Measurement of mRNA Decay in Mouse Embryonic Fibroblasts
小鼠胚胎成纤维细胞中mRNA Decay的测量
发布: 2016年07月05日第6卷第13期 DOI: 10.21769/BioProtoc.1858 浏览次数: 11542
评审: Ivan ZanoniAnonymous reviewer(s)
下载 PDF 
Q&A
参见作者原研究论文
The authors used this protocol in:
Apr 2015
Abstract
mRNA stability control is a critical step in the post-transcriptional regulation of gene expression. Actinomycin D, an antibiotic initially used as an anti-cancer drug, has turned out to be a convenient tool for studying the turnover rates of transcripts in cells, due to its inhibition of mRNA synthesis. Here, we describe a protocol for the measurement of mRNA decay after adding actinomycin D into the medium of stable fibroblast cell lines derived from wild-type and tristetraprolin (TTP)-deficient mouse embryonic fibroblast (MEF) cultures, as well as a protocol for determining the relative transcript abundance using semi-quantitative real-time RT-PCR. Northern blotting or NanoString n-Counter are alternative methods to measure mRNA abundance, which is quantified using a phosphorimager in the former case. This protocol is suitable for studying primary cultured cells and stable cell lines derived from transgenic mice and their respective controls, and provides for direct comparisons of mRNA decay rates in otherwise identical cells with and without the gene of interest.
Keywords: MRNA decay (mRNA的衰变)
Mouse embryonic fibroblasts (小鼠胚胎成纤维细胞)
Actinomycin D (actinomycin D)
Real-time RT-PCR (实时RT-PCR)
Materials and Reagents
- 60-mm sterile Petri dish (e.g., BD Biosciences, Falcon®, catalog number: 353002 )
Note: Currently, it is “Corning, Falcon®, catalog number: 353002”. - T-75 tissue culture flask (e.g., BD Biosciences, Falcon®, catalog number: 353136 )
Note: Currently, it is “Corning, Falcon®, catalog number: 353136”. - 50 ml sterile conical tube (e.g., BD Biosciences, Falcon®, catalog number: 352070 )
Note: Currently, it is “Corning, Falcon®, catalog number: 352070”. - 384-well microplate (e.g., BioExpress, catalog number: T-6062-1 )
- 1.7 ml RNase-free, DNase-free Posi-Click tubes (Denville Scientific Inc., catalog number: C2170 )
- Mouse wild-type (WT) and TTP-deficient stable fibroblast cell lines (Lai WS et al., 2006)
- 1x Phosphate-buffered saline (PBS) without calcium and magnesium
- 0.05% trypsin/EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300 )
- Fetal bovine serum defined (FBS) (GE Healthcare, HyCloneTM, catalog number: SH30070.03 )
- Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11965-092 )
- Penicillin-Streptomycin 10,000 U/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
- L-glutamine 200 mM (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-081 )
- Recombinant mouse tumor necrosis factor (TNF) (R&D Systems, catalog number: 410-MT )
- Actinomycin D (Sigma-Aldrich, catalog number: A4262 )
- Illustra RNAspin MiniRNA isolation kit (Sigma-Aldrich, GE Healthcare, catalog number: 25-0500-72 )
- SuperScript First-Strand Synthesis System (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18080-051 )
- Power SYBR Green master mix (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4368702 )
- Mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
- DEPC water (Baltimore Bioworks, catalog number: WA-137-500 )
- Primers for transcripts of interest
- 70% ethanol
- Complete medium (see Recipes)
- Serum-starving medium (see Recipes)
Equipment
- 37 °C, 5% CO2 forced-air incubator (e.g., Thermo Fisher Scientific, FormaTM, model: 3110 )
- Centrifuge with swinging-bucket rotor and adaptors for 50-ml conical tubes
- ABI Prism 7900HT Real-Time PCR System and Sequence Detection System (Applied Biosystems, model: 7900HT ) or similar
- Vortex-Genie-2 (Scientific Industries, catalog number: SI-0236 ) or similar
- Nanodrop 2000c spectrophotometer (Thermo Scientific, model: 2000c )
- Desktop centrifuge (e.g., Eppendorf, model: 5417R )
- DNA Engine Peltier Thermal Cycler (Bio-Rad Laboratories, MJ research, catalog number: PTC-200 ) or similar
- Cell scraper (Corning, Costar, catalog number: 3010 )
Software
- GraphPad Prism software (GraphPad Software, model: version 6.0)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Qiu, L. Q., Lai, W. S., Stumpo, D. J. and Blackshear, P. J. (2016). Measurement of mRNA Decay in Mouse Embryonic Fibroblasts. Bio-protocol 6(13): e1858. DOI: 10.21769/BioProtoc.1858.
Download Citation in RIS Format
分类
免疫学 > 免疫细胞功能 > 综合
您对这篇实验方法有问题吗？
在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。
提问指南
+ 问题描述
写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。
发布问题
