Mir 16 5p

Manufactured by Thermo Fisher Scientific

Sourced in United States

MiR-16-5p is a microRNA (miRNA) molecule. miRNAs are small, non-coding RNA molecules that play a role in gene expression regulation. The core function of MiR-16-5p is to bind to target mRNA molecules and regulate their translation or stability.

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Close spelling variants of this product

MiRNA mimic (hsa-miR-15a-5p and hsa-miR-16-5p) Has-miR-16-5p MiR-16

4 protocols using mir 16 5p

Quantifying miRNA Expression via qPCR

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Total cell RNA, including miRNAs were isolated with an miRNeasy Mini Kit (Qiagen, Hilden, Germany). RNA concentrations were determined with the DeNovix DS-11 spectrophotometer (DeNovix Inc., Wilmington, DE, USA). cDNA was generated using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), and miRNA expression was quantified via real-time quantitative PCR using a TaqMan Universal PCR Master Mix (Applied Biosystems) and the BioRad CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Specific primers for hsa-miR-130a-3p were obtained from Applied Biosystems. Expression of miR-130a was normalized to the expression of either U6 small nuclear RNA (snRNA) or miR-16-5p (Applied Biosystems), and the expression of these RNAs was similar in all treatment conditions. The data were then further normalized by setting the control values to one.

Hammond C.L., Roztocil E., Gonzalez M.O., Feldon S.E, & Woeller C.F. (2021). MicroRNA-130a Is Elevated in Thyroid Eye Disease and Increases Lipid Accumulation in Fibroblasts Through the Suppression of AMPK. Investigative Ophthalmology & Visual Science, 62(1), 29.

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Identifying Suitable Reference Genes for miR-21 Analysis

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To test for suitable reference genes in the miR-21 analysis, 7 different miRNAs were validated in initial experiments in 12 smokers and 12 non-smokers collected from the same cohort: U6 small nuclear RNA (U6 snRNA), snRNA+small nucleolar RNA (RNU) 44, RNU 48, miR-425-5p, miR-16 -5p, Caenorhabditis elegans (let) -7a-5p and let-7g-5p (Assay ID: 001973, 001094, 001006, 001516, 000391, 000377, 002282, respectively, Applied Biosystems), The tested miRNAs were chosen based on manufactures recommendations and reports from the literature [22] [23] (link)[24] [25] (link)[26] U6 snRNA, RNU 44 and RNU 48 were not expressed in our test samples. Due to higher observed cycle threshold (Ct) mean for let-7a and let7g (34.2 and 33.3, respectively), as compared to miR-425 (32.7) and miR-16 (25.8), and less stable expressed miR-16 as compared to miR-425 (SD 1.14, correlation of variation (CV) 4.4% vs. SD 0.96, CV 2.4%, respectively), mir-425 was chosen as the reference gene to normalize miR-21 expression.
Additionally, for this validation experiment, no influence on expression by cigarette smoke was essential for the choice.

Opstad T.B., Brusletto B.S., Arnesen H., Pettersen A.Å, & Seljeflot I. (2017). Cigarette smoking represses expression of cytokine IL-12 and its regulator miR-21-An observational study in patients with coronary artery disease. Immunobiology, 222(2).

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Profiling Novel miRNA Expression in Hypertension

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RT-qPCR was performed to determine the novel miRNA expression levels in an independent sample of 881 normotensive, screen-detected hypertensive and known hypertensive male and female participants, as determined by next generation sequencing. Briefly, using the TaqMan Advanced miRNA cDNA synthesis kit (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA), 2 µl of total RNA was converted into cDNA through sequential conduction of poly-A tailing, adaptor ligation, reverse transcription and miR-Amp steps as per the manufacturer’s recommendation. The miRNA expression levels were then determined using TaqMan miRNA Assay primers (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA) on the QuantStudio 7 Flex real-time PCR instrument (Life Technologies, Carlsbad, CA, USA). In order to determine relative miRNA expression in each sample, the 2^−ΔCt method was used whilst the 2^−ΔΔCt method was used to compute fold change differences in miRNA expression between the study groups using miR-16-5p (ThermoFisher Scientific, Waltham, MA, USA) as the endogenous control [19 (link)].

Matshazi D.M., Weale C.J., Erasmus R.T., Kengne A.P., Davids S.F., Raghubeer S., Davison G.M, & Matsha T.E. (2021). Two novel microRNAs and their association with absolute blood pressure parameters in an urban South African community. Molecular Biology Reports, 48(3), 2553-2560.

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Determination of miRNA Expression by RT-qPCR

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In order for miRNA expression to be determined using RT-qPCR, the extracted RNA had to be converted to cDNA first and this conversion was done using the TaqMan Advanced miRNA cDNA synthesis kit (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, United States) as per the manufacturer’s recommendations. In summary, by sequentially conducting poly-A tailing, adaptor ligation, reverse transcription and miR-Amp steps, we converted 2 μl of total RNA into cDNA, which was the starting material in the succeeding PCR step used to determine miRNA expression levels. This PCR analysis was conducted on the QuantStudio 7 Flex real-time PCR instrument (Life Technologies, Carlsbad, CA, United States) using TaqMan miRNA Assay primers (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, United States). The determination of relative miRNA expression in a sample was done using the 2^–ΔCt, whilst fold change differences in miRNA expression between the study groups were computed with the use of the 2^–ΔΔCt method. In order to normalize miRNA quantification, miR-16-5p (ThermoFisher Scientific, Waltham, MA, United States) was used as the endogenous control (Livak and Schmittgen, 2001 (link)).

Matshazi D.M., Weale C.J., Erasmus R.T., Kengne A.P., Davids S.F., Raghubeer S., Davison G.M, & Matsha T.E. (2021). Circulating Levels of MicroRNAs Associated With Hypertension: A Cross-Sectional Study in Male and Female South African Participants. Frontiers in Genetics, 12, 710438.

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