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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 Min Young Kim
JL Jong Joo Lee
Chul Geun Kim Chul Geun Kim

发布: 2016年01月20日第6卷第2期 DOI: 10.21769/BioProtoc.1715 浏览次数: 8883

评审: Antoine de MorreeZhen Shi

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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) search Phage display (噬菌体展示) search Requiem (REQ/DPF2) (安魂曲（请求/ dpf2）) search CDNA library screening (cDNA文库筛选) search

Materials and Reagents

  1. X-Omat AR film (Eastman Kodak Company, catalog number: 0572842 )
  2. Eppendorf tubes
  3. 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.
  4. XL1-Blue cells (Agilent Technologies, catalog number: 200403 )
  5. XLOR cell (Agilent Technologies, catalog number: 200403 )
  6. 10% bovine calf serum (GE Healthcare, HycloneTM, catalog number: SH30073.03 )
  7. RPMI 1640 medium (Life Technologies, GibcoTM, catalog number: 11875-093 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11875-093”.
  8. Ultraspec-RNA isolating system (Biotech, catalog number: BL-10050 )
  9. Biotinylated oligo (dT) probe (50 pmol/μl) (Promega Corporation, catalog number: Z5261 )
  10. cDNA synthesis kit (Agilent Technologies, catalog number: 200403 )
  11. λZAP II express phage vector (Agilent Technologies, catalog number: 200403 )
  12. Nitrocellulose membrane (immobilon-NC membrane) (EMD Millipore Corporation, catalog number: N8395 )
  13. Ribonucleic acid from torula yeast (RNA type VI) (Sigma-Aldrich, catalog number: R6625 )
  14. [α-32P]-labeled G8-RNA ligand (BMS)
  15. ExAssist helper phage (Agilent Technologies, catalog number: 200253 )
  16. IPTG [≥ 99% (TLC), ≤ 0.1% Dioxan] (Sigma-Aldrich, catalog number: I6758 )
  17. HEPES (pH 7.9)
  18. KCl
  19. 0.1% (w/v) Ficoll 400-DL
  20. 0.01% polyvinyl-pyrolidon PVP-40
  21. MnCl2
  22. ZnCl2
  23. EDTA
  24. DTT
  25. Tryptone
  26. NaCl
  27. Yeast Extract
  28. DW
  29. MgSO4
  30. Tris-HCl (pH 7.5)
  31. Gelatin
  32. Screening buffer (see Recipes)
  33. LB plates (see Recipes)
  34. SM buffer (see Recipes)

Equipment

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

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

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.

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分类

分子生物学 > 蛋白质 > 表达

分子生物学 > RNA > RNA-蛋白质相互作用

生物化学 > 蛋白质 > 相互作用 > 噬菌体展示

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