A549 cells were transfected with 10 nM miRNAs by reverse transfection. After 72 h, immunoprecipitation was performed using a microRNA Isolation Kit, Human Ago2 (Wako Pure Chemical Industries, Ltd., Osaka, Japan) as described previously [47 (link),48 (link)]. Expression levels of miR-150-5p and miR-150-3p were analyzed by qRT-PCR. MiRNA data were normalized to expressions of miR-16-5p, miR-21-5p, and miR-26a, which were not affected by miR-150-5p and miR-150-3p.
Human ago2
Manufactured by Fujifilm
Sourced in Japan
The Human Ago2 is a laboratory equipment product from Fujifilm. It is a key component in molecular biology research, responsible for the central function of RNA interference (RNAi). The Ago2 protein plays a crucial role in the RNAi pathway, mediating the silencing of target genes. This product is designed to facilitate researchers in studying and understanding the mechanisms of gene regulation through RNAi technology.
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Lab products found in correlation
7 protocols using human ago2
1
Profiling miR-150-5p and miR-150-3p
2
Incorporation of miR-149 into RISC
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To confirm that exogenous miR-149-5p or miR-149-3p were incorporated into RISC, we performed immunoprecipitation assays using a microRNA Isolation Kit, Human Ago2 (Wako, Osaka, Japan) as described previously [19 (link)]. The expression levels of miRNAs bound to Ago2 were measured by TaqMan RT-qPCR. The miRNA expression data were normalized to the expression of miR-26a (product ID: 000404; Applied Biosystems), which was not affected by miR-149-5p or miR-149-3p expression.
3
Transfection and Immunoprecipitation of miR-148a
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PANC‐1 and SW1990 cells growing on 6‐well plates were transfected with 10 nmol/L pre‐miR‐148a. After reverse transfection, immunoprecipitation was carried out using a microRNA Isolation kit, Human Ago2 (Wako, Osaka, Japan). The procedure was as described previously.13 , 14
4
Profiling miR-150 Isoform Expression
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SAS cells were transfected with 10nM miRNA by Reverse transfection. After 48h, immunoprecipitation was performed using a microRNA isolation kit, Human Ago2 (Wako, Osaka, Japan) according to the manufacturer's protocol. Expression levels of miR-150-5p or miR-150-3p were measured by RT-qPCR methods. Detection of miRNA data were normalized to the expression of miR-26a (assay ID: 000404; Applied Biosystems), which was not affected by miR-150-5p and miR-150-3p.
5
Investigating miRNA Incorporation into RISC
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We investigated whether the passenger strand of miRNA was incorporated into RNA‐induced silencing complex (RISC). We used a miRNA Isolation Kit, Human Ago2 (Wako, Osaka, Japan) to prepare a high purity fraction of microRNA based on an immunoprecipitation method using a high affinity anti‐Human Ago2 monoclonal antibody. The procedure was carried out according to the manufacturer's protocol.
6
Measuring miRNA Incorporation into RISC
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For the measurement of incorporated miRNAs into RISC in PDAC cells, we applied Ago2 immunoprecipitation by using a microRNA Isolation Kit, Human Ago2 (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The number of Ago2-conjugated miRNAs were assessed by qRT-PCR assay. The experimental procedure has been described in previous studies [32 (link),33 (link),41 (link)].
7
Isolation and Analysis of Ago2-Bound miRNAs
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Argonaute 2 (AGO2)-bound miRNA isolation by immunoprecipitation. PC3 cells were transfected with 10 nM miRNA by reverse transfection and plated in 10-cm plates at 1x10 5 cells/ml. After 48 h, immunoprecipitation was performed using a microRNA Isolation kit, Human Ago2 (Wako, Osaka, Japan) according to the manufacturer's protocol. Expression levels of miRNAs bound to Ago2 were measured by TaqMan RT-qPCR. miRNA expression data were normalized to the expression of miR-26a (product ID: 000404; Applied Biosystems), which was not affected by miR-150-5p and miR-150-3p.
Western blot analysis. Immunoblotting was conducted with diluted monoclonal anti-SPOCK1 antibodies (1:100 dilution; sc-398782; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and with diluted anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (1:1,000 dilution; ab8245; Abcam, Cambridge, UK) as a loading control. The procedures were performed as previously described (16) .
Western blot analysis. Immunoblotting was conducted with diluted monoclonal anti-SPOCK1 antibodies (1:100 dilution; sc-398782; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and with diluted anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (1:1,000 dilution; ab8245; Abcam, Cambridge, UK) as a loading control. The procedures were performed as previously described (16) .
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