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In vitro Reconstitution Assay of miRNA Biogenesis by Arabidopsis DCL1
拟南芥DCL1催化miRNA合成的体外实验

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Abstract

microRNAs (miRNAs) are small non-coding RNAs, regulating most if not all, biological processes in eukaryotic organisms. miRNAs are initially processed from primary transcripts (pri-miRNAs) to produce miRNA precursors (pre-miRNAs), that are further processed into miRNA and its complementary strands (miRNA/*). In Arabidopsis, and possibly other plants, the processing from pri-miRNAs to pre-miRNAs and from pre-miRNAs to miRNA/* are both implemented through Dicer-like 1 (DCL1) complexes. Recently, we demonstrated isolation of DCL1 complexes of unprecedented quality from in planta. We further successfully reconstituted DCL1 cleavage assays in vitro that were able to fully recapitulate in vivo miRNA biogenesis. Here we provide a detailed protocol of DCL1 reconstitution assays. The protocol comprises three major parts (Figure 1): 1) Preparation of pri- and pre-miRNA transcripts (Procedures A-C); 2) Purification of the recombinant Arabidopsis DCL1 machinery from Nicotiana benthamiana (N. benthamiana) through immunoprecipitation (IP) (Procedures D and E); and 3) in vitro processing of radioisotope-labeled pri- or pre-miRNAs using the isolated DCL1 complexes (Procedure F). It is our desire that the protocol be a powerful tool for the RNAi community to study mechanistic issues or to develop RNA silencing technologies.

Keywords: Microprocessor(微处理器), Pri-miRNA(miRNA初级转录产物), MiRNA(miRNA), Dicer(Dicer), Arabidopsis(拟南芥)

Materials and Reagents

  1. Five to six-week old plants of N. benthamiana
  2. Agrobacterium strain: ABI (Zhang et al., 2006)
  3. Plasmid [pBA-2Flag-4Myc-DCL1, pBA-6Myc-HYL1, pBA-6Myc-SE. Detailed approach could refer to the Online Methods from Zhu et al. (2013). Negative control plasmid: pBA]
  4. G-Tube® snap cap siliconized microcentrifuge tubes (VWR International, catalog number: 22179-004 )
  5. Anti-FLAG® M2 magnetic beads (Sigma-Aldrich, catalog number: M8823 )
  6. 3x Flag peptide (NH2-MDYKDHDGDYKDHDIDYKDDDDK-COOH) (Sigma-Aldrich, catalog number: F4799 )
  7. DNase (Promega Corporation, catalog number: M6101 )
  8. 3,000 Ci/mmol 10 mCi/ml [γ-32P]-ATP (PerkinElmer, catalog number: BLU502A100UC )
  9. SuperaseIn RNase inhibitor (Ambion, catalog number: N8080119 )
  10. T7 RNA polymerase (Ambion, catalog number: 18033019 )
  11. Calf intestine alkaline phosphatase (NEB, catalog number: M0290S )
  12. Phenol: chloroform: isoamyl alcohol (25:24:1) (Life Technologies, InvitrogenTM, catalog number: 15593049 )
  13. NaAc (Sigma-Aldrich, catalog number: S7670 )
  14. 5 M ammonium acetate (Ambion, catalog: AM9071 )
  15. GlycoBlue (Ambion, catalog number: AM9516 )
  16. Decade marker (Ambion, catalog number: AM7778 )
  17. Anti-c-Myc Agarose Affinity Gel (Sigma-Aldrich, catalog number: A7470 )
  18. EDTA-free protease inhibitor cocktail (Roche Diagnostics, catalog number: 05892953001 )
  19. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P2714 )
  20. HEPES (Sigma-Aldrich, catalog number: H3375 )
  21. Spermidine (Sigma-Aldrich, catalog number: S2626 )
  22. DTT (DL-Dithiothreitol) (Sigma-Aldrich, catalog number: 43817 )
  23. MgCl2 (Sigma-Aldrich, catalog number: M8266 )
  24. NTP (Thermo scientific, catalog number: R0481 )
  25. Deionized formamide (Ambion, catalog number: AM9342 )
  26. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  27. Xylene cyanol (Sigma-Aldrich, catalog number: X4126 )
  28. EDTA (Sigma-Aldrich, catalog number: V900106 )
  29. SDS (Sigma-Aldrich, catalog number: L3771 )
  30. DEPC (Sigma-Aldrich, catalog number: V900882 )
  31. KCl (Sigma-Aldrich, catalog number: V900068 )
  32. Tris-HCl (Sigma-Aldrich, catalog number: V900312 )
  33. Triton X-100 (Sigma-Aldrich, catalog number: V900502 )
  34. PMSF (Sigma-Aldrich, catalog number: 78830 )
  35. NaCl (Sigma-Aldrich, catalog number: V900058 )
  36. ATP (Thermo scientific, catalog number: R1441 )
  37. GTP (Thermo scientific, catalog number: R1461 )
  38. Glycerol (Sigma-Aldrich, catalog number: V900122 )
  39. 0.1 M Glycine-HCl (Sigma-Aldrich, catalog number: 55097-5ML-F )
  40. 5x transcription buffer (see Recipes)
  41. RNA loading buffer (see Recipes)
  42. RNA elution buffer (see Recipes)
  43. DEPC H2O (see Recipes)
  44. RNA dissolve buffer (see Recipes)
  45. Chloroform: isoamyl alcohol (24:1) (see Recipes)
  46. IP buffer (see Recipes)
  47. Protease inhibitor cocktail stock (see Recipes)
  48. TBS buffer (see Recipes)
  49. 3x Flag elution buffer stock (4 mg/ml) (see Recipes)
  50. Washing buffer (see Recipes)
  51. Assay buffer (see Recipes)
  52. Fixing buffer (see Recipes)

Equipment

  1. Compact UV lamp (UVP, model: UVGL-25 )
  2. Fluor-Coated TLC Plate (10 x 10 cm) (Ambion, catalog number: AM10110 )
  3. Geiger counter (Medcom, model: CRM-100 )
  4. Vertical electrophoresis system (Whatman, model: V15.17 )
  5. Thermomixer C (Eppendorf, model: 5382000023 )
  6. Gel dryer (Bio-Rad Laboratories, model: 583 )
  7. Gel analyzer Quantity One (Bio-Rad Laboratories, version: 4.6.9 )
  8. DynaMagTM-2 (Life technologies, model: 12321D )
  9. Rugged Rotator (Glas-Col, model: 099A MR1512 )
  10. PolyATtract® System 1000 Magnetic Separation Stand (Promega Corporation, model: Z541A )
  11. Phospho imager (PharosFXTM plus system)

Procedure

Note: Please see Figure 1 for a schematic diagram of the six steps described below.


Figure 1. A schematic diagram of procedure

  1. Preparation of pri- and pre-miRNA transcripts (Han et al., 2014)
    1. Substrates of pri- and pre-miRNA are transcribed under the T7 promoter in vitro using PCR-generated templates. In this case, the forward PCR primer should contain T7 promoter sequences (TAATACGACTCACTATAG) to drive transcription directly from the PCR product.
    2. Prepare in vitro transcription reaction as follows:

      Reagent
      Volume
      Final conc.
      Purified PCR product (100 ng)
      - µl

      5x transcription buffer
      4 µl

      Superase-In
      1 µl
      1 U/μl
      T7 RNA polymerase
      2 µl

      Add H2O to final volume 20 µl
      20 µl

      Note: In vitro transcription kits are also available from other commercial vendors.

    3. Briefly spin down. Incubate at 37 °C for 3 h or overnight.
    4. Add 1 μl DNase. Mix and leave the tube at 37 °C for 30 min.
    5. Add 20 μl RNA loading buffer to the transcribed RNA.
    6. Heat the tubes for 2 min at 95 °C. Cool down for 2 min on ice.
    7. Briefly spin down the tubes at 4 °C. Keep the samples on ice until loading.
    8. Meanwhile, prepare a 6% urea-polyacrylamide gel.
    9. Before loading the samples, flush the wells of the 6% urea-polyacrylamide gel with 1x TBE running buffer to remove the diffused urea from the wells.
    10. Load the RNA samples on 6% urea-polyacrylamide gel and run at 350 V (20 V/cm) until bromophenol blue reaches the bottom of the gel.
    11. Remove one of the glass plates from the gel.
    12. Apply UV shadowing to locate the positions of transcripts and then cut out the gel slice of single bands of pri- or pre-miRNAs band.
      UV Shadowing: Remove the gel from the glass plates used for the electrophoresis and place it on a sheet of plastic wrap. Then position the plastic wrap and gel on top of a fluor-coated TLC plate (attention: Avoid skin and eye exposure to UV light). Using a hand-held UV monitor, identify the bands that exhibit distinguishable shadow and mark the locations of the bands of interest if they are to be cut of the gel for further purification.
    13. Put the gel slice into a 1.5 ml tube with 350 μl of RNA elution buffer.
    14. Incubate the tube overnight at 42 °C with 1,200 rpm shaking using Thermomixer C.
    15. Centrifuge the mixture at 4 °C for 10 min.
    16. Transfer the supernatant (about 300 μl) to a fresh tube.
    17. Add 100 μl of RNA elution buffer into the old tube with the gel slice, vortex and centrifuge for 5 min.
    18. Pool the supernatant into the supernatant previously collected (step A16).
    19. Add 1 μl of glycogen blue and 1 ml of 99.7% ice cold ethanol to the total supernatant.
    20. Mix and place the tube overnight at -20 °C or for 30 min at -80 °C.
    21. Centrifuge the tube at 16,000 x g for 15 min at 4 °C.
    22. Wash the blue pellet with 500 μl of 75% ice cold ethanol, then centrifuge at 12,000 x g for 5 min at 4 °C and remove the ethanol.
    23. Quick-spin the tube and carefully remove the residual ethanol.
    24. Air-dry the pellet for 5 min.
    25. Re-suspend the RNA pellet in 21 μl of DEPC H2O. Measure the concentration of RNA and calculate molarity. Normally, 1 μg 150 nt RNA approximately equals to 20 pmol.
    26. Adjust the final concentration of RNA sample to 5 μM (5 pmol/μl or 250 ng/μl).
    27. Store the RNA at -80 °C.

  2. Removing 5' phosphates of pri- and pre-miRNAs
    1. Mix following in a fresh tube at 4 °C: 1 μl RNA (5 pmol), 2 μl of 10x NEB buffer 3, 2 μl of NEB Calf Intestine Alkaline Phosphatase (CIP), 1 μl of Superase-In (final concentration 1 U/μl), DEPC H2O 14 μl.
    2. Incubate at 37 °C for 30 min.
    3. Add 220 μl of DEPC H2O to the above reaction mixture.
    4. Add 240 μl of phenol: chloroform: isoamyl: alcohol (25:24:1) and vortex for 30 sec. Centrifuge for 5 min at RT and collect 210 μl aqueous phase.
    5. Add 210 μl chloroform: isoamyl alcohol (24:1) and vortex for 30 sec. Centrifuge for 5 min at RT and collect 170 μl aqueous phase.
    6. Add 510 μl of 99.7% ice cold ethanol, 17 μl 3 M NaAc (pH 5.2) and 1 μl of glycogen blue. Mix and leave the tube overnight at -20 °C or 30 min at -80 °C.
    7. Centrifuge at 16,000 x g for 15 min at 4 °C.
    8. Remove the supernatant carefully.
    9. Wash the blue pellet with 500 μl of 75% ice cold ethanol, then centrifuge at 12,000 x g for 5 min at 4 °C and remove the ethanol.
    10. Air-dry the pellet for 5 min.
    11. Re-suspend the RNA pellet in 4.5 μl of DEPC H2O.

  3. 5' end radioactive labeling of pri-and pre-miRNAs (this step must be performed in a radioisotope-designated space and with the appropriate safety training)
    1. Mix the following in a fresh tube at 4 °C: 4.5 μl of RNA (from step B), 1 μl of 10x NEB T4 kinase buffer, 3 μl of [γ-32P]-ATP (3,000 Ci/mmol 10 mCi/ml), 0.5 μl of Superase-In (final concentration 1 U/μl) and 1 μl of NEB T4 kinase.
    2. Incubate the reaction mixture at 37 °C for 2 h.
    3. Add 230 μl of DEPC H2O to the above reaction mixture.
    4. Add 240 μl of phenol: chloroform: isoamyl alcohol (25:24:1) and vortex for 30 sec. Centrifuge for 5 min at RT and take 210 μl aqueous phase.
    5. Add 140 μl of 5 M-ammonium acetate, 1 μl of glycogen blue and 1,050 μl of 99.7% ice cold ethanol. Mix and leave the tube overnight at -20 °C or 30 min at -80 °C.
    6. Centrifuge at 16,000 x g for 15 min at 4 °C.
    7. Remove the supernatant carefully.
    8. Wash the blue pellet with 500 μl of 75% ice cold ethanol, then centrifuge at 12,000 x g for 5 min at 4 °C and remove the ethanol.
    9. Air-dry the pellet for 5 min.
    10.  Add 20 μl RNA loading buffer into the tube to dissolve the pellet. Here it can be stored at -20 °C.
    11. Prepare a 6% urea-polyacrylamide gel.
    12. Heat the tubes for 2 min at 95 °C. Cool down for 2 min on the ice.
    13. Briefly spin down the tubes.
    14. Flush the wells using the running buffer to remove the diffused urea.
    15. Load the RNA samples on 6% urea-PAGE gel and run at 350 V (20 V/cm) until bromophenol blue reaches the bottom of the gel.
    16. Remove one of the glass plates from the gel. Make sure to mark the position and orientation of the gel so that the gel can be aligned with the film once the film is developed. Wrap the gel with plastic wrap and place a phospho imager on the gel for 30 min. The radiolabeled transcript will appear as a strong band on the developed film.
    17.  Align the photo with the gel and cut out the gel slice containing the labeled transcript. Put the gel slice in a 1.5 ml fresh tube with 350 μl of RNA elution buffer.
    18. Incubate the tube overnight at 42 °C with 1,200 rpm shaking using Thermomixer C.
    19. Transfer the supernatant (about 300 μl) to a fresh tube.
    20. Add 100 μl of RNA elution buffer into the old tube with the gel slice and vortex.
    21. Transfer the supernatant into the previous supernatant.
    22. Add 1 μl of glycogen blue and 1 ml of 99.7% ice cold ethanol into total supernatant.
    23. Mix and place the tube overnight at -20 °C or 30 min at -80 °C.
    24. Centrifuge the tube at 16,000 x g for 15 min at 4 °C.
    25. Wash the blue pellet with 500 μl of 75% ice cold ethanol, then centrifuge at 12,000 x g for 5 min at 4 °C and remove the ethanol.
    26. Quick-spin the tube and carefully remove the residual ethanol.
    27. Air-dry the pellet for 5 min. Be careful not to over-dry the pellet.
    28. Count cpm and re-suspend radiolabeled RNA in 20 μl RNA dissolving buffer. Denature the radiolabeled RNA in a 95 °C heating block for 2 min, and then take the entire 95 °C block out and fold the RNA by slowly cooling down the heat block to room temperature. Store the radiolabeled RNA at -20 °C.

  4. Heterologous expression of Arabidopsis DCL1 complex in N. benthamiana
    1. Infiltrate N. benthamiana leaves with OD600 = 1 mixture of Agrobacterium tumefaciens ABI strains harboring pBA-2Flag-4Myc-DCL1, pBA-6Myc-HYL1, pBA-6Myc-SE, at OD600 of 0.8, 0.1, and 0.1 respectively. As a negative control we use A. tumefaciens ABI strains harboring an empty vector-pBA only.
    2. After two days, the infiltrated leaves are collected, ground in liquid nitrogen, and stored at -80 °C.

  5. Immunoprecipitation of DCL1 microprocessor
    1. Make sure to cool down the centrifuges and rotors to 4 °C.
    2. Re-suspend the N. benthamiana powder sample in 5 volumes of the IP Buffer (typically 3 g of sample in 15 ml of IP buffer); keep on ice and protected from light (use aluminum foil to wrap the tube) until the sample is completely thawed. Transfer the solution to the 15 ml conical tubes.
    3. Centrifuge the samples at 16,000 x g for 15 min at 4 °C. Transfer all the supernatant into a new chilled 15 ml tube.
    4. Centrifuge at 4 °C for 15 min at 16,000 x g. Transfer all the supernatant into another new chilled 15 ml tube.
    5. During the centrifugation steps prepare anti-Flag antibody for immunoprecipitation. Add 400 μl bed volume Anti-FLAG® M2 magnetic beads (for 3 g plant tissue) into a new 2 ml Eppendorf tube on ice.
    6. Load the Eppendorf tube into the magnetic separator, DynaMagTM-2 to collect the beads. Remove the storage buffer quickly.
    7. Release the tube from DynaMagTM-2 and remove antibodies that are not conjugated to the DynaMag beads by incubation the Anti-FLAG® M2 magnetic beads with one bed volume of 0.1 M Glycine-HCl (pH 3.0).
    8. Invert the tube for 1.5 min (exactly).
    9. Load the Eppendorf tube into DynaMagTM-2, separate and remove the Glycine-HCl buffer quickly.
    10. Quickly equilibrate the Anti-FLAG® M2 magnetic beads with 1 ml TBS buffer three times using DynaMagTM-2.
    11. Wash the beads with 1 ml IP buffer for three times using DynaMagTM-2.
    12. Completely remove the IP buffer and add 400 μl new IP buffer into the tube to re-suspend the beads.
    13. Add the equilibrated beads into 15 ml tube of step E4.
    14. Then rotate the 15 ml tube at 4 °C for 2 h using a Rugged Rotator.
    15. While doing the first step immunoprecipitation with the Anti-FLAG® M2 magnetic beads, prepare anti-Myc antibody for the second-step immunoprecipitation.
    16. Add 120 μl bed volume of anti-c-myc agarose affinity gel into a new 1.5 ml tube. Then, wash the gel with 1 ml IP buffer.
    17. Spin the tube at 1,600 x g for 1min at 4 °C. Discard the supernatant.
    18. Repeat the washing for two more times.
    19. Take the majority of the washing buffer out and put anti-c-myc agarose affinity gel on ice.
    20. At the end of step E14, prepare 3x Flag peptide solution for elution of Flag-Myc-tagged DCL1 from the Anti-FLAG® M2 magnetic beads.
    21. Add 30 μl 3x Flag elution buffer stock (4 mg/ml) into 1.2 ml IP buffer to make a final concentration 100 μg/ml of 3x Flag peptide. Mix well and put it on ice.
    22. After step E14 is done, load the 15 ml tube (step E14) on the PolyATtract® System 1000 Magnetic Separation Stand for a few seconds, then slowly pour the supernatant to a trash can.
    23. Wash the wall of 15 ml conical tube with IP buffer until the supernatant is not green anymore, typically three times. For this, use the magnetic stand.
    24. Add 2 ml of IP buffer to the conical tube.
    25. Transfer all the beads carefully to a clean 2 ml Eppendorf tube.
    26. Load the 2 ml tube into DynaMagTM-2, and remove the IP buffer.
    27. Add 600 μl 3x Flag elution buffer into the 2 ml tube, incubate in rotation for 30 min at 4 °C.
    28. Load the 2 ml tube into DynaMagTM-2. Transfer the supernatant (1st elution solution) to the 1.5 ml Eppendorf tube containing anti-c-myc agarose affinity gel (step E19). Then, start second IP incubating the second tube in rotation at 4 °C.
    29. Add another 600 μl 3x Flag elution buffer to the anti-flag magnetic beads and perform a second round of elution for 30 min at 4 °C.
    30. Collect the second elution of the anti-Flag IP by placing the 2 ml Eppendorf tube into DynaMagTM-2. Pool this second elution with the first one in the 1.5 ml tube containing anti-c-myc agarose affinity gel (step E28).
    31. Continue the second IP step by rotating the 1.5 ml tube for 1.5 h at 4 °C.
    32. Centrifuge the 1.5 ml tube containing anti-c-myc agarose affinity gel (step E30) at 1,600 x g for 3 min at 4 °C. Remove the supernatant.
    33. Wash out the non-specific binding from the anti-c-myc agarose affinity gel by adding 1 ml IP buffer and inverting the tube 6-7 times. Centrifuge at 4 °C for 3 min at 1600 x g and remove the buffer.
    34. Re-suspend the affinity gel in 1 ml of IP buffer and aliquot it into eight new 1.5 ml siliconized tubes.
    35. Spin down the eight tubes at 1,600 x g for 3 min at 4 °C.
    36. Remove the buffer as complete as possible.
    37. Keep the eight tubes on ice for in vitro DCL1 reconstitution assay, as these purified DCL1 complexes are ready for in vitro assays.
      Note: We prepare the fresh immunoprecipitate immediately before each assay.

  6. In vitro DCL1 reconstitution assay (this step must be performed in a radioisotope-designated space and with the appropriate safety training)
    1. Carefully add 14 μl of fresh assay buffer and 1 μl radiolabeled pri-miRNA (approximately 2 x 103 cpm) into the eight tubes containing the immunoprecipitate. Gently re-suspend the agarose gel with the reaction mixture.
    2. Incubate the tube for 1 h at 37 °C with 900 rpm shaking using Thermomixer C.
    3. Gently mix the reaction every 5 min.
    4. After 1 h, add 170 μl of RNA elution buffer to stop assay.
    5. Add 200 μl of phenol: chloroform: isoamyl alcohol (25:24:1) and vortex for 30 sec.
    6. Centrifuge for 5 min at RT.
    7. Transfer 150 μl upper aqueous phase to the siliconized tubes.
    8. Add 20 μl of 3 M NaAc (pH 5.2), 1 µl of glycogen-blue, and 1 ml of 100% ice cold ethanol. Invert the tubes for several times and leave it overnight at -20 °C.
    9. Centrifuge at 16,000 x g for 15 min at 4 °C.
    10. Discard the supernatant carefully.
    11. Wash the blue pellet with 500 μl of 75% ice cold ethanol, then centrifuge at 12,000 x g for 5 min at 4 °C and remove the ethanol.
    12. Quick spin down the tubes, remove the residue ethanol using pipette.
    13. Air-dry the pellet for 5 min.
    14. Suspend pellets with 20 µl RNA loading buffer. The sample can now be stored at -20 °C.
    15. Boil the tubes for 1 min at 95 °C. Cool down for 2 min on the ice.
    16. Briefly spin down the tubes.
    17. Prepare a 15% urea-polyacrylamide gels, and flush the wells of gel to remove the diffused urea before loading the samples.
    18. Load the assay samples and RNA marker on 15% urea-polyacrylamide gels. The RNA marker should be 5′-end labeled according to the manufacturer’s protocol with [γ-32P]-ATP.
    19. Run at 350 V (20 V/cm) for approximately one hour until bromophenol blue reaches the bottom of the gel.
    20. Disassemble the gel cast.
    21. Place the gel into a tank containing fixing buffer for 30 min.
    22. Dry the gel with a Gel Dryer under the gradient cycle at 80 °C for 2-4 h.
    23. Cover the dried gel with plastic wrap and place a phosphate screen on the gel for 2 h or overnight.
    24. The processed RNA products will appear as a strong band on phospho imager (PharosFXTM Plus System) and quantified with Quantity One Version 4.6.9 according to the manufacturer’s instructions.

Representative data

Representative data could refer to the Figures 2, 3, 4, 5, 6, 7 and 8 from Zhu et al. (2013), as well as supplementary Figures 2, 3, 4, 5, 6 and 9.

Notes

  1. The E and F steps should be performed successively in one day.
  2. The C and F steps must be performed in a radioisotope-designated space and with the appropriate safety training.

Recipes

  1. 5x transcription buffer
    400 mM HEPES (pH 7.5)
    10 mM spermidine
    200 mM DTT
    125 mM MgCl2 and 20 mM of each NTP
  2. RNA loading buffer
    95% deionized formamide
    0.025% bromophenol blue
    0.025% xylene cyanol
    5 mM EDTA and 0.025% SDS
  3. RNA elution buffer
    0.3 M NaAc (pH 5.2) and 2% SDS
  4. DEPC H2O
    Add 1 ml of DEPC to 1 L of H2O, shake vigorously and autoclave.
  5. RNA dissolve buffer
    100 mM KCl
    30 mM Tris-HCl (pH 7.5)
  6. Chloroform: isoamyl alcohol (24:1)
    Chloroform 96 ml
    Isoamyl alcohol 4 ml
  7. IP buffer
    40 mM Tris-HCl (pH 7.5)
    300 mM KCl
    5 mM MgCl2
    5 mM DTT
    0.2 mM EDTA (pH 8.0)
    0.2% Triton X-100
    1 mM PMSF
    2% glycerol
    0.3% (vol/vol) protease inhibitor cocktail stock
    1 tablet per 25 ml IP buffer of EDTA-free protease inhibitor cocktail
  8. Protease inhibitor cocktail stock
    Protease inhibitor cocktail (about 89 mg) is dissolved in 10 ml ddH2O
  9. TBS buffer
    50 mM Tris HCl (pH 7.4)
    150 mM NaCl
  10. 3x Flag elution buffer stock (4 mg/ml)
    Add 4 mg 3x Flag peptide into 1 ml IP buffer
  11. Washing buffer
    20 mM Tris-HCl (pH 7.5)
    1 mM DTT
    4 mM MgCl2 and 100 mM KCl
  12. Assay buffer
    20 mM Tris-HCl (pH 7.5)
    4 mM MgCl2
    1 mM DTT
    10 mM ATP
    2 mM GTP
    2 U/μl Supernase-In
  13. Fixing buffer
    40% ethanol
    10% acetic acid and 5% glycerol

Acknowledgments

This work was supported by grants from the US National Science Foundation (NSF) CAREER (MCB-1253369), the US National Institutes of Health (R21AI097570) and the Welch foundation (A-1777) to X.Z, and also supported by Chinese Universities Scientific Fund (2014RC006) to H.Z.

References

  1. Han, J., Lee, Y., Yeom, K. H., Kim, Y. K., Jin, H. and Kim, V. N. (2004). The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev 18(24): 3016-3027.
  2. Zhang, X., Henriques, R., Lin, S. S., Niu, Q. W. and Chua, N. H. (2006). Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc 1(2): 641-646.
  3. Zhu, H., Zhou, Y., Castillo-Gonzalez, C., Lu, A., Ge, C., Zhao, Y. T., Duan, L., Li, Z., Axtell, M. J., Wang, X. J. and Zhang, X. (2013). Bidirectional processing of pri-miRNAs with branched terminal loops by Arabidopsis Dicer-like1. Nat Struct Mol Biol 20(9): 1106-1115.

简介

微小RNA(miRNA)是小的非编码RNA,其调节真核生物中的大多数(如果不是全部)生物过程。 miRNA最初从初级转录物(pri-miRNA)加工以产生miRNA前体(前-miRNA),其进一步加工成miRNA及其互补链(miRNA/*)。在拟南芥和可能的其他植物中,从pri-miRNA到pre-miRNA和从pre-miRNA到miRNA/*的加工都通过Dicer样1(DCL1)复合物实现。最近,我们证明了从植物中以前的质量的DCL1复合物的分离。我们进一步成功地重建了能够完全重现体内 miRNA生物发生的DCL1切割测定。在这里我们提供DCL1重建测定的详细协议。该方案包括三个主要部分(图1):1)pri-miRNA和pre-miRNA转录物的制备(方法A-C); 2)通过免疫沉淀(IP)纯化来自本塞姆氏烟草(本塞姆氏烟草)的重组拟南芥DCL1机器(程序D和E);和3)使用分离的DCL1复合物(步骤F)在放射性同位素标记的pri-miRNA或pre-miRNA的体外处理。这是我们的愿望,协议是RNAi社区研究机械问题或开发RNA沉默技术的强大工具。

关键字:微处理器, miRNA初级转录产物, miRNA, Dicer, 拟南芥

材料和试剂

  1. 五至六周龄的N的植物。 本bentiana
  2. 农杆菌菌株:ABI(Zhang等人,2006)
  3. 质粒[pBA-2Flag-4Myc-DCL1,pBA-6Myc-HYL1,pBA-6Myc-SE。 详细方法可以参考来自Zhu等人的在线方法(2013)。 阴性对照质粒:pBA]
  4. G-Tube 卡帽硅化微量离心管(VWR International,目录号:22179-004)
  5. 抗FLAG磁珠(Sigma-Aldrich,目录号:M8823)
  6. 3x Flag肽(NH2-MDYKDHDGDYKDHDIDYKDDDDK-COOH)(Sigma-Aldrich,目录号:F4799)
  7. DNase(Promega Corporation,目录号:M6101)
  8. 3,000 Ci/mmol 10mCi/ml [γ-32 P] -ATP(PerkinElmer,目录号:BLU502A100UC)
  9. Superase In RNA酶抑制剂(Ambion,目录号:N8080119)
  10. T7 RNA聚合酶(Ambion,目录号:18033019)
  11. 小牛肠碱性磷酸酶(NEB,目录号:M0290S)
  12. 苯酚:氯仿:异戊醇(25:24:1)(Life Technologies,Invitrogen TM,目录号:15593049)
  13. NaAc(Sigma-Aldrich,目录号:S7670)
  14. 5M乙酸铵(Ambion,目录:AM9071)
  15. GlycoBlue(Ambion,目录号:AM9516)
  16. 十进制标记(Ambion,目录号:AM7778)
  17. 抗c-Myc琼脂糖亲和凝胶(Sigma-Aldrich,目录号:A7470)
  18. 无EDTA蛋白酶抑制剂混合物(Roche Diagnostics,目录号:05892953001)
  19. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P2714)
  20. HEPES(Sigma-Aldrich,目录号:H3375)
  21. 亚精胺(Sigma-Aldrich,目录号:S2626)
  22. DTT(DL-二硫苏糖醇)(Sigma-Aldrich,目录号:43817)
  23. MgCl 2(Sigma-Aldrich,目录号:M8266)
  24. NTP(Thermo scientific,目录号:R0481)
  25. 去离子甲酰胺(Ambion,目录号:AM9342)
  26. 溴酚蓝(Sigma-Aldrich,目录号:B0126)
  27. 二甲苯Cyanol(Sigma-Aldrich,目录号:X4126)
  28. EDTA(Sigma-Aldrich,目录号:V900106)
  29. SDS(Sigma-Aldrich,目录号:L3771)
  30. DEPC(Sigma-Aldrich,目录号:V900882)
  31. KCl(Sigma-Aldrich,目录号:V900068)
  32. Tris-HCl(Sigma-Aldrich,目录号:V900312)
  33. Triton X-100(Sigma-Aldrich,目录号:V900502)
  34. PMSF(Sigma-Aldrich,目录号:78830)
  35. NaCl(Sigma-Aldrich,目录号:V900058)
  36. ATP(Thermo scientific,目录号:R1441)
  37. GTP(Thermo scientific,目录号:R1461)
  38. 甘油(Sigma-Aldrich,目录号:V900122)
  39. 0.1M甘氨酸-HCl(Sigma-Aldrich,目录号:55097-5ML-F)
  40. 5x转录缓冲液(见配方)
  41. RNA加载缓冲液(参见配方)
  42. RNA洗脱缓冲液(参见配方)
  43. DEPC H 2 O(参见配方)
  44. RNA溶解缓冲液(见配方)
  45. 氯仿:异戊醇(24:1)(参见配方)
  46. IP缓冲区(参见配方)
  47. 蛋白酶抑制剂混合物(见配方)
  48. TBS缓冲区(请参阅配方)
  49. 3x Flag洗脱缓冲液原液(4mg/ml)(见配方)
  50. 洗涤缓冲液(见配方)
  51. 测试缓冲区(参见配方)
  52. 修复缓冲区(请参阅配方)

设备

  1. 紧凑型UV灯(UVP,型号:UVGL-25)
  2. 氟涂覆的TLC板(10×10cm)(Ambion,目录号:AM10110)
  3. 盖革计数器(Medcom,型号:CRM-100)
  4. 垂直电泳系统(Whatman,型号:V15.17)
  5. Thermomixer C(Eppendorf,型号:5382000023)
  6. 凝胶干燥器(Bio-Rad Laboratories,型号:583)
  7. 凝胶分析仪Quantity One(Bio-Rad Laboratories,版本:4.6.9)
  8. DynaMag TM -2(Life technologies,型号:12321D)
  9. 坚固的旋转器(Glas-Col,型号:099A MR1512)
  10. PolyATtract System 1000磁力分离架(Promega公司,型号:Z541A)
  11. Phospho成像仪(PharosFX TM plus系统)

程序

注意:请参见图1,了解以下六个步骤的示意图。


图1.过程示意图

  1. 制备pri和pre-miRNA转录物(Han等人,2014)
    1. pri和pre-miRNA的底物在T7启动子下在体外使用PCR产生的模板转录。 在这种情况下,正向PCR 引物应含有T7启动子序列(TAATACGACTCACTATAG) 驱动直接从PCR产物转录
    2. 按照以下步骤准备体外转录反应:

      试剂

      最终浓度。
      纯化的PCR产物(100ng)
      - 微博

      5x转录缓冲液
      4微升

      Superase-In
      1微升
      1 U /μl
      T7 RNA聚合酶
      2微升

      将H sub 2 O加到最终体积20μl
      20微升

      注意:其他商业供应商也提供体外转录试剂盒。

    3. 短暂旋转。 在37℃孵育3小时或过夜
    4. 加入1μlDNA酶。 混合并在37℃下离心30分钟
    5. 加入20微升RNA加载缓冲液到转录的RNA。
    6. 将管在95℃下加热2分钟。 在冰上冷却2分钟。
    7. 在4℃下短暂旋转试管。 保持样品在冰上,直到加载
    8. 同时,制备6%的脲 - 聚丙烯酰胺凝胶
    9. 在装载样品前,冲洗6% 脲 - 聚丙烯酰胺凝胶用1x TBE运行缓冲液除去 扩散的尿素
    10. 加载RNA样品在6% 脲 - 聚丙烯酰胺凝胶并在350V(20V/cm)下运行直至溴酚 蓝色到达凝胶的底部
    11. 从凝胶中取出一块玻璃板。
    12. 应用UV阴影定位誊本的位置然后 切除pre-miRNA或pre-miRNAs条带的单个条带的凝胶切片。
      UV阴影:从用于的玻璃板上除去凝胶 电泳并将其放置在一片塑料包装上。 然后定位 在氟涂覆的TLC板的顶部上的塑料包装和凝胶(注意: 避免皮肤和眼睛暴露在紫外线下)。 使用手持式UV监视器, 识别具有可区分阴影和标记的带 如果要切割凝胶的话,感兴趣带的位置 进一步净化。
    13. 将凝胶切片放入带有350μlRNA洗脱缓冲液的1.5ml管中
    14. 使用Thermomixer C在42℃下用1200rpm振荡孵育试管过夜
    15. 将混合物在4℃离心10分钟
    16. 将上清液(约300μl)转移到新管中
    17. 加入100微升RNA洗脱缓冲液到老管用凝胶切片,涡旋和离心5分钟
    18. 将上清液混合到先前收集的上清液中(步骤A16)
    19. 向总上清液中加入1μl糖原蓝和1ml 99.7%冰冷的乙醇
    20. 混合并置于-20℃下过夜或在-80℃下放置30分钟
    21. 在4℃下以16,000×g离心管15分钟。
    22. 用500μl75%冰冷的乙醇洗涤蓝色沉淀,然后 在4℃下以12,000×g离心5分钟,并除去乙醇。
    23. 快速旋转管,小心除去残留的乙醇
    24. 空气干燥沉淀5分钟。
    25. 将RNA沉淀重悬在21μl的DEPC H 2 O中。 测量 浓度和计算摩尔浓度。 通常,1微克150 nt RNA 大约等于20pmol。
    26. 将RNA样品的最终浓度调整为5μM(5 pmol /μl或250 ng /μl)
    27. 将RNA储存于-80℃

  2. 去除pri和pre-miRNA的5'磷酸酯
    1. 在4℃下,在新管中混合:1μlRNA(5 pmol),2μl10x NEB缓冲液3,2μlNEB小牛肠碱性磷酸酶(CIP),1 μl的Superase-In(终浓度1U /μl),DEPC H 2 O14μl。
    2. 在37℃孵育30分钟。
    3. 向上述反应混合物中加入220μlDEPC H sub 2 O
    4. 加入240μl苯酚:氯仿:异戊基:醇(25:24:1)和 涡旋30秒。 在室温离心5分钟,收集210μl水   相。
    5. 加入210μl氯仿:异戊醇(24:1)并涡旋   30秒。 在室温离心5分钟,收集170μl水 阶段
    6. 加入510μl的99.7%冰冷的乙醇,17μl3M NaAc(pH 5.2)和1μl的糖原蓝。 混合并在-20℃下离心过夜 ℃或30分钟,在-80℃
    7. 在4℃下以16,000xg离心15分钟。
    8. 小心除去上清液。
    9. 用500μl75%冰冷的乙醇洗涤蓝色沉淀,然后 在4℃下以12,000×g离心5分钟,并除去乙醇
    10. 空气干燥沉淀5分钟。
    11. 将RNA沉淀重悬于4.5μlDEPC H 2 O中。

  3. 5'端对pri-和pre-miRNA的放射性标记(该步骤必须在放射性同位素指定空间中进行,并进行适当的安全培训)
    1. 将以下物质在4℃的新管中混合:4.5μlRNA(来自步骤B),1   μl的10×NEB T4激酶缓冲液,3μl的[γ-32 P] -ATP(3,000Ci/mmol 10 mCi/ml),0.5μlSuperase-In(终浓度1U /μl)和1μl NEB T4激酶
    2. 将反应混合物在37℃下孵育2小时。
    3. 向上述反应混合物中加入230μlDEPC H 2 O
    4. 加入240μl苯酚:氯仿:异戊醇(25:24:1)和 涡旋30秒。 在室温离心5分钟,取210μl水 相。
    5. 加入140微升5 M醋酸铵,1微升糖原蓝   和1,050μl的99.7%冰冷乙醇。 混合并离开管 在-20℃下过夜或在-80℃下30分钟。
    6. 在4℃下以16,000xg离心15分钟。
    7. 小心除去上清液。
    8. 用500μl75%冰冷的乙醇洗涤蓝色沉淀,然后 在4℃下以12,000×g离心5分钟,并除去乙醇
    9. 风干球团5分钟。
    10.  向管中加入20μlRNA加样缓冲液以溶解沉淀。 这里可以存储在-20°C
    11. 制备6%的脲 - 聚丙烯酰胺凝胶
    12. 将管在95℃下加热2分钟。 在冰上冷却2分钟。
    13. 短暂地向下旋转管。
    14. 使用运行缓冲液冲洗孔以除去扩散的尿素。
    15. 将RNA样品装载在6%尿素-AGE凝胶上并在350V(20V/cm)下运行,直到溴酚蓝到达凝胶底部。
    16. 从凝胶中取出一块玻璃板。 请务必标记 位置和取向,使得凝胶可以对准 该膜一旦被显影。 用塑料包装包裹凝胶 在凝胶上放置磷光成像器30分钟。 放射性标记 转录物将在显影胶片上显示为强带
    17.  将照片与凝胶对齐,切出含有的凝胶切片 标记的转录物。 将凝胶切片放入一个1.5毫升的新鲜管350微升   的RNA洗脱缓冲液
    18. 使用Thermomixer C在42℃下用1200rpm振荡孵育试管过夜
    19. 将上清液(约300μl)转移到新管中
    20. 加入100微升RNA洗脱缓冲液到老管用凝胶切片和涡流
    21. 将上清液转移到之前的上清液中
    22. 向总上清液中加入1μl糖原蓝和1ml 99.7%冰冷乙醇
    23. 混合并在-20℃下放置过夜,或在-80℃下放置30分钟
    24. 在4℃下以16,000×g离心管15分钟。
    25. 用500μl75%冰冷的乙醇洗涤蓝色沉淀,然后 在4℃下以12,000×g离心5分钟,并除去乙醇。
    26. 快速旋转管,小心除去残留的乙醇
    27. 风干球团5分钟。 小心不要过度干燥颗粒。
    28. 计数cpm和重悬20μLRNA溶解的放射性标记的RNA 缓冲。 使放射性标记的RNA在95℃加热块中变性2 min,然后取出整个95°C的块,慢慢折叠RNA   将加热块冷却至室温。 储存放射性标记   RNA在-20°C。

  4. 拟南芥DCL1复合体在N中的异源表达。 本bentiana
    1. 渗透 N。 本生烟叶具有土壤杆菌的OD 600 = 1混合物   包含pBA-2Flag-4Myc-DCL1,pBA-6Myc-HYL1, pBA-6Myc-SE,OD 600分别为0.8,0.1和0.1。 作为否定 控制我们使用 A。 包含空载体pBA的根瘤土壤杆菌 ABI菌株 只有。
    2. 两天后,收集浸润的叶子,在液氮中研磨,并储存在-80℃

  5. 免疫沉淀DCL1微处理器
    1. 确保将离心机和转子冷却至4°C
    2. 在5体积的IP中重新悬浮本塞姆氏烟草粉末样品 缓冲液(通常为3g样品在15ml IP缓冲液中); 保持在冰和 防止光照(使用铝箔包裹管),直到 样品完全解冻。 转移溶液到15毫升锥形
    3. 在4℃下以16,000×g离心样品15分钟。 将所有上清液转移至新的冷却的15ml管中。
    4. 在4℃下以16,000×g离心15分钟。 将所有上清液转移到另一新的冷却的15ml试管中
    5. 在离心步骤期间制备抗Flag抗体 免疫沉淀。 加入400μl床体积抗FLAG M2磁珠 (对于3g植物组织)放入新的2ml Eppendorf管中。
    6. 将Eppendorf管装入磁分离器DynaMag TM -2以收集珠子。 快速删除存储缓冲区。
    7. 从DynaMag TM -2释放管,并除去不是的抗体   通过孵育抗-FLAG M2偶联到DynaMag珠上 磁珠与一床体积的0.1M甘氨酸-HCl(pH 3.0)。
    8. 将管反转1.5分钟(完全)。
    9. 将Eppendorf管装入DynaMag TM -2,快速分离并除去甘氨酸-HCl缓冲液。
    10. 使用DynaMag TM -2,用1ml TBS缓冲液快速平衡抗FLAG M2磁珠3次。
    11. 用1ml IP缓冲液洗涤珠子三次,使用DynaMag -2。
    12. 完全删除IP缓冲区,并添加400μl新的IP缓冲液到管中以重悬浮珠。
    13. 将平衡的珠子添加到步骤E4的15ml管中。
    14. 然后使用Rugged Rotator在4℃下旋转15ml管2小时。
    15. 在用抗-FLAG M2磁珠进行第一步免疫沉淀时,制备用于第二步的抗Myc抗体 免疫沉淀。
    16. 加入120微升床体积的抗c-myc琼脂糖亲和凝胶到一个新的1.5毫升管。 然后,用1ml IP缓冲液洗涤凝胶
    17. 在4℃下在1,600×g下旋转管1分钟。 弃去上清液。
    18. 重复洗涤两次。
    19. 取大部分洗涤缓冲液,并将抗c-myc琼脂糖亲和凝胶置于冰上
    20. 在步骤E14结束时,制备3×Flag肽溶液 Flag-Myc标记的DCL1从抗-FLAG M2磁珠的洗脱。
    21. 加入30μl3×Flag洗脱缓冲液储备液(4 mg/ml)到1.2 ml IP 缓冲液,使最终浓度为100μg/ml的3×Flag肽。 混合 把它放在冰上。
    22. 在步骤E14之后,加载15ml (步骤E14)在PolyATtract 系统1000磁分离 站立几秒钟,然后缓慢倒入上清液到垃圾桶 可以。
    23. 用IP缓冲液洗涤15 ml锥形管的壁,直到 上清液不再是绿色,通常是三次。 为了这, 使用磁性支架。
    24. 加入2毫升IP缓冲液到锥形管。
    25. 将所有珠子小心地转移到一个干净的2毫升Eppendorf管
    26. 将2ml管装入DynaMag TM -2,并取出IP缓冲液
    27. 加入600μl3×Flag洗脱缓冲液到2ml管中,在4℃下旋转孵育30分钟。
    28. 将2ml管装入DynaMag TM -2。 将上清液(1μL洗脱溶液)转移到含有抗c-myc的1.5ml Eppendorf管中 琼脂糖亲和凝胶(步骤E19)。 然后,开始第二个IP孵化 第二管在4℃下旋转
    29. 添加另外600μl3x标志 洗脱缓冲液至抗标磁珠并进行第二次 在4℃洗脱30分钟
    30. 收集第二次洗脱 的抗Flag IP,将2ml Eppendorf管放入DynaMag TM -2。   将第二次洗脱与第一次洗脱在1.5ml管中 含有抗c-myc琼脂糖亲和凝胶(步骤E28)。
    31. 通过在4℃下旋转1.5ml管1.5小时继续第二IP步骤。
    32. 离心含有抗c-myc琼脂糖亲和力的1.5ml管 凝胶(步骤E30)以1,600×g在4℃下3分钟。 除去上清液。
    33. 洗掉来自抗c-myc琼脂糖的非特异性结合 通过加入1ml IP缓冲液并倒置管6-7次来制备亲和凝胶。 在4℃下以1600×g离心3分钟并除去缓冲液。
    34. 将亲和凝胶重悬在1ml IP缓冲液中,并分装到8个新的1.5ml硅化试管中。
    35. 在4℃下,在1,600×g下旋转8根管子3分钟。
    36. 尽可能完整地删除缓冲区。
    37. 保持八管在冰上在体外 DCL1重建测定,   因为这些纯化的DCL1复合物准备用于体外测定。
      注意:我们在每次测定前立即准备新鲜的免疫沉淀物。

  6. 体外 DCL1重建测定(该步骤必须在放射性同位素指定空间内进行,并进行适当的安全培训)
    1. 小心加入14μl新鲜的测定缓冲液和1μl放射性标记 pri-miRNA(约2×10 3 cfu)加入含有8个管中 免疫沉淀。 轻轻地重新悬浮琼脂糖凝胶 反应混合物。
    2. 使用Thermomixer C在37℃,900rpm摇动下孵育管1小时
    3. 每5分钟轻轻混匀反应。
    4. 1小时后,加入170μlRNA洗脱缓冲液终止测定
    5. 加入200μl苯酚:氯仿:异戊醇(25:24:1)并涡旋30秒。
    6. 在室温离心5分钟。
    7. 将150μl上层水相转移到硅化试管中。
    8. 加入20微升3 M NaAc(pH 5.2),1微升糖原蓝,和1毫升 100%冰冷乙醇。 倒置管几次,离开它 在-20℃过夜。
    9. 在4℃下以16,000xg离心15分钟。
    10. 小心弃去上清液。
    11. 用500μl75%冰冷的乙醇洗涤蓝色沉淀,然后 在4℃下以12,000×g离心5分钟,并除去乙醇
    12. 快速旋转管,用移液管除去残余乙醇
    13. 空气干燥沉淀5分钟。
    14. 用20μlRNA加样缓冲液悬浮沉淀。 样品现在可以储存在-20°C
    15. 将管在95℃下煮沸1分钟。 在冰上冷却2分钟。
    16. 简短地向下旋转管。
    17. 准备15%的尿素 - 聚丙烯酰胺凝胶,冲洗凝胶的孔以除去扩散的尿素,然后装载样品。
    18. 将测定样品和RNA标记物装载在15%脲 - 聚丙烯酰胺上 凝胶。 RNA标记应该根据5'-end标记 使用[γ- 32 P] -ATP的制造商方案
    19. 在350V(20V/cm)下运行约1小时,直到溴酚蓝到达凝胶底部
    20. 拆卸凝胶。
    21. Place the gel into a tank containing fixing buffer for 30 min.
    22. Dry the gel with a Gel Dryer under the gradient cycle at 80 °C for 2-4 h.
    23. Cover the dried gel with plastic wrap and place a phosphate screen on the gel for 2 h or overnight.
    24. The processed RNA products will appear as a strong band on phospho imager (PharosFXTM Plus System) and quantified with Quantity One Version 4.6.9 according to the manufacturer’s instructions.

Representative data

Representative data could refer to the Figures 2, 3, 4, 5, 6, 7 and 8 from Zhu et al. (2013), as well as supplementary Figures 2, 3, 4, 5, 6 and 9.

Notes

  1. The E and F steps should be performed successively in one day.
  2. The C and F steps must be performed in a radioisotope-designated space and with the appropriate safety training.

Recipes

  1. 5x transcription buffer
    400 mM HEPES (pH 7.5)
    10 mM spermidine
    200 mM DTT
    125 mM MgCl2 and 20 mM of each NTP
  2. RNA loading buffer
    95% deionized formamide
    0.025% bromophenol blue
    0.025% xylene cyanol
    5 mM EDTA and 0.025% SDS
  3. RNA elution buffer
    0.3 M NaAc (pH 5.2) and 2% SDS
  4. DEPC H2O
    Add 1 ml of DEPC to 1 L of H2O, shake vigorously and autoclave.
  5. RNA dissolve buffer
    100 mM KCl
    30 mM Tris-HCl (pH 7.5)
  6. Chloroform: isoamyl alcohol (24:1)
    Chloroform 96 ml
    Isoamyl alcohol 4 ml
  7. IP buffer
    40 mM Tris-HCl (pH 7.5)
    300 mM KCl
    5 mM MgCl2
    5 mM DTT
    0.2 mM EDTA (pH 8.0)
    0.2% Triton X-100
    1 mM PMSF
    2% glycerol
    0.3% (vol/vol) protease inhibitor cocktail stock
    1 tablet per 25 ml IP buffer of EDTA-free protease inhibitor cocktail
  8. Protease inhibitor cocktail stock
    Protease inhibitor cocktail (about 89 mg) is dissolved in 10 ml ddH2O
  9. TBS buffer
    50 mM Tris HCl (pH 7.4)
    150 mM NaCl
  10. 3x Flag elution buffer stock (4 mg/ml)
    Add 4 mg 3x Flag peptide into 1 ml IP buffer
  11. Washing buffer
    20 mM Tris-HCl (pH 7.5)
    1 mM DTT
    4 mM MgCl2 and 100 mM KCl
  12. Assay buffer
    20 mM Tris-HCl (pH 7.5)
    4 mM MgCl2
    1 mM DTT
    10 mM ATP
    2 mM GTP
    2 U/μl Supernase-In
  13. Fixing buffer
    40% ethanol
    10% acetic acid and 5% glycerol

Acknowledgments

This work was supported by grants from the US National Science Foundation (NSF) CAREER (MCB-1253369), the US National Institutes of Health (R21AI097570) and the Welch foundation (A-1777) to X.Z, and also supported by Chinese Universities Scientific Fund (2014RC006) to H.Z.

References

  1. Han, J., Lee, Y., Yeom, K. H., Kim, Y. K., Jin, H. and Kim, V. N. (2004). The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev 18(24): 3016-3027.
  2. Zhang, X., Henriques, R., Lin, S. S., Niu, Q. W. and Chua, N. H. (2006). Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc 1(2): 641-646.
  3. Zhu, H., Zhou, Y., Castillo-Gonzalez, C., Lu, A., Ge, C., Zhao, Y. T., Duan, L., Li, Z., Axtell, M. J., Wang, X. J. and Zhang, X. (2013). Bidirectional processing of pri-miRNAs with branched terminal loops by Arabidopsis Dicer-like1. Nat Struct Mol Biol 20(9): 1106-1115.
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引用:Wang, T., Castillo-González, C., You, L., Li, R., Wen, L., Zhu, H. and Zhang, X. (2015). In vitro Reconstitution Assay of miRNA Biogenesis by Arabidopsis DCL1. Bio-protocol 5(8): e1454. DOI: 10.21769/BioProtoc.1454.
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