ qRT-PCR miRNA Detection Kit from Invitrogen (Carlsbad, CA, USA). The primers were designed using the OligoArchitect™ Online service (https://www.sigmaaldrich.com/china-mainland/zh/technical-documents/articles/biology/oligoarchitect-online.html) and miRprimer (https://sourceforge.net/projects/mirprimer/), respectively (Table 2) and synthesized by GenePharma (Shanghai, China).

Fan G., Gu Y., Zhang J., Xin Y., Shao J., Giampieri F, & Battino M. (2019). Transthyretin Upregulates Long Non-Coding RNA MEG3 by Affecting PABPC1 in Diabetic Retinopathy. International Journal of Molecular Sciences, 20(24), 6313.

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Hepatitis C miRNA Profiling Protocol

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Serum samples from 15 patients with hepatitis C were collected and the miRNA was purified using the TaqMan miRNA ABC Purification Kit (Thermo Fisher Scientific, USA) according to the instruction. The cDNA was prepared using the TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific, USA), following the manufacturer's instruction. PCR amplification was performed on QS 1 plus and ABI 7500 using the cDNA as template and adding hsa-miR-122 or hsa-miR-16 primer/probe mixtures. Sample #1 was set as the reference sample and hsa-miR-16 as the reference gene.

Wang Z., Yi J., Yu Q., Liu Y., Zhang R., Zhang D., Yang W., Xu Y, & Chen Y. (2023). Performance evaluation of QuantStudio 1 plus real-time PCR instrument for clinical laboratory analysis: A proof-of-concept study. Practical Laboratory Medicine, 36, e00330.

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Exosomal miRNA Profiling by NGS

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The high-throughput sequencing service and subsequent bioinformatics analysis were provided by BGI Biotech (Shenzhen, China). Briefly, miRNA was purified from exosome total RNA using the TaqMan ABC miRNA Purification Kit (Thermo Scientific, USA) following the manufacturer’s instructions. RNA libraries were generated, and sequencing was performed using BGISEQ-500. The differentially expressed miRNA was identified by BGI with the value of log2-Ratio > 1 and Q value < 0.001. Gene ontology and KEGG pathway analyses were based on NCBI.

Lv P.Y., Gao P.F., Tian G.J., Yang Y.Y., Mo F.F., Wang Z.H., Sun L., Kuang M.J, & Wang Y.L. (2020). Osteocyte-derived exosomes induced by mechanical strain promote human periodontal ligament stem cell proliferation and osteogenic differentiation via the miR-181b-5p/PTEN/AKT signaling pathway. Stem Cell Research & Therapy, 11, 295.

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