Mir 181b 5p

Manufactured by Thermo Fisher Scientific

MiR-181b-5p is a microRNA (miRNA) product offered by Thermo Fisher Scientific. MiRNAs are small, non-coding RNA molecules that play important regulatory roles in gene expression. The MiR-181b-5p product can be used for various research applications involving the study of this specific miRNA.

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MiR-181b

3 protocols using mir 181b 5p

Quantitative Analysis of miRNA-181b Expression

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Tissues were homogenized using TissueLyser II (Qiagen) according to manufacturer/s instructions. RNA isolation was using TRIzol Reagent according to the manufacturer’s instructions. Subsequent RT-qPCR was performed using a High-Capacity cDNA Reverse Transcription Kit (4368813, Applied Biosystems). For SyberGreen-based assay GoTaq qPCR Master Mix (A6001, Promega) was used, and for TaqMan Universal Master Mix II, UNG (4440038, Life Technologies) was used. Expression of mRNAs and miRNA expression were normalized to β-actin or U6 (Aglient, AriaMx Real Time PCR System). Specific primers including miR-181b-5p (#001098), U6 (#001973), human-HPRT (Hs05647472_cn), human-primary-miR-181b1 (Hs03302966_pri), human-primary-miR-181b2 (Hs03302963_pri), mouse-HPRT (Mm00522878_cn), mouse-primary-miR-181b1 (Mm03307120_pri) and mouse-primary-miR-181b2 (Mm03307414_pri) were purchased from Life Technologies. Changes in expression were calculated using delta delta Ct method.

Zhou H., Yang D., Cheng H.S., McCoy M.G., Pérez-Cremades D., Haemmig S., Wong D., Chen L, & Feinberg M.W. (2022). miR-181b regulates vascular endothelial aging by modulating a MAP3K3 signaling pathway. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 36(6), e22353.

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Quantifying miRNA and mRNA Expression

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Tissues were homogenized using TissueLyser II (Qiagen) according to manufacturer’s instructions. For RNA isolation TRIzol reagent (Invitrogen) or RNeasy kit (Qiagen) was used based on manufacturers protocol. Subsequent RT-qPCR was performed using High-Capacity cDNA Reverse Transcription kit (4368813, Applied Biosystems). For SyberGreen-based assay GoTaq qPCR Master Mix (A6001, Promega) was used; and for TaqMan Universal Master Mix II, UNG (4440038, Life Technologies) was used. Expression of mRNAs and miRNA expression were normalized to Hprt, β-actin, or U6 (Aglient, AriaMx Real-Time PCR System). Specific primers including Mir181b-5p (#001098), U6 (#001973), human-primary-miR-181a1b1 (#Hs03302966_pri), human-primary-miR-181a2 (#Hs03302889_pri), human-primary-Mir181b1 (#Hs03302963_pri), for TaqMan system were purchased from Life Technologies. Changes in expression were calculated using deltadelta Ct method. Primer sequences are described in (Supplementary file 2).

Yang D., Haemmig S., Zhou H., Pérez-Cremades D., Sun X., Chen L., Li J., Haneo-Mejia J., Yang T., Hollan I, & Feinberg M.W. (2021). Methotrexate attenuates vascular inflammation through an adenosine-microRNA-dependent pathway. eLife, 10, e58064.

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RNA Isolation and RT-qPCR Analysis

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Total RNA from intima, PBMCs, and BMCs were isolated using TRIzol Reagent according to the manufacturer’s instructions. Subsequent RT-qPCR was performed using a High-Capacity cDNA Reverse Transcription Kit (4368813, Applied Biosystems). For SyberGreen-based assay, GoTaq qPCR Master Mix (A6001, Promega) was used, and for TaqMan Universal Master Mix II, UNG (4440038, Life Technologies) was used. Expression of mRNAs and miRNA expression were normalized to β-actin or U6 (Aglient, AriaMx Real Time PCR System). Specific primers including miR-181b-5p (#001098), U6 (#001973), snoRNA234 (ID# 001234), human-Hprt (Hs05647472_cn), human-primary-miR-181b1 (Hs03302966_pri), human-primary-miR-181b2 (Hs03302963_pri), mouse-Hprt (Mm00522878_cn), mouse-primary-miR-181b1 (Mm03 307120_pri) and mouse-primary-miR-181b2 (Mm03307414_pri) were purchased from Life Technologies. VE-Cadherin forward primer CACTGCTTTGGGAGCCTTC and reverse primer GGGGCAGCGATTCATTTTTCT were used to detect VE-Cadherin mRNA expression. GAPDH primers are forward AGGTCGGTGTGAACGGATTTG and reverse TGTAGACCATGTAGTTGAGGTCA. Changes in expression were calculated using deltadelta Ct method. Primer sequences are described in (Supplemental Table SII).

Yang D., Haemmig S., Chen J., McCoy M., Cheng H.S., Zhou H., Pérez-Cremades D., Cheng X., Sun X., Haneo-Mejia J., Vellarikkal S.K., Gupta R.M., Barrera V, & Feinberg M.W. (2022). Endothelial cell-specific deletion of a microRNA accelerates atherosclerosis. Atherosclerosis, 350, 9-18.

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