The Ago2-miRNA-co-IP Assay to Study TGF- β1 Mediated Recruitment of miRNA to the RISC in CFBE Cells.
The Ago2-miRNA-co-IP Assay to Study TGF- β1 Mediated Recruitment of miRNA to the RISC in CFBE Cells.
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DOI：
10.3791/61571
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发表时间：
2020-07-31
期刊：
影响因子：
--
通讯作者：
Swiatecka-Urban A
中科院分区：
文献类型：
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作者：
Mitash N;Donovan JE;Swiatecka-Urban A
Micro(mi)RNAs are short, non-coding RNAs that mediate the RNA interference (RNAi) by post-transcriptional mechanisms. Specific miRNAs are recruited to the cytoplasmic RNA induced silencing complex (RISC). Argonaute2 (Ago2), an essential component of RISC, facilitates binding of miRNA to the target-site on mRNA, followed by cleaving the miRNA-mRNA duplex with its endonuclease activity. RNAi is mediated by a specific pool of miRNAs recruited to RISC, and thus is referred to as the functional pool. The cellular levels of many miRNAs are affected by the cytokine Transforming Growth Factor-β1 (TGF-β1). However, little is known about whether the TGF-β1 affects the functional pools of these miRNAs. The Ago2-miRNA-co-IP assay, discussed in this manuscript, is designed to examine effects of TGF-β1 on the recruitment of miRNAs to RISC and it helps to determine whether changes in the cellular miRNA levels correlate with changes in the RISC-associated, functional pools. The general principles of the assay are as follows. Cultured cells treated with TGF-β1 or vehicle control are lysed and the endogenous Ago2 is immunoprecipitated with immobilized anti-Ago2 antibody, and the active miRNAs complexed with Ago2 are isolated with a RISC immunoprecipitation (RIP) assay kit. The miRNAs are identified with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) using miRNA-specific stem-looped primers during reverse transcription, followed by PCR using miRNA-specific forward and reverse primers, and TaqMan hydrolysis probes.
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影响因子：
5.6
作者：
Mitash, Nilay;Mu, Fangping;Swiatecka-Urban, Agnieszka
通讯作者：
Swiatecka-Urban, Agnieszka
影响因子：
5.5
作者：
Bebok, Z;Collawn, JF;Clancy, JP
通讯作者：
Clancy, JP
DOI：
10.1165/rcmb.2004-0012oc
发表时间：
2004-08-01
影响因子：
6.4
作者：
Hentchel-Franks, K;Lozano, D;Clancy, JP
通讯作者：
Clancy, JP
影响因子：
4.5
作者：
Janas, Maja M.;Wang, Bingbing;Novina, Carl D.
通讯作者：
Novina, Carl D.
miRNA 的茎环 RT-qPCR。
影响因子：
--
作者：
Kramer, Martha F
通讯作者：
Kramer, Martha F