Hsa mir 214

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Hsa-miR-214 is a microRNA (miRNA) molecule that is a part of the human genome. miRNAs are small, non-coding RNA molecules that play a role in the regulation of gene expression. The core function of Hsa-miR-214 is to serve as a genetic regulator, influencing the translation and stability of target messenger RNAs (mRNAs).

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Hsa-miR-214-3p (2 citations)

4 protocols using hsa mir 214

Isolation and detection of miRNA isoforms

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Total RNA in the cells or bones and total miRNAs in serum or exosomes were isolated using TRIzol (Life technologies) reagent and miRNeasy Serum/Plasma Kit (Qiagen) or mirVana miRNA Isolation Kit (Ambion) according to the manufacturer's protocol, respectively. After purification, the total RNA was treated with TURBO DNase (Ambion) and reverse transcribed into first-strand cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems). We used 100 ng cDNA per PCR. The TaqMan primer-probe combinations for pri-miRNAs, pre-miRNAs and miRNAs were products of Ambion. The primer for mRNAs were purchased from Shanghai Integrated Biotech Solutions Co., Ltd (Supplementary Table 6). For detection of 3′ end uridylated miR-214-3p isoforms, the following miRNA assays were used: hsa-miR-214-3p (Ambion, ID 002306), hsa-miR-214-3p-AAA (custom order: 5′-ACAGCAGGCACAGACAGGCAGUAAA-3′, hsa-miR-214-3p-UUU (custom order: 5′-ACAGCAGGCACAGACAGGCAGUUUU-3′). Real-time PCR reactions were performed using the 96-well ABI Prism 7900 HT Sequence detection system (Applied Biosystems). Please see Supplementary Table 5 for the raw data for all the real-time PCR analysis.

Li D., Liu J., Guo B., Liang C., Dang L., Lu C., He X., Cheung H.Y., Xu L., Lu C., He B., Liu B., Shaikh A.B., Li F., Wang L., Yang Z., Au D.W., Peng S., Zhang Z., Zhang B.T., Pan X., Qian A., Shang P., Xiao L., Jiang B., Wong C.K., Xu J., Bian Z., Liang Z., Guo D.A., Zhu H., Tan W., Lu A, & Zhang G. (2016). Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation. Nature Communications, 7, 10872.

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Dual Luciferase Assay for MTHFR 3'UTR

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The dual luciferase assay was performed using the dual-luciferase reporter assay system (psiCHECK-2 vector, Promega). A 850-bp fragment of the MTHFR 3' UTR was amplified from genomic DNA using the following primer sequences: forward, 5 ‫׳‬ -GGACTAGTGGTTGTTGCCAACTAAGCCC-3 ‫׳‬ ; reverse, 5 ‫׳‬ -TTCAAGCTTTCCAGGGAGTGATGACAGAG-3 ‫׳‬ . The PCR product was inserted into the psiCHECK-2 vector downstream of the luciferase gene sequence. All of the constructs were verified by DNA sequencing. According to the MTHFR genotypes, constructed vectors were termed psiCHECK-AA and psiCHECK-GG, respectively. HEK-293 cells were plated in 96-well clusters. Lipofectamine 2000 was used to transfect the cells with 80 ng of psiCHECK-AA or psiCHECK-GG reporter constructs,(Invitrogen, USA)hsa-miR-214 or controls (all at 30 nM, Ambion, USA), and 80 ng of a psiCHECK-2 luciferase vector. After 48 hoftransfection, the cells were washed twice and lysed with passive lysis buffer. Firefly luciferase activity was determined using the dual-luciferase reporter assay system and a luminometer (Promega), as we previously described [27] . The relative luciferase activity was calculated by normalizing the firefly luciferase activity against the internal control luciferase activity.

Chen Q., Qin R., Fang Y., Li H, & Liu Y. (2015). A Functional Variant at the miR-214 Binding Site in the Methylenetetrahydrofolatereductase Gene Alters Susceptibility to Gastric Cancer in a Chinese Han Population. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 36(2).

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Quantitative RT-PCR Analysis of miRNAs

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Ten nanograms of total RNA were reverse transcribed using Taqman microRNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s instructions, as previously described [51 (link)]. The obtained cDNA was amplified using the following Taqman MicroRNA assays: hsa-miR-22, hsa-miR-24, hsa-miR-99a, hsa-miR-194, hsa-miR-214, hsa-miR-335, hsa-miR-339, hsa-miR-708 (Life Technologies, Carlsbad, CA, USA).
To normalize quantitative Real-Time PCR reactions, parallel reactions were run on each sample for RNU48 snRNA. The reactions were performed in triplicate and changes in the target miRNA content relative to RNU48 were determined using the comparative Ct method to calculate changes in Ct, and, ultimately, fold and percent change. An average Ct value for each RNA was obtained for replicate reactions.

Incorvaia L., Fanale D., Badalamenti G., Brando C., Bono M., De Luca I., Algeri L., Bonasera A., Corsini L.R., Scurria S., Iovanna J.L., Russo A, & Bazan V. (2020). A “Lymphocyte MicroRNA Signature” as Predictive Biomarker of Immunotherapy Response and Plasma PD-1/PD-L1 Expression Levels in Patients with Metastatic Renal Cell Carcinoma: Pointing towards Epigenetic Reprogramming. Cancers, 12(11), 3396.

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miRNA Mimics Profiling in Breast Cancer Cell Lines

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JIMT-1 cell line was procured from DSMZ (Brauenschweig, Germany) and grown in 1 : 1 DMEM/F12 (both from Mediatech, Inc., Manassas, VA, USA) media supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals Inc., Lawrenceville, GA, USA), 1% glutaMAX (Gibco, Paisly, UK), 10 μg ml⁻¹ insulin (Sigma Chemicals, Perth, WA, Australia) and 1% penicillin–streptomycin (Lonza BioWhittaker, Walkersville, MD, USA). The KPL-4 cell line was a kind gift from Professor Junichi Kurebayashi (Kawasaki Medical School, Japan) and was grown in DMEM supplemented with 10% FBS, 1% glutaMAX and 1% penicillin–streptomycin. Both cell lines were kept at 37 °C in 5% CO₂ and routinely tested for mycoplasma infections using the ATCC Mycoplasma detection kit (ATCC, Manassas, VA, USA).
Twenty different human mirVANA miRNA Mimics (hsa-miR-892a, hsa-miR-380-5p, hsa-miR-125b-2*, hsa-miR-363*, hsa-miR-940, hsa-miR-214, hsa-miR-34b*, hsa-miR-665, hsa-miR-593, hsa-miR-29c, hsa-miR-555, hsa-miR-885-3p, hsa-miR-567, hsa-miR-297, hsa-miR-187-3p, hsa-miR-124a-1, hsa-miR-326, hsa-miR-601, hsa-miR-506 and hsa-miR-708) and the mirVANA miRNA mimic negative control #1 were purchased from Life Technologies (Grand Island, NY, USA).

Nygren M.K., Tekle C., Ingebrigtsen V.A., Mäkelä R., Krohn M., Aure M.R., Nunes-Xavier C.E., Perälä M., Tramm T., Alsner J., Overgaard J., Nesland J.M., Borgen E., Børresen-Dale A.L., Fodstad Ø., Sahlberg K.K, & Leivonen S.K. (2014). Identifying microRNAs regulating B7-H3 in breast cancer: the clinical impact of microRNA-29c. British Journal of Cancer, 110(8), 2072-2080.

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