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UPF1 RNA Immunoprecipitation from Mini-μ Construct–expressing Cells

Mini-μ 载体-表达细胞中的UPF1 RNA 免疫沉淀

David Zünd David Zünd
Oliver Mühlemann Oliver Mühlemann

发布: 2014年04月05日第4卷第7期 DOI: 10.21769/BioProtoc.1086 浏览次数: 10966

评审: Anonymous reviewer(s)

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Abstract

UPF1, an RNA helicase and a core factor of nonsense-mediated mRNA decay (NMD), interacts with RNA independently of the sequence context. To investigate the influence of translation on the association of UPF1 with specific reporter transcripts, UPF1 RNA immunoprecipitations (RIPs) are performed from Hela cells that either express a normally translated immunoglobulin-µ (Ig-µ) reporter (mini µ) or a version with a stable stem loop in the 5' UTR (SL mini µ) that efficiently inhibit translation initiation (Zund et al., 2013). Both the cloning of the SL mini µ reporter construct and the UPF1 RIP experiment are described in detail.

Keywords: RNA-IP (RNA-IP) search RIP (RIP) search NMD (NMD) search Nonsense-mediated mRNA decay (无义介导的mRNA衰变) search RNA helicase UPF1 (RNA解旋酶Upf1) search

Materials and Reagents

  1. Hela cells
  2. XL-10 Gold Ultracompetent Cells (Agilent Technologies, Stratagene, catalog number: 200315 )
  3. Dulbecco’s Modified Eagle Media (DMEM) (powder, high glucose) (Life Technologies, catalog number: 52100-039 )
  4. MQ-water (pure water from the ELGA system)
  5. Fetal Calf Serum (FCS) (BioConcept, Amimed, catalog number: 2-01F30-I )
  6. Penicillin-Streptomycin solution (P/S) (1 unit/ml) (BioConcept, Amimed, catalog number: 4-01F00-H )
  7. Trypsin-EDTA (T/E) (BioConcept, Amimed, catalog number: 5-51F00-H )
  8. DreamFectTM (OZ Biosciences, catalog number: DF45000 )
  9. KpnI (New England Biolabs, catalog number: R0142S )
  10. SL Oligo 1 (Microsynth AG)
    Oligo sequence: 5'-CGGGTTCCGTCCAAGCACTGTTGAAGCAGGAAACCCGGGTTGCTAGTCGATCGACTAG-CAACCCGGGTTTCCTGCTTCAACAGTGCTTGGACGGAACCCCGATCGTAC-3'
  11. SL Oligo 2 (Microsynth AG)
    Oligo sequence: 5'-GATCGGGGTTCCGTCCAAGCACTGTTGAAGCAGGAAACCCGGGTTGCTAGTCGATCG-ACTAGCAACCCGGGTTTCCTGCTTCAACAGTGCTTGGACGGAACCCGGTAC-3'
  12. T4 DNA Ligase (New England Biolabs, catalog number: M0202S )
  13. T4 Polynucleotide Kinase (PNK) (New England Biolabs, catalog number: M0201L )
  14. Alkaline phosphatase calf intestinal (CIAP) (Promega Corporation, catalog number: M182A )
  15. 100x HaltTM Protease Inhibitor Cocktail (Thermo Fisher Scientific, catalog number: 1861279 )
  16. RiboLock RI RNase Inhibitor (40 U/µl) (Thermo Fisher Scientific, catalog number: EO0381 )
  17. RNase I (cloned, 100 U/µl) (Life Technologies, Ambion®, catalog number: AM2294 )
  18. Turbo DNase (Life Technologies, Ambion®, catalog number: AM2238 )
  19. Glycogen for molecular biology (Roche Diagnostics, catalog number: 10 901 393 001 )
  20. Pre-stained Protein Ladder (broad range) (New England Biolabs, catalog number: P7710S )
  21. Goat anti-UPF1 Antibody (G-α-RENT1) (Bethyl Laboratories, catalog number: A300-038A )
  22. Goat anti-rabbit IgG (polyclonal) (Bio-Rad Laboratories, catalog number: 172-1053 )
  23. Rabbit anti-actin (polyclonal) (Sigma-Aldrich, catalog number: A5050 )
  24. AffiniPure Goat Anti-mouse IgM (µ chain specific) (Jackson ImmunoResearch Laboratories, catalog number: 115-005-020 )
  25. IRDye 800CW Donkey anti-Rabbit (LI-COR, catalog number: 926-32213 )
  26. IRDye 800CW Donkey anti-Goat (LI-COR, catalog number: 926-32214 )
  27. Dynabeads® Protein G (Life Technologies, catalog number: 10004D )
  28. Wizard® SV Gel and PCR Clean-Up System (Promega Corporation, catalog number: A9282 )
  29. Affinity Script Multi-Temp Reverse Transcriptase (Agilent, catalog number: 600105 )
  30. Random hexamer primers (Microsynth AG)
  31. Brilliant III Ultra-Fast qPCR Master mix (Agilent, catalog number: 600880 )
  32. Chloroform
  33. Isopropanol
  34. DMEM-/- (see Recipes)
  35. DMEM+/+ (see Recipes)
  36. Phosphate-bufferd saline (PBS) (pH 7.4) (see Recipes)
  37. Hypotonic gentle lysis buffer (pH 7.5) (RNase-free) (see Recipes)
  38. Wash buffer (pH 7.5) (RNase-free) (see Recipes)
  39. Net-2 buffer (pH 7.5) (RNase-free) (see Recipes)
  40. Hybridization buffer (pH 7.5) (see Recipes)
  41. 2x SDS loading buffer (pH 6.8) (see Recipes)
  42. TRI-reagent (see Recipes)
  43. Tris buffered saline (pH 7.6) (TBS) (see Recipes)
  44. TBS-Tween milk (see Recipes)
  45. Bjerrum transfer buffer (see Recipes)
  46. DEPC treated water/buffer (see Recipes)
  47. Turbo DNase mix (see Recipes)

Equipment

  1. Pure water system: PURELAB Priama (Prima 7) and PURELABULTRA (Ultra Genetic) (ELGA LabWater)
  2. CO2 incubator (BINDER GmbH, model: 9140-0047 )
  3. Clear-viewTM Snap-Cap microtubes (1.5 ml, natural, low retention) (Sigma-Aldrich, catalog number: T4816-250EA )
  4. Multiply®-Pro 0.2Ml Biosphere® (Sarstedt AG, catalog number: 72.727 )
  5. Filter Tips (10 µl, 20 µl and 200 µl) (Axon Lab AG, catalog numbers: AL60X10 , AL60X20 , AL60X200 )
  6. Filter Tips (1,250 µl) (Greiner Bio-One GmbH, catalog number: 7.750.261 )
  7. CountessTM automated cell counter (Life Technologies, model: C10227 )
  8. CountessTM cell counting chamber slides (Life Technologies, catalog number: C10283 )
  9. 15 cm tissue culture dishes (TPP Techno Plastic Products, catalog number: 93150 )
  10. GP Millipore express® PLUS Membrane (0.22 µm) (500 ml Funnel, 45 mm Neck Size) (EMD Millipore, catalog number: SCGPT05RE )
  11. NanoDrop 2000 (Thermo Fisher Scientific)
  12. Heat block, Thermomixer® compact (Eppendorf)
  13. DynaMagTM-2 magnet (Life Technologies, catalog number: 12321D )
  14. Eppendorf centrifuge 5415R with rotor F45-24-11 (Eppendorf, catalog numbers: 022621459 and 022636502 )
  15. Lab cycler gradient equipped with Thermoblock 96 (SensoQuest GmbH, models: 011-101 and 012-103 )
  16. Blotting paper (ALBET Lab Science, catalog number: BP 002 46579 )
  17. Corbett Rotor-Gene® 6000 (QIAGEN)
  18. Corbett CAS-1200 (QIAGEN)
  19. SE260 Mighty Small II Deluxe Mini vertical electrophoresis unit (Hoefer, model: SE260-10A-1.5 )
  20. Optitran BA-S 85 reinforced nitrocellulose membrane (GE Healthcare, Whatman, catalog number: 10 439 196 )
  21. Semi-dry transfer unit with built-in power supply TE77XP (Hoefer, model: TE77XP )
  22. Odyssey® infrared imaging system (LI-COR)

Procedure

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© 2014 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Zünd, D. and Mühlemann, O. (2014). UPF1 RNA Immunoprecipitation from Mini-μ Construct–expressing Cells. Bio-protocol 4(7): e1086. DOI: 10.21769/BioProtoc.1086.

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分类

生物化学 > RNA > RNA-蛋白质相互作用

分子生物学 > RNA > RNA 检测

生物化学 > 蛋白质 > 免疫检测

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