构建RNA结合蛋白cDNA表达文库以筛选与RNA结合的蛋白

Min Young Kim; Jong Joo Lee; Chul Geun Kim

doi:10.21769/BioProtoc.1715

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Peer-reviewed

Identification of RNA-binding Proteins by RNA Ligand-based cDNA Expression Library Screening

构建RNA结合蛋白cDNA表达文库以筛选与RNA结合的蛋白

Min Young Kim

JL Jong Joo Lee

Chul Geun Kim email

发布: 2016年01月20日第6卷第2期 DOI: 10.21769/BioProtoc.1715 浏览次数: 8089

评审: Antoine de MorreeZhen Shi

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Cover of Nucleic Acids Research, featuring study using the protocol.

Jun 2014

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Abstract

We previously reported when a portion of the Requiem (REQ/DPF2) messenger ribonucleic acid (mRNA) 3’ untranslated region (3’UTR), referred to as G8, was overexpressed in K562 cells, β-globin expression was induced, suggesting that the 3’UTR of REQ mRNA plays a physiological role (Kim et al., 2014). To identify trans-acting factors that bind to the REQ 3’UTR, we describe the RNA ligand based cDNA expression library screening method. This protocol could be adapted to detect specific RNA-protein interactions. Following this method, we identified six positive clones in the initial round of screening and four pure clones after sib-screening. This protocol was originally published in Kim et al. (2014).

Keywords: RNA-protein interaction (RNA和蛋白质的相互作用)

Phage display (噬菌体展示)

Requiem (REQ/DPF2) (安魂曲（请求/ dpf2）)

CDNA library screening (cDNA文库筛选)

Materials and Reagents

X-Omat AR film (Eastman Kodak Company, catalog number: 0572842 )
Eppendorf tubes
K562 cells
Note: Phagemid-based K562 cDNA expression libraries were constructed by isolating mRNA from cells with an Ultraspec-RNA isolating system and a biotinylated oligo (dT) probe.
XL1-Blue cells (Agilent Technologies, catalog number: 200403 )
XLOR cell (Agilent Technologies, catalog number: 200403 )
10% bovine calf serum (GE Healthcare, Hyclone^TM, catalog number: SH30073.03 )
RPMI 1640 medium (Life Technologies, Gibco^TM, catalog number: 11875-093 )
Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 11875-093”.
Ultraspec-RNA isolating system (Biotech, catalog number: BL-10050 )
Biotinylated oligo (dT) probe (50 pmol/μl) (Promega Corporation, catalog number: Z5261 )
cDNA synthesis kit (Agilent Technologies, catalog number: 200403 )
λZAP II express phage vector (Agilent Technologies, catalog number: 200403 )
Nitrocellulose membrane (immobilon-NC membrane) (EMD Millipore Corporation, catalog number: N8395 )
Ribonucleic acid from torula yeast (RNA type VI) (Sigma-Aldrich, catalog number: R6625 )
[α-32P]-labeled G8-RNA ligand (BMS)
ExAssist helper phage (Agilent Technologies, catalog number: 200253 )
IPTG [≥ 99% (TLC), ≤ 0.1% Dioxan] (Sigma-Aldrich, catalog number: I6758 )
HEPES (pH 7.9)
KCl
0.1% (w/v) Ficoll 400-DL
0.01% polyvinyl-pyrolidon PVP-40
MnCl₂
ZnCl₂
EDTA
DTT
Tryptone
NaCl
Yeast Extract
DW
MgSO₄
Tris-HCl (pH 7.5)
Gelatin
Screening buffer (see Recipes)
LB plates (see Recipes)
SM buffer (see Recipes)

Equipment

Refrigerated Eppendorf centrifuge (Hanil, catalog number: Union 55R )
Heat block (Thermolyne, catalog number: DB17615 )
Tissue culture CO₂ incubators set at 37 °C (HERAcell, catalog number: HERAcell^® 240 )
Developer (TAEAHN, catalog number: TM-90S )
Vortex (JEIO TECH, catalog number: VM-96B )
Deep freezer (Forma Scientific, catalog number: 917 )
Shaker (SLB, catalog number: SLRM-3 )

Procedure

English

中文翻译

文章信息

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如何引用

Kim, M. Y., Lee, J. J. and Kim, C. G. (2016). Identification of RNA-binding Proteins by RNA Ligand-based cDNA Expression Library Screening. Bio-protocol 6(2): e1715. DOI: 10.21769/BioProtoc.1715.