Taqman pri mirna assay

Manufactured by Thermo Fisher Scientific

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TaqMan Pri-miRNA Assays are a set of molecular biology tools designed to quantify the expression of primary microRNA (pri-miRNA) transcripts. These assays employ real-time PCR technology to enable sensitive and specific detection of pri-miRNA molecules within a sample. The core function of TaqMan Pri-miRNA Assays is to facilitate the measurement and analysis of pri-miRNA expression levels.

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TaqMan assays (1810 citations) TaqMan miRNA assays (726 citations) MiRNA assay (20 citations) Hsa-mir-181a-1 and -2 TaqMan® Pri-miRNA Assays (2 citations)

24 protocols using taqman pri mirna assay

Quantification of miRNA and mRNA in Wound Tissues

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Total RNA from human LCM tissues and from cells of wound tissues was extracted using the miRNeasy Mini kit (Qiagen) and that from cultured cells was extracted using TRIzol reagents (ThermoFisher Scientific).
Quantification of miRNAs by qRT-PCR was performed as previously described^{20 (link)}. MiRNA expression levels were normalized between different samples on the basis of the values of RNU48 RNA. The mature miR-132 expression was quantified using the TaqMan miRNA assay (TM000457, ThermoFisher Scientific). The primary miR transcripts were quantified using the TaqMan Pri-miRNA assay (Hs03303111_pri, ThermoFisher Scientific).
In order to quantify mRNAs, total RNA was reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific). RASA1 mRNAs were quantified by TaqMan gene expression assays (Integrated DNA Technologies). Target gene expression levels were normalized between samples to the internal control 18 S rRNA (forward: 5′-CGGCTACCACATCCAAGGAA-3′; reverse: 5′-GCTGGAATTACCGCGGCT-3′; probe: 5′-FAM-TGCTGGCACCAGACTTGCCCTC-TAMRA-3′).

Li X., Li D., Wikstrom J.D., Pivarcsi A., Sonkoly E., Ståhle M, & Landén N.X. (2017). MicroRNA-132 promotes fibroblast migration via regulating RAS p21 protein activator 1 in skin wound healing. Scientific Reports, 7, 7797.

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Quantification of FUT8-AS1, NRAS, and miRNAs

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Total RNA was isolated from indicated tissues and cells by Trizol reagent (Invitrogen, Thermo Fisher Scientific) in accordance with the manufacturer’s manual. The first strand cDNA was generated using the RNA and the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). Real-time PCR was performed using TB Green^® Premix Ex Taq™ II (Takara) on StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) with the primers 5’-GGCTCCTTGCTACTTTTAGGG-3’ (forward) and 5’-TGGGGGGGGTCTTTCTCTTC-3’ (reverse) for FUT8-AS1, 5’-GAAATACGCCAGTACCGAATG-3’ (forward) and 5’-TTCTCCTCCAGGGAAGTCAG-3’ (reverse) for NRAS, 5’-GTCGGAGTCAACGGATTTG-3’ (forward) and 5’-TGGGTGGAATCATATTGGAA-3’ (reverse) for GAPDH. GAPDH was used as endogenous control for the quantification of FUT8-AS1 and NRAS expression. For the quantification of miRNAs and pri-miRNAs expression, real-time PCR was carried out using the TaqMan™ Advanced miRNA Assay (Thermo Fisher Scientific) and TaqMan™ Pri-miRNA Assay (Thermo Fisher Scientific) respectively on StepOnePlus Real-Time PCR System in accordance with the manufacturer’s manuals.

Chen X.J., Liu S., Han D.M., Han D.Z., Sun W.J., Zhao X.C., Liang J.Q, & Yu L. (2021). FUT8-AS1 Inhibits the Malignancy of Melanoma Through Promoting miR-145-5p Biogenesis and Suppressing NRAS/MAPK Signaling. Frontiers in Oncology, 10, 586085.

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Profiling Mouse Tissue miRNA and mRNA

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Total RNA was extracted from crushed frozen tissues of 12-week-old male mice and cultured using RNAiso Plus (Takara Bio, Shiga, Japan). The miReasy Mini Kit (QIAGEN, Hilden, Germany) was used according to the manufacturer's instructions. Serum small RNAs were isolated using the miRNeasy Serum/Plasma Advanced Kit (QIAGEN). Genomic DNA was digested using Dnase I (QIAGEN), and cDNA was synthesized using M-MuLV Reverse Transcriptase (New England BioLabs, Ipswich, MA) and ReverTra Ace (TOYOBO, Osaka, Japan) for miRNAs and mRNAs/pri-miRNAs, respectively. Quantitative real-time PCR was performed using FastStart Universal Probe Master (Roche, Basel, Switzerland) with the TaqMan MicroRNA Assay (Thermo Fisher Scientific) for mature miRNAs, according to the manufacturer's recommendations. THUNDERBIRD Next SYBR qPCR Mix (TOYOBO) was used with primer sets for pri-miRNAs and Actb (S1 Table ). The TaqMan Pri-miRNA Assay was used for the quantitative analysis of pri-miRNAs (Thermo Fisher Scientific). Rnu6 and Actb were used as internal controls to normalize the miRNA and pri-miRNA levels, respectively. A Caenorhabditis elegans MIR39 mimic (Thermo Fisher Scientific) was used to normalize serum sRNA levels [11] (link). All analyses were performed using the comparative Ct method with a StepOnePlus real-time PCR system (Thermo Fisher Scientific).

Ogasawara T., Ito S., Ogashira S., Hoshino T., Sotomaru Y., Yoshiko Y, & Tanimoto K. (2024). The expression of MIR125B transcripts and bone phenotypes in Mir125b2-deficient mice. PloS one, 19(7).

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Quantification of miR-4284 Expression

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Extraction of total mRNA was performed with mirVana miRNA Isolation Kit (Thermo Fisher Scientific). The miR-4284 expression level was assessed by miRNA-specific TaqMan Pri-miRNA Assay (Hs04227316_pri; Thermo Fisher Scientific) and normalized by RNU48 Control miRNA Assay (001006; Thermo Fisher Scientific). 1 μg of total RNA was prepared for reverse-transcription using Superscript II First-Strand Synthesis kit (Thermo Fisher Scientific) at the manufacturer's recommendation. Levels of GAPDH mRNA were used for normalization. Primers used in the current study are presented in Table 1.

Miao X., Li Z., Zhang Q., & Wang T. (2021). MicroRNA-4284 inhibits colon cancer epithelial-mesenchymal transition by down-regulating Perilipin 5. STEMedicine, 2(6), e85-e85.

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Quantitative Analysis of miR-122 Expression

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Real-time PCR analysis for miR-122 was performed using a TaqMan miRNA Kit (Applied Biosystems). The U6 endogenous control was used for normalization. Pri-miR-122 was detected using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and a TaqMan pri-miRNA Assay. Actin was used as an internal standard gene. Relative expression was quantified using the comparative threshold cycle (Ct) method.

Hu J., Xu Y., Li C., Hao J., Peng S., Chu X., Zhang D., Xu D, & Meng S. (2015). A cross-talk between Hepatitis B virus and host mRNAs confers viral adaptation to liver. Scientific Reports, 5, 10572.

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Quantitative Analysis of miR-122 and pri-miR-122

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Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Real-time PCR analysis for miR-122 was performed using a TaqMan miRNA kit (Applied Biosystems, Foster City, CA, USA). The U6 endogenous control was used for normalization. miR-122 primary transcript (pri-miR-122) was detected using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) and a TaqMan pri-miRNA assay. Actin was used as an internal standard gene. Real-time fluorescence quantitative PCR was performed using SYBR green premix reagent (TaKaRa Bio Inc., Shiga, Japan). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard for quantification. Primers used in RT-PCR were chemically synthesized and are shown in Supplementary Table S1, S2 and S3. Relative expression was quantified using the comparative threshold cycle (CT) method.

Li C., Deng M., Hu J., Li X., Chen L., Ju Y., Hao J, & Meng S. (2016). Chronic inflammation contributes to the development of hepatocellular carcinoma by decreasing miR-122 levels. Oncotarget, 7(13), 17021-17034.

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Quantitative Analysis of miRNA and mRNA Expression

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Total RNA was isolated using TRIzol reagent (Invitrogen) as recommended by the manufacturer. Reverse transcription of 1 µg of total RNA was performed using Superscript II reverse transcriptase (Invitrogen) and 250 ng of random hexamer primer, according to the manufacturer’s instructions. Real-time PCR was performed using TaqMan Universal PCR master mix (Applied Biosystems) and the TaqMan Pri-miRNA Assay (hsa-mir-576) for quantification of the primary transcript or TaqMan MicroRNA Assay (hsa-miR-576-3p) for quantification of miR-576-3p. Gene-specific primers were used to quantify SEC24B and GAPDH using the TaqMan system. For quantification of STING and IFNβ, real-time qPCR was performed using gene-specific primers, iQ SYBR green supermix (Bio-Rad) according to the manufacturer’s instructions, and the Bio-Rad CFX96 real-time PCR machine. The results were normalized to β-actin and GAPDH. Each PCR was performed in triplicate, and standard deviations were calculated. The amount of RNA is expressed in relative units with respect to mock treated samples.

Yarbrough M.L., Zhang K., Sakthivel R., Forst C.V., Posner B.A., Barber G.N., White M.A, & Fontoura B.M. (2014). Primate-Specific miR-576-3p Sets Host Defense Signaling Threshold. Nature communications, 5, 4963.

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Quantitative Analysis of miRNA and mRNA Expression

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RNA Extraction, Reverse Transcription, and qRT-PCR

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Total RNA was extracted from frozen tissues and culturing cells with the TRIzol reagent (Invitrogen) in accordance with the manufacturer’s protocol. Reverse transcription was performed with the M-MLV Reverse Transcriptase (Invitrogen) to produce the first strand cDNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out with the SYBR Premix Ex Taq II (Takara, Dalian, China) on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) in accordance with the manufacturer’s instructions. glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control for lncRNA and mRNA. The primers used are as follows: for lncRNA-HEIH, 5'-CCTCTTGTGCCCCTTTCT-3' (forward) and 5'-AGGTCTCATGGCTTCTCG-3' (reverse); for Bcl-xL, 5'-CGTGGAAAGCGTAGACAAG-3' (forward) and 5'-AAGAGTGAGCCCAGCAGAA-3' (reverse); and for GAPDH, 5'-GGTCTCCTCTGACTTCAACA-3' (forward) and 5'-GTGAGGGTCTCTCTCTTCCT-3' (reverse). The expression of miR-939 and pri-miR-939 was measured by qRT-PCR as above described, using TaqMan MicroRNA Assay (Applied Biosystems) and TaqMan PrimiRNA Assay (Applied Biosystems) respectively, in accordance with the manufacturer’s protocols. The expression of target genes was calculated using the comparative Ct method.

Cui C., Zhai D., Cai L., Duan Q., Xie L, & Yu J. (2017). Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL. Cancer Research and Treatment : Official Journal of Korean Cancer Association, 50(3), 992-1008.

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Quantification of miRNA and Primary Transcript

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Total RNA from frozen tissue specimens and cultured cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA quantity and quality were determined by a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington DE). Furthermore, mature miRNA levels were measured by using an NCode miRNA First-strand cDNA synthesis and real-time PCR Kit (Invitrogen, Carlsbad, CA). U6 snRNA (RNU6B) served as an endogenous control. Expression of the primary miR-622 transcript was analysed using a TaqMan Pri-miRNA assay (Assay ID Hs03304667_pri; Applied Biosystems). Primer sets are listed in Supplementary Table 5. Real-time PCR was performed on the Applied Biosystems 7300 Real-Time PCR system using SYBR Green dye (Applied Biosystems, Foster City, CA) as described by the manufacture. All determinations were performed in triplicate and in at least three independent experiments. The 2^−ΔΔCt method was applied to estimate relative transcript levels.

Liu H., Liu Y., Liu W., Zhang W, & Xu J. (2015). EZH2-mediated loss of miR-622 determines CXCR4 activation in hepatocellular carcinoma. Nature Communications, 6, 8494.

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