The Ago2-miRNA-co-IP Assay to Study TGF- β1 Mediated Recruitment of miRNA to the RISC in CFBE Cells

Summary

Here, we describe the Ago2-miRNA-co-IP assay designed to quantify an active pool of specific miRNAs induced by TGF-β1 in human bronchial epithelial CFBE41o- cells. This assay provides functional information on the recruitment of miRNA to the RNA induced silencing complex, quantified by qRT-PCR using specific miRNA primers and TaqMan hydrolysis probes.

Abstract

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.

Introduction

Transforming Growth Factor-β1 (TGF-β1) is a multifunctional cytokine that can change the expression of many micro(mi)RNAs^1,2,3. The total cellular level of a particular miRNA does not correlate with its inhibitory potential because only a specific fraction of the miRNA is incorporated into RNA induced silencing complex (RISC) to perform RNA interference (RNAi)³. Only up to 10% of each miRNA is RISC-associated and participates in RNAi^4,5. Next, the RNAi process involves binding of the ....

Protocol

1. Preparation before experiment

1. Seeding cells
   1. Prepare 10% collagen I solution in Minimal Essential Medium (MEM) and add 500 µL to each 24 mm cell culture filter in a 6-well plate. Distribute to cover the entire surface of the filter by rotating gently by hand. Incubate filters under the UV light in the laminar flow hood at room temperature for 30 minutes (min), followed by incubation in cell culture incubator at 37 °C for 1 h.
   2. Prepare cell culture medium (MEM.......

Discussion

The Ago2-miRNA-co-IP assay is designed to investigate the active pool of miRNAs in response to TGF-β1 treatment. The active or RISC-associated miRNAs are important to understand their inhibitory potential for the target mRNA⁴. Panshin et al. recently showed that the immunoprecipitation efficiency of Ago2 and miRNAs may depend on the protocol¹⁶. There are several differences between the protocol here and the above published data. The protocol here was optimized for CFBE.......

Materials

Name	Company	Catalog Number	Comments
100 mM dNTPs (with dTTPs)	Applied Biosystems	4366596
10x Reverse Transcription Buffer (RT Buffer)	Applied Biosystems	4366596
2-propanol	Fisher BioReagents	BP2618-1
2x Laemmli Sample Buffer	Bio-Rad	1610737
7300 Real Time PCR System	Applied Biosystems	4345240
Anti-Ago2 antibody (anti-EIF2C2), mouse monoclonal against human Ago2	Medical & Biological Laboratories Co. Ltd	RN003M
Bovine Albumin Fraction V (7.5% solution)	Thermo Scientific	15260037
Collagen I (Purecol-Type I Bovie collagen solution)	Advanced Biometrix	50005-100mL
DL-Dithiothreitol (DTT)	Sigma	646563-.5ML
DTT	Sigma-Aldrich	646563
Ethanol	Deacon Laboratories	64175
Fetal Bovine Serum	ATLANTA Biologicals	S10350
Goat Anti-Mouse IgG	Bio-Rad	1706516
L-glutamine (200 mM Solution; 29.20 mg/mL)	Corning	25-005-Cl
Mini cell scrapers United Biosystems	Thermo Fisher	MCS-200
Minimal Essential Medium	Thermo Fisher Scientific	11095-080
miRNA specific stem looped RT primers	Applied Biosystems	4427975
Mouse IgG2 control	Dako, Glostrup, Denmark	A0424
MultiScribe Reverse Transcriptase, 50 U/µL	Applied Biosystems	4366596
Nano Drop ND-1000 Spectrophotometer	NanoDrop Technologies, Inc.	E112352
Nuclease-free water	Ambion	AM9937
Opti-MEM (1x) Reduced Serum Medium	Gibco by Life Technologies	11058-021
PBS	Gibco	14190250
Penicillin-streptomycin, Sterile	Sigma-Aldrich	P0781
Pierce Protease Inhibitor Tablets, EDTA-Free	Thermo Scientific	A32955
Protein G agarose beads (Pierce Protein G Plus Agarose)	Thermo Scientific	22851
Puromycin	InvivoGen	ant-pr-1
RiboCluster Profiler RIP-Assay Kit for microRNA	Medical & Biological Laboratories Co. Ltd	RN1005
RNase Inhibitor, 20 U/µL	Applied Biosystems	4366596
TaqMan 2x Universal PCR master mix without AmpErase UNG	Applied Biosystems	4427975
TaqMan miRNA single tube Assay (20x) containing miRNA specific forward/reverse primers and probe	Applied Biosystems	4427975 (assay ID #002278, #002146, and #000477)
TGF-beta1	Sigma	T1654
Transwell filters (24 mm)	Corning Life Sciences Plastic	3412
Veriti 96 Well Thermal Cycler (Model:9902)	Applied Biosystems	4375786