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Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation
通过蔗糖梯度分级分离准确、高效分析mRNA的翻译   

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Abstract

The efficiency with which proteins are produced from mRNA molecules can vary widely across transcripts, cell types, and cellular states. Methods that accurately assay the translational efficiency of mRNAs are critical to gaining a mechanistic understanding of post-transcriptional gene regulation. One way to measure translational efficiency is to determine the number of ribosomes associated with an mRNA molecule, normalized to the length of the coding sequence. The primary method for this analysis of individual mRNAs is sucrose gradient fractionation, which physically separates mRNAs based on the number of bound ribosomes. Here, we describe a streamlined protocol for accurate analysis of mRNA association with ribosomes. Compared to previous protocols, our method incorporates internal controls and improved buffer conditions that together reduce artifacts caused by non-specific mRNA–ribosome interactions. Moreover, our direct-from-fraction qRT-PCR protocol eliminates the need for RNA purification from gradient fractions, which greatly reduces the amount of hands-on time required and facilitates parallel analysis of multiple conditions or gene targets. Additionally, no phenol waste is generated during the procedure. We initially developed the protocol to investigate the translationally repressed state of the HAC1 mRNA in S. cerevisiae, but we also detail adapted procedures for mammalian cell lines and tissues.

Keywords: Translation( 翻译), Gene regulation(基因调控), Ribosome(核糖体), Polysome analysis(多核糖体分析), Sucrose gradient fractionation(蔗糖梯度分级分离), Reproducibility(可重现性)

Background

The translation of mRNA into protein is a highly regulated process that can occur at different rates depending on the gene, cellular context, or environment. Each step of translation–initiation, elongation, and termination–can be a point of regulation that ultimately affects the number of ribosomes associated with an mRNA (Dever and Green, 2012; Hinnebusch and Lorsch, 2012). Because the time between consecutive initiation events is usually shorter than the time required for elongation, most mRNAs are associated with more than one ribosome at a time to form ‘polysome’ structures (Warner et al., 1963). Thus, the ability to count the number of ribosomes per mRNA molecule provides an assay for the overall translation state of mRNA. Traditionally, this counting has been achieved by sucrose gradient fractionation (also sometimes called polysome analysis), in which mRNAs are separated by ultracentrifugation based on their size/shape and then quantified (Mašek et al., 2011). The detection of mRNAs in gradient fractions can either be done for individual mRNAs by RNA blotting or qRT-PCR, or for the entire transcriptome by microarrays or high-throughput RNA sequencing (Arava et al., 2003; Floor and Doudna, 2016). In this way, the absolute number of ribosomes associated with individual mRNA molecules can be determined. An alternative method for assaying translation is ribosome-footprint profiling, in which short fragments of mRNA that are protected from RNase digestion by ribosomes are captured and subjected to high-throughput sequencing (Ingolia et al., 2009). When combined with total RNA sequencing to determine mRNA abundances, ribosome-profiling data can measure the translational efficiencies of mRNAs in a genome-wide manner. However, ribosome profiling provides only a relative measure of translational efficiency that can be biased by RNA-abundance measurements (Weinberg et al., 2016). In addition, ribosome profiling is not well suited to the study of low-abundance mRNAs or when only a small number of mRNAs are of interest. For these reasons, sucrose gradient fractionation remains an important tool for the analysis of translational efficiency.

We present an adaption of this widely used technique that incorporates key features that improve accuracy and reduce hands-on time. mRNA polysome analysis by sucrose gradient fractionation is completed in three steps: lysate preparation, sucrose gradient fractionation, and RNA-abundance analysis. Our protocol was initially developed to streamline the analysis of multiple RNAs in parallel, but in the process of protocol development we also carefully optimized each step to ensure that the assay provided an accurate and reproducible measure of ribosome association. We developed the protocol for the budding yeast S. cerevisiae (Di Santo et al., 2016) but since then have also applied it to a wide variety of human and mouse cell lines and even whole mouse tissues (Odegaard et al., 2016). A key feature of our protocol is the inclusion of heparin in the lysis buffer, which reduces non-specific interactions between mRNA and ribosomes that can otherwise lead to artefactual co-sedimentation of untranslated mRNAs with polysomes. We also incorporate a reliable control for untranslated RNA: an un-capped exogenous RNA that is spiked into the lysate prior to ultracentrifugation. For the RNA analysis step we adapted a qRT-PCR kit previously used for cell lysates to work directly with gradient fractions, thus eliminating the time-consuming RNA purification steps used in all previous polysome analysis protocols. Measuring RNA abundances directly from crude gradient fractions not only reduces time requirements and hands-on manipulations but also eliminates generation of phenol waste. Finally, to control for variations in RT-PCR efficiencies among fractions (which differ in sucrose concentration and macromolecular composition), we spike in an equal amount of artificial RNA to each fraction just after collection to serve as a normalization reference. In summary, our protocol–presented in detail below–contains a collection of improvements and internal controls that together provide an accurate, streamlined assay for polysome analysis.

Materials and Reagents

  1. STAGE 1: Lysate preparation
    Materials
    Yeast
    1. Inoculation loop (Fisher Scientific, catalog number: 22-363-604 )
    2. 50 ml conical tube (Corning, Falcon®, catalog number: 352098 )
    3. 1.5 ml siliconized G-tube (Bio Plas, catalog number: 4165SL )
    4. 0.45 micron filters (Pall, catalog number: 60206 )
    5. Cell lifter (Corning, catalog number: 3008 )

    Mammalian cells
    1. 6-well plate (Corning, catalog number: 3516 ) or 10-cm cell culture dish (Corning, catalog number: 353803 )
    2. Cell lifter (Corning, catalog number: 3008 )
    3. 15 ml conical tube (Corning, Falcon®, catalog number: 352096 )
    4. 1.5 ml siliconized G-tube (Bio Plas, catalog number: 4165SL )

    Tissues
    1. 50 ml conical tubes (Corning, Falcon®, catalog number: 352098 )
    2. 5 ml centrifuge tubes (Eppendorf, catalog number: 0030119401 )
    3. 1.5 ml siliconized G-tubes (Bio Plas, catalog number: 4165SL )

    Reagents
    1. Liquid nitrogen (LN2)
    2. Appropriate culturing media (e.g., YPD for S. cerevisiae; DMEM, FBS and additives for mammalian cell lines)
    3. 1x phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
    4. Luciferase RNA (Promega, catalog number: L4561 )
      Note: Store aliquotted 100 ng/μl stock at -80 °C.
    5. HEPES (Sigma-Aldrich, catalog number: H4034 )
    6. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: 68475 )
    7. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
    8. Heparin (Sigma-Aldrich, catalog number: H3149 )
      Note: Store 10 mg/ml stock solution at 4 °C.
    9. Triton X-100 solution (Sigma-Aldrich, catalog number: 93443 )
    10. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779 )
      Note: Store filtered and aliquotted 1 M stock solution at -20 °C.
    11. Cycloheximide (AMRESCO, catalog number: 94271 )
    12. Superase-IN (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2696 )
      Note: Store filtered and aliquotted 100 mg/ml stock solution at -20 °C.
    13. cOmplete, mini, EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, catalog number: 11836170001 )
    14. Sucrose (Sigma-Aldrich, catalog number: S5016 )
      Note: Store solution at 4 °C, see Recipes section.
    15. Lysis buffer (see Recipes)
    16. 10% sucrose solution (see Recipes)
    17. 50% sucrose solution (see Recipes)

  2. STAGE 2: Sucrose gradient fractionation
    Materials
    1. 50 ml SteriFlip (EMD Millipore, catalog number: SCGP00525 )
    2. Open top polyclear centrifuge tubes (Seton Scientific, catalog number: 7030 )
    3. SW41 marker block (included with fractionator)
    4. 60 ml syringe (BD, catalog number: 309653 )
    5. Stainless steel syringe needle, noncoring point, ~10 inches, ~12 gauge (Sigma-Aldrich, Cadence Science, catalog number: Z116971 )
    6. Short caps (Biocomp, catalog number: 105-514-6 )
    7. Tube rack (Beckman Coulter, catalog number: 331313 )
    8. 2 ml tubes w/screw caps (USA Scientific, catalog number: 1420-8700 )
    9. Cling film or Parafilm

  3. STAGE 3: mRNA analysis
    Materials
    1. qPCR plates (RPI, catalog number: 141328 )
    2. qPCR film (Bio-Rad Laboratories, catalog number: MSB1001 )
    3. PCR tubes

    Reagents
    1. Cells-to-Ct kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1728 )
    2. Superase-In (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2696 )
    3. XenoRNA (Thermo Fisher Scientific, InvitrogenTM, catalog number: 4386995 , part of control kit)
      Note: Store in small aliquots at -80 °C.
    4. TaqMan Gene Expression Master mix (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4369016 )
    5. Primer/Probe qPCR assays for genes of interest (Thermo Fisher Scientific, Ac00010014_a1 (XenoRNA) and Mr03987587_mr (Luciferase))

Equipment

  1. Pipette (e.g., P1000, P10)
  2. 250 ml baffled flask (Corning, PYREX®, catalog number: 4444-250 )
  3. 2 L baffled flask (Corning, PYREX®, catalog number: 4444-2L )
  4. Filtration system (Restek, catalog number: KT676001-4035 )
  5. Coors porcelain mortar and pestle (Sigma-Aldrich, catalog numbers: Z247472 and Z247510)
    Manufacturer: CoorsTek, catalog numbers: 60316 and 60317 .
  6. Dounce, tissue grinder (DWK Life Sciences, WHEATON, catalog number: 357538 ) [optional]
  7. Tabletop cold centrifuge (Eppendorf, model: 5424 R )
  8. SW41 Ti rotor (Beckman Coulter, model: SW 41 Ti , catalog number: 331362)
  9. Ultracentrifuge (Beckman Coulter, model: L8-80M )
  10. Gradient station (Biocomp, catalog number: 153-001 )
  11. Fraction collector (Gilson, catalog number: FC 203B )
  12. BIORAD EM-1 Econo UV monitor (Bio-Rad Laboratories, model: EM-1 EconoTM )
  13. NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Thermo Scientific, model: NanoDropTM 2000 , catalog number: ND-2000)
  14. CFX96 Touch Real-Time PCR detection system (Bio-Rad Laboratories, model: CFX96 TouchTM, catalog number: 1855195 )

Software

  1. Gradient Profiler V2’ software

Procedure

The protocol is divided into three stages (see Figure 1):
Stage 1 Lysate Preparation
1A. Growth and harvesting of cells (a. yeast, b. cells lines, c. tissues)
1B. Sample preparation
Stage 2 Sucrose Gradient Fractionation
2A. Gradient preparation
2B. Ultracentrifugation
2C. Fractionation
Stage 3 mRNA Analysis
3A. DNase treatment and control RNA spike-in
3B. Reverse transcription
3C. Real-time PCR
The recommended workflow timing is:

Note: Stage 2A requires an incubation of 30-60 min and is carried out prior to Stage 1B to optimize timing.


Figure 1. Workflow schematic of the sucrose polysome gradient protocol from multiple cell types. This illustrates the overall steps of the procedure to analyze RNA distribution across a polysome gradient.

  1. STAGE 1: Lysate Preparation
    1. PART 1A. Growth and harvesting of cells
      Yeast
      1. Day 0
        1. Inoculate cells from plate into 50 ml YPD in a 250 ml baffled flask.
        2. Grow overnight to saturation.
      2. Day 1
        1. Dilute overnight culture into 400 ml YPD at an OD600 of 0.05 in a 2 L baffled flask.
          Note: If growing multiple cultures, stagger culture growth slightly (30 min difference between first and last cultures is sufficient).
        2. When culture reaches an OD600 of 0.5-0.6, harvest cells by rapid filtration:
          1) Pour YPD on filter prior to pouring culture.
          2) Pour entire culture down the side of the filtration vessel, taking care to avoid pouring foam that will collect on top of the filter.
          3) As the last liquid pours through, quickly remove the clamp and top of the filter unit, scrape cells from the filter quickly but gently using a cell lifter, and submerge into conical containing liquid nitrogen. The total time between the last liquid flowing through the filter and the cells being submerged in liquid nitrogen should not exceed 5 sec.
          Note: Cell scraper should be pre-chilled in liquid nitrogen.
        3. Place conical with pellet in a -80 °C freezer and allow liquid nitrogen to boil off.
          Note: Leave cap loosely tightened.
        4. Lyse cells by grinding with a mortar and pestle.
          1) Pre-chill mortar and pestle with liquid nitrogen (~2-3 min) in an ice bucket.
          2) Pour out any residual LN2 from the mortar.
          3) Add the cell pellet to the mortar.
          4) Gently pour ~1-5 ml of LN2 on the cell pellet.
              Note: Adding too much LN2 will significantly increase processing times.
          5) Grind with a pestle to break apart cells until all LN2 boils off, then grind the dry powder for an additional ~1-2 min.
              
          Note: After evaporation of all the liquid nitrogen in the mortar, the pellet reaches a powder-like consistency quickly, 1-2 min. No benefits are gained from further grinding. From experience, there are no adverse effects of grinding for too long.
          6) Re-suspend the cell powder in liquid nitrogen and carefully pour back into the original conical tube.
        5. Place the conical tube in a -80 °C freezer and allow liquid nitrogen to boil off.
          Note: Leave cap loosely tightened.
        6. Pause point.
        7. Proceed to Stage 2A.

      Mammalian cells
      1. Day 0
        1. Plate cells as required by experiment.
          Note: The procedure has been successfully applied to various mammalian cell lines cultured in 6-well plates, 10-cm dishes, and 15-cm dishes, with a harvested range of 106 to 107 cells. Confluency at time of harvesting should be avoided by controlling plating density it is important to consider the effects of cell manipulation on translation. Over-confluence, depleted nutrients or serum, or media changes can induce quick translational responses. Using a stable cell line is recommended over transiently transfected cells to ensure reproducibility.
        2. Incubate cells under optimal growth conditions.
      2. Day 1
        1. Add cycloheximide (CHX) to media at a final concentration of 100 μg/ml, incubate for 10 min at 37 °C. This step can be omitted .
          Note: While CHX pre-treatment in growth media is optional, we recommend adding CHX to the PBS and lysis buffer to prevent ribosome run-off during harvesting.
        2. Pre-chill PBS and lysis buffer on ice and add additives (see Recipes).
        3. Transfer tissue culture dish to an ice bucket.
        4. Aspirate media.
        5. Wash the dish twice with 10 ml ice-cold PBS.
        6. Scrape cells thoroughly and quickly in 5 ml of ice-cold PBS.
        7. Transfer cell suspension to a 15 ml conical tube.
        8. Centrifuge for 5 min at 4 °C at 500 x g, discard supernatant.
        9. Flash freeze cell pellet and store at -80 °C.
        10. Proceed to Stage 2A. 

      Mammalian tissue
      1. Day 0
        1. Dissect out a whole tissue sample.
        2. Wash tissue with ice-cold PBS prior to freezing in liquid nitrogen.
      2. Day 1
        1. Break apart and lyse tissue by grinding with a mortar and pestle.
          1) Pre-chill mortar and pestle with liquid nitrogen (~2-3 min) in an ice bucket.
          2) Pour out any residual LN2 from the mortar.
          3) Add the frozen tissue to the mortar.
          4) Pour ~1-5 ml of LN2 on the frozen tissue.
              Note: Adding too much LN2 will significantly increase processing times.
          5) Grind with a pestle to break apart cells until all LN2 boils off, then grind the dry powder for an additional ~1-2 min.
              
          Note: After evaporation of all the liquid nitrogen in the mortar, the pellet reaches a powder-like consistency quickly, 1-2 min. No benefits are gained from further grinding. From experience, there are no adverse effects of grinding for too long.
          6) Re-suspend the cell powder in liquid nitrogen and pour back into the original conical tube.
        2. Place the conical tube in a -80 °C freezer and allow liquid nitrogen to boil off.
          Note: Leave cap loosely tightened.
        3. Pause point
        4. Proceed to Stage 2A 

      Mammalian cells
      1. Day 0
        1. Plate cells as required by experiment.
          Note: The procedure has been successfully applied to various mammalian cell lines cultured in 6-well plates, 10-cm dishes, and 15-cm dishes, with a harvested range of 106 to 107 cells. Confluency at time of harvesting should be avoided by controlling plating density it is important to consider the effects of cell manipulation on translation. Over-confluence, depleted nutrients or serum, or media changes can induce quick translational responses. Using a stable cell line is recommended over transiently transfected cells to ensure reproducibility.
        2. Incubate cells under optimal growth conditions.
      2. Day 1
        1. Add cycloheximide (CHX) to media at a final concentration of 100 μg/ml, incubate for 10 min at 37 °C. This step can be omitted .
          Note: While CHX pre-treatment in growth media is optional, we recommend adding CHX to the PBS and lysis buffer to prevent ribosome run-off during harvesting.
        2. Pre-chill PBS and lysis buffer on ice and add additives (see Recipes).
        3. Transfer tissue culture dish to an ice bucket.
        4. Aspirate media.
        5. Wash the dish twice with 10 ml ice-cold PBS.
        6. Scrape cells thoroughly and quickly in 5 ml of ice-cold PBS.
        7. Transfer cell suspension to a 15 ml conical tube.
        8. Centrifuge for 5 min at 4 °C at 500 x g, discard supernatant.
        9. Flash freeze cell pellet and store at -80 °C.
        10. Proceed to Stage 2A.

      Mammalian tissue
      1. Day 0
        1. Dissect out a whole tissue sample.
        2. Wash tissue with ice-cold PBS prior to freezing in liquid nitrogen.
      2. Day 1
        1. Break apart and lyse tissue by grinding with a mortar and pestle.
          1) Pre-chill mortar and pestle with liquid nitrogen (~2-3 min) in an ice bucket.
          2) Pour out any residual LN2 from the mortar.
          3) Add the frozen tissue to the mortar.
          4) Pour ~1-5 ml of LN2 on the frozen tissue.
              Note: Adding too much LN2 will significantly increase processing times.
          5) Grind with a pestle to break apart cells until all LN2 boils off, then grind the dry powder for an additional ~1-2 min.
              
          Note: After evaporation of all the liquid nitrogen in the mortar, the pellet reaches a powder-like consistency quickly, 1-2 min. No benefits are gained from further grinding. From experience, there are no adverse effects of grinding for too long.
          6) Re-suspend the cell powder in liquid nitrogen and pour back into the original conical tube.
        2. Place the conical tube in a -80 °C freezer and allow liquid nitrogen to boil off.
          Note: Leave cap loosely tightened.
        3. Pause point
        4. Proceed to Stage 2A 
    2. PART 1B: Sample preparation (Day 2)
      Note: ALL following steps are done on an ice block or in a 4 °C cold room.
      Yeast procedure
      1. Thaw grinded powder on ice for 5 min.
        Prematurely adding lysis buffer can cause it to freeze.
      2. In the meantime, pre-label four siliconized microcentrifuge tubes per sample:
        Re-suspended powder
        Clarified undiluted lysate
        Clarified diluted lysate
        Aliquoted lysate (multiple tubes)
        Note: You will also need three 0.6 ml tubes per sample containing 90 μl ddH2O.
      3. Add 1 ml lysis buffer to the cell powder in each conical.
      4. Swirl each tube to lightly mix, then fully re-suspend by pipetting up and down using a P1000.
      5. Transfer entire tube contents to pre-labeled ‘re-suspended’ 1.7 ml tubes.
      6. Spin for 10 min at 1,300 x g at 4 °C.
      7. Transfer clarified lysate (~800 μl) into ‘clarified undiluted’ labeled 1.7 ml tubes. Clarified lysate should have a translucent appearance with a white/yellow hue.
      8. Transfer 10 μl into 90 μl water to spec on NanoDrop for RNA concentration, which serves as a proxy for total lysate concentration.
        Notes:
        1. Triton X-100 interferes with reading so dilution is needed.
        2. Blank will have 10 μl lysis buffer + 90 μl ddH2O.
      9. Dilute all lysates to 25 OD260 U/ml (1 μg/μl RNA) with lysis buffer.
      10. Spec the diluted lysate to ensure that all samples are within ~5% of each other.
      11. Add exogenous uncapped Luciferase RNA (Promega) to a final concentration of 100 ng/ml.
      12. Aliquot 150 μl into 1.7 ml tubes.
      13. Store lysates not immediately needed for experiment at -80 °C.
      14. Proceed to Stage 2B.

      Mammalian cells procedure
      1. Thaw cell pellet on ice.
      2. Resuspend cell pellet in 100 μl lysis buffer per 106 cells.
      3. Transfer lysate to a 1.7 ml tube.
      4. Incubate for 10 min on ice, mix by pipetting up and down.
        Note: Optimal lysis time and detergent concentration may vary depending on the cell type. Check cell lysis under a microscope with phase contrast at different times during lysis. Triton can be substituted by other detergents such as NP-40 or mechanical lysis using a dounce homogenizer.
      5. Centrifuge for 10 min at 4 °C at 12,000 x g.
      6. Transfer clarified lysate into a ‘clarified undiluted’ labeled 1.7 ml tube. 
      7. Dilute 10 μl of lysate into 90 μl water to spec on NanoDrop for RNA concentration, which serves as a proxy for total lysate concentration (see Note 8).
      8. Dilute all samples to the same concentration by adding an appropriate amount of lysis buffer.
        Note: We recommend diluting to ~20-100 μg/ml. Adjust concentrations and measure by spec to ensure all samples are within 5% of each other.
      9. Spike in exogenous uncapped Luciferase RNA to a final concentration of 100 ng/ml. 
      10. Aliquot 200-500 μl into 1.7 ml tubes and store the remaining lysate (input) at -80 °C.
      11. Proceed to stage 2B.

      Tissue procedure
      1. Weigh 50 mg (one scoop) of frozen powder into LN2 chilled 5 ml Eppendorf tubes.
        1. Act quickly to avoid tube warming up.
        2. Dip tubes into LN2 and shake to separate powder frequently (main 50 ml conical).
      2. Let to ‘thaw’ to 4 °C in ice before adding lysis buffer (LB).
      3. Add 50-100 μl of lysis buffer per mg of powder.
      4. Pipette up and down to mix, vortex vigorously, and let sit on ice.
        1. Allow Triton X-100 to lyse lipids for 5-10 min after adding LB before spinning.
        2. Vortex again.
      5. Spin 750 x g for 10 min at 4 °C.
      6. Separate supernatant into a new tube. 
        Note: Whole tissue samples: Lipid-rich samples must be carefully prepared to avoid lipid contamination. As such, we recommend taking the middle 75% of the clarified lysate after centrifugation to avoid disturbing the top lipid layer or bottom insoluble material.
      7. Spin 12,000 x g for 10 min at 4 °C.
      8. Separate supernatant into a new tube.
        Note: Take 75% liquid from the middle.
      9. Transfer 10 μl of supernatant into 90 μl water to spec on NanoDrop for RNA concentration, which serves as a proxy for total lysate concentration (see Note 8).
      10. Dilute all samples to 100 ng/μl RNA in lysis buffer containing Heparin.
      11. Add exogenous uncapped Luciferase RNA (Promega) to a final concentration of 100 ng/ml, then vortex to mix.
      12. Aliquot 250 μl into 1.7 ml tubes and store the remaining lysate (input) at -80 °C. 
      13. Proceed to Stage 2B.

  2. STAGE 2: Sucrose Gradient Fractionation (Day 2)
    1. PART 2A. Gradient preparation
      The set up should be done at room temperature and prior to the second step of lysate preparation. While gradients are cooling to 4 °C, prepare and clarify the lysates. 
      1. Prepare sucrose solutions
        1. Aliquot 40 ml of pre-filtered sucrose solutions (stored at 4 °C, see Recipes section) into a conical tube, and let warm to room temperature.
        2. Add DTT, cycloheximide, and Superase-IN to sucrose solutions, then mix by gentle rotation.
      2. Prepare lysis buffer and put on ice to cool to 4 °C.
      3. Mark Polyclear centrifuge tubes using the SW41 Ti marker block by drawing a line on each tube at the top marker block line.
      4. Using a stripette, fill centrifuge tubes with 10% sucrose solution (see Recipes) up to ~2 mm above the marked line.
      5. Fill up a 50 ml syringe with the 50% sucrose solution (see Recipes) slowly (to avoid bubbles). Attach the cannula and expel any air by holding the syringe vertically (with the cannula pointing up).
      6. Holding the tube such that the marked line is at eye level, quickly and vertically insert the cannula into the bottom of the tube (avoiding the 50% sucrose solution leaking into the 10% solution).
      7. Slowly expel the 50% sucrose solution while maintaining the bottom of the cannula ~5 mm below the meniscus. When the meniscus of the interphase layer reaches the marked line, stop expelling and quickly pull out the cannula.
      8. Cap each tube (taking care to avoid any air pockets).
      9. Using a P1000, pipette out any residual sucrose that was pushed out through the cap’s hole.
      10. Place tubes into the gradient maker tube holder (that has been pre-leveled using the manufacturer-supplied level).
      11. Using the gradient maker station software, run the ‘14S short 10-50%’ program (see Note 1 for program information).
      12. Transfer the tubes to the cold room (but do not remove caps yet) while you prepare the lysates.

      13. At this point, turn on the ultra-centrifuge to allow it to pre-cool to 4 °C.
    2. PART 2B. Ultracentrifugation
      1. Gently remove caps from 10-50% sucrose gradients. 
      2. Slide sucrose-gradient tubes into rotor buckets.
      3. Remove (X + 100) μl from the top of each gradient, where X is the amount of lysate you will load (typically 100 μl but up to 600 μl is acceptable).
        Note: The downstream RNA analysis steps of this protocol work best for sucrose gradients performed using < 100 μg of lysate (based on A260 units). For higher loading of lysates some scaling up and optimization of the downstream steps of the protocol may be required.
      4. Slowly layer 100-600 μl of lysate on top of the gradient. The lysate should form a visible and neat layer. 
        Note: Save at least 10 μl of lysate as the ‘Input’ fraction for downstream qRT-PCR analysis.
      5. Weigh each gradient tube in a balance and carefully adjust the weight of each tube, if needed, by adding lysis buffer. Equilibrate the bucket pairs facing each other on the rotor: 1-4, 2-5 and 3-6. 
      6. Cap the buckets.
      7. Attach buckets to the SW41 Ti rotor.
      8. Gently lower the rotor into the centrifuge, and lightly spin the rotor by hand to ensure that all buckets are connected properly.
      9. Enter centrifugation settings:
        Vacuum–ON
        Temp–4 °C
        Speed–36,000 rpm (160,000 x g)
        Time–2.5 h 
        Acceleration–1
        De-acceleration–7
      10. Start the centrifuge and ensure that it reaches the desired speed. The centrifuge may pause acceleration at 3,000 rpm until the vacuum is fully engaged.
        Note: On an SW41 Ti rotor, 36,000 rpm corresponds to 160,000 x g at rav. If you are using a different rotor, please refer to the manual to use the correct speed.
    3. PART 2C. Fractionation
      Read and follow manufacturer’s instructions. We recommend contacting the local Biocomp representative for an advanced tutorial. 
      1. During the spin, turn on the Bio-Rad Econo UV monitor to warm up and label and chill screw-cap tubes.
        Notes:
        1. Allow the Bio-Rad Econo UV Monitor to warm up for at least 2 h before setting the zero.
        2. During centrifugation: Pre-label 16 screw cap tubes (USA Scientific) per gradient, cover with cling film or Parafilm to prevent dust/RNase contamination and store in at 4 °C.
      2. Turn on the Gilson fraction collector, the Biocomp gradient station, the computer and open ‘Gradient Profiler V2’ software.
      3. Set the zero UV reading with clean water Bio-Rad Econo UV monitor. 
      4. Ensure that UV readout is stable, not fluctuating.
      5. Remove rotor from centrifuge, place rotor tubes on rack, and place in cold room.
        Note: Do not remove screw cap until needed for fractionation.
      6. Fractionate gradients into 2 ml screw-cap tubes using the following settings:
        Note: If at any point the Econo-UV monitor light turns red, pull up the piston, release the air valve, and repeat the zeroing with water.
        Speed:
        0.30 mm/sec
        Total distance:
        75 mm
        Number of fractions:
        15
        Distance/fraction:
        5.00 mm
        Volume/fraction:
        0.71 ml
      7. Store fractions in the cold room until the entire set of samples have been fractionated.
      8. Flash freeze all tubes and store at -80 °C.

  3. STAGE 3: mRNA Analysis (Day 3)
    1. PART 3A. DNase treatment and control RNA spike-in
      1. Thaw fraction tubes, input tubes, and Cells-to-Ct stop solution. 
      2. Dilute the input samples 30-fold by adding 6 μl to 174 μl RNase-free water, then put on ice.
      3. Prepare a Master mix of lysis solution containing the following (per sample):
        9.9 μl
        Cells-to-Ct lysis buffer
        0.1 μl
        Cells-to-Ct lysis buffer
        0.1 μl
        XenoRNA
      4. Per gradient, prepare 16 PCR tubes (to be used for 15 fractions plus the input) containing 10.1 μl lysis solution master mix.
      5. Add 1 μl of each fraction (or input) directly into the lysis master mix (i.e., not to the tube wall), then pipette up and down 2-3 times. 
      6. Invert tubes several times to mix gently, then briefly spin down.
      7. Incubate at room temperature for 5 min, then put on ice (during this incubation you can put the fraction tubes back into -80 °C freezer).
      8. Pipet 1 μl Cells-to-Ct stop solution directly into each PCR tube (i.e., not to the tube wall).
      9. Invert tubes several times to mix gently, then briefly spin down.
      10. Incubate at room temperature for 2 min, then put on ice.
    2. PART 3B. Reverse transcr
      1. Prepare RT Master mix containing the following (per sample):
        5 μl
        2x Cells-to-Ct RT buffer
        0.5 μl
        20x Cells-to-Ct RT enzyme mix
      2. Use P10 to distribute 5.5 μl RT master mix to PCR tubes.
      3. Use multichannel P10 to add 4.5 μl of lysate.
      4. Perform RT reaction in a thermocycler with the following program: 37 °C for 1 h, 95 °C for 5 min, 4 °C forever.
      5. Dilute each RT reaction by adding 50 μl water and mixing thoroughly.
      6. Store at -20 °C or proceed directly to PCR.
    3. PART 3C. Quantitative real-time PCR protocol
      Every fraction is analyzed with qPCR technical duplicates for each probe.
      1. Program instrument for TaqMan assay:
        1. Probes are labeled with FAM dye and nonfluorescent quencher.
        2. Cycling conditions: 50 °C for 2 min (UDG incubation), 95 °C for 10 min (enzyme activation), 40x [95 °C for 15 sec + 60 °C for 1 min] (PCR).
      2. Mix 2x TaqMan Gene Expression Master mix by swirling the bottle, mix 20x assays by vortexing briefly and centrifuging; keep all solutions on ice.
      3. For each gene-of-interest (including the Xeno and Luciferase controls), prepare a TaqMan PCR Cocktail containing (for each qPCR reaction) 5 μl 2x TaqMan Gene Expression Master MIX + 0.5 μl 20x TaqMan assay (gene specific).
      4. Use a P10 to distribute 5.5 μl of PCR cocktail into a real-time PCR plate at room temperature.
      5. Use a multichannel P10 to add 4.5 μl of RT reaction for each qPCR reaction, mix by pipetting.
      6. Cover the plate carefully and briefly centrifuge (~800 x g for a few seconds).
      7. Place reactions in a real-time PCR instrument and start the run.

Data analysis

  1. Per sample, gather raw Cq information from the qPCR machine for:
    1. Xeno
    2. Luciferase
    3. Actin (or other well-translated gene)
    4. Additional genes of interest
  2. Assemble values by fraction numerical order.
  3. Average Cq values from technical duplicates. Also calculate the difference between replicates and repeat qPCR reactions for any samples a difference greater than 0.5 Cq units (see Note 2).
  4. Calculate mRNA abundance in each fraction relative to the input (Pfaffl, 2001) taking into account differences in qRT-PCR efficiency calculated by normalizing to XenoRNA Cq values:



  5. Convert relative RNA abundances to the percent of total detected RNA:



  6. For each gradient, generate line plots with fraction numbers on the x axis and ‘Percent of total mRNA’ for each target on the y axis.

Note: All of the above analysis should be automated in a spreadsheet. In this way, the researcher only needs to copy and paste Cq values to receive all abundance information, quality control metrics, and polysome plots (see Note 2 for troubleshooting). Figure 2 shows an example of the data analysis with this procedure.


Figure 2. Representative data generated from this polysome gradient analysis protocol. The top panel shows the A260 absorbance trace of a fractionated yeast lysate with the species of ribosome associated with each peak annotated. The bottom panel shows a representative plot of the relative distribution of RNA associated with each fraction of the gradient as analyzed by qRT-PCR. Represented is a translationally repressed mRNA (orange), a well-translated mRNA (blue) and the uncapped luciferase RNA (green), which serves as a control for non-specific interactions. The well-translated mRNA is mainly polysomic and sediment deep in the gradient toward the bottom of the tube. Both the translationally repressed mRNA and the exogenous control RNA are not associated to ribosomes and remain in the top fractions.  

Notes

  1. Fractionation program setup:
    Gradient Master program for 10-50% sucrose gradient:
    1. 05/85/35
    2. 01/77/0
    3. 04/86/35
    4. 03/86.5/35
    5. 20/81/14
    6. 07/86/20
    Sequence of steps: abcbdbabcbdbef
  2. Ideally the averaged Cq values for XenoRNA will be roughly the same in all fractions and input (since equal amounts of XenoRNA were added to samples before qRT-PCR). In practice, we allow a range of up to 1 Cq value; a larger range indicates issues with qRT-PCR efficiency in some fractions, which may reflect an overly concentrated or ‘dirty’ lysate. Consider repeating the experiment if > 5% of the uncapped RNA is found associating with polysomes. If a fraction is significantly different in all probe’s Cq values, there was likely a problem introduced at the RT step. If a fraction is significantly different in one probe’s Cq values, there was likely a problem introduced at the qPCR step.

Recipes

  1. Lysis buffer (made fresh each time)
    20 mM HEPES-KOH (pH 7.4)
    5 mM MgCl2
    100 mM KCl
    200 μg/ml Heparin
    1% Triton X-100
    2 mM DTT
    100 μg/ml cycloheximide
    20 U/ml Superase-IN
    cOmplete mini EDTA-free Protease Inhibitor Cocktail (1 tablet per 10 ml solution)
  2. 10% sucrose solution
    Base: 20 mM HEPES-KOH (pH 7.4), 5 mM MgCl2, 100 mM KCl, 10% sucrose
    Filter sterilize. Store at 4 °C for > 2 weeks
    Additives added fresh each time (final concentration): 2 mM DTT, 100 μg/ml cycloheximide, 20 U/ml Superase-IN
  3. 50% sucrose solution
    Base: 20 mM HEPES-KOH (pH 7.4), 5 mM MgCl2, 100 mM KCl, 50% sucrose
    Filter sterilize. Store at 4 °C for > 2 weeks
    Additives added fresh each time (final concentration): 2 mM DTT, 100 μg/ml cycloheximide, 20 U/ml Superase-IN
    Volumes: 1 gradient = 12 ml total volume (~6 ml 10% sucrose solution, ~6 ml 50% sucrose solution)  For 6 gradients (which can be spun simultaneously in SW41 Ti rotor), 40 ml of each sucrose solution is sufficient

Acknowledgments

We acknowledge Jonathan Weissman, Raul Andino, Keith Yamamoto, and Alan Frankel for generously sharing equipment. This work was supported by the UCSF Program for Breakthrough Biomedical Research (funded in part by the Sandler Foundation) and by an NIH Director’s Early Independence Award (DP5OD017895).

References

  1. Arava, Y., Wang, Y., Storey, J. D., Liu, C. L., Brown, P. O. and Herschlag, D. (2003). Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae . Proc Natl Acad Sci U S A 100(7): 3889-3894.
  2. Dever, T. E. and Green, R. (2012). The elongation, termination, and recycling phases of translation in eukaryotes. Cold Spring Harb Perspect Biol 4(7): a013706.
  3. Di Santo, R., Aboulhouda, S. and Weinberg, D. E. (2016). The fail-safe mechanism of post-transcriptional silencing of unspliced HAC1 mRNA. Elife 5.
  4. Floor, S. N. and Doudna, J. A. (2016). Tunable protein synthesis by transcript isoforms in human cells. Elife 5.
  5. Hinnebusch, A. G. and Lorsch, J. R. (2012). The mechanism of eukaryotic translation initiation: new insights and challenges. Cold Spring Harb Perspect Biol 4(10).
  6. Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. and Weissman, J. S. (2009). Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324(5924): 218-223.
  7. Mašek, T., Valasek, L. and Pospisek, M. (2011). Polysome analysis and RNA purification from sucrose gradients. Methods Mol Biol 703: 293-309.
  8. Odegaard, J. I., Lee, M. W., Sogawa, Y., Bertholet, A. M., Locksley, R. M., Weinberg, D. E., Kirichok, Y., Deo, R. C. and Chawla, A. (2016). Perinatal licensing of thermogenesis by IL-33 and ST2. Cell 166(4): 841-854.
  9. Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9): e45.
  10. Warner, J. R., Knopf, P. M. and Rich, A. (1963). A multiple ribosomal structure in protein synthesis. Proc Natl Acad Sci U S A 49: 122-129.
  11. Weinberg, D. E., Shah, P., Eichhorn, S. W., Hussmann, J. A., Plotkin, J. B. and Bartel, D. P. (2016). Improved ribosome-footprint and mRNA measurements provide insights into dynamics and regulation of yeast translation. Cell Rep 14(7): 1787-1799.

简介

从mRNA分子产生蛋白质的效率可以在转录本,细胞类型和细胞状态之间广泛变化。准确测定mRNA翻译效率的方法对获得对转录后基因调控的机理理解至关重要。测量翻译效率的一种方法是确定与mRNA分子相关的核糖体的数目,归一化为编码序列的长度。分析单个mRNA的主要方法是蔗糖梯度分级,其基于结合核糖体的数目物理分离mRNA。在这里,我们描述了精确分析与核糖体的mRNA相关性的简化方案。与以前的方案相比,我们的方法结合内部控制和改进的缓冲条件,共同减少由非特异性mRNA - 核糖体相互作用引起的伪像。此外,我们的直接分数qRT-PCR方案消除了从梯度部分中RNA纯化的需要,这大大减少了所需的手动时间量,并促进了多个条件或基因靶标的并行分析。此外,在该过程中不产生苯酚废物。我们最初开发了协议来研究S-HAC1 mRNA的翻译抑制状态。但是我们还详细介绍了哺乳动物细胞系和组织的适应程序。
【背景】将mRNA翻译成蛋白质是一种高度调节的过程,其可以以不同的速率发生,这取决于基因,细胞环境或环境。翻译起始,延伸和终止的每个步骤可以是最终影响与mRNA相关的核糖体数量的调节点(Dever和Green,2012; Hinnebusch和Lorsch,2012)。因为连续起始事件之间的时间通常短于延长所需的时间,因此大多数mRNA一次与多于一个的核糖体相关联以形成“多聚体”结构(Warner等人,1963) 。因此,每个mRNA分子计数核糖体数量的能力提供了mRNA的整体翻译状态的测定。传统上,这种计数是通过蔗糖梯度分级(有时也称为多聚体分析)实现的,其中通过基于其大小/形状的超离心将mRNA分离,然后进行定量(Mašek等人,2011) 。可以通过RNA印迹或qRT-PCR或通过微阵列或高通量RNA测序对整个转录组进行梯度级分中的mRNA的检测(Arava等人,2003; Floor和Doudna,2016)。以这种方式,可以确定与各个mRNA分子相关的核糖体的绝对数量。用于测定翻译的替代方法是核糖体 - 足迹分析,其中被RNA核酸酶消化保护的mRNA的短片段被捕获并进行高通量测序(Ingolia等人,2009) 。当与总RNA测序结合以确定mRNA丰度时,核糖体分析数据可以以全基因组方式测量mRNA的翻译效率。然而,核糖体分析仅提供可通过RNA丰度测量(Weinberg等人,2016)偏见的翻译效率的相对量度。此外,核糖体分析不太适合低丰度mRNA的研究,或者只有少量的mRNA受到关注。由于这些原因,蔗糖梯度分馏仍然是分析翻译效率的重要手段。
我们提出了这种广泛使用的技术的适应性,其中包含提高精度和减少实际操作时间的关键功能。蔗糖梯度分离的mRNA多聚体分析分三个步骤完成:裂解物制备,蔗糖梯度分级和RNA丰度分析。我们的协议最初是为了简化多个RNAs的并行分析而开发的,但是在协议开发过程中,我们也仔细地优化了每个步骤,以确保测定提供了核糖体关联的准确和可重复的测量。我们开发出芽酵母S的方案。啤酒酵母(Di Santo等人,2016),但是从那时起,它也已经将其应用于各种各样的人和小鼠细胞系甚至全小鼠组织(Odegaard等人。,2016)。我们的方案的一个关键特征是在裂解缓冲液中包含肝素,这减少了mRNA和核糖体之间的非特异性相互作用,否则可能会导致与多聚核糖体的非翻译mRNA的假象共沉淀。我们还对非翻译RNA进行了可靠的控制:在超速离心之前将其加入裂解物中的未加帽的外源性RNA。对于RNA分析步骤,我们调整了先前用于细胞裂解物的qRT-PCR试剂盒,直接与梯度级分一起使用,从而消除了所有以前的多聚体分析方案中使用的耗时RNA纯化步骤。直接从粗梯度分数测量RNA丰度不仅减少了时间需求和动手操作,而且消除了苯酚废物的产生。最后,为了控制部分(蔗糖浓度和大分子组成不同)中RT-PCR效率的变化,我们在收集之后将等量的人造RNA加入到每个级分作为标准化参考。总而言之,我们的协议 - 在下面详细介绍 - 包含一系列改进和内部控制,它们一起提供准确,精细化的多发症分析测定。

关键字:翻译, 基因调控, 核糖体, 多核糖体分析, 蔗糖梯度分级分离, 可重现性

材料和试剂

  1. 阶段1:溶菌剂制剂
    材料
    的 酵母
    1. 接种环(Fisher Scientific,目录号:22-363-604)
    2. 50ml锥形管(Corning,Falcon ®,目录号:352098)
    3. 1.5ml硅化G管(Bio Plas,目录号:4165SL)
    4. 0.45微米过滤器(Pall,目录号:60206)
    5. 电池升降机(Corning,目录号:3008)

    哺乳动物细胞
    1. 6孔板(Corning,目录号:3516)或10-cm细胞培养皿(Corning,目录号:353803)
    2. 电池升降机(Corning,目录号:3008)
    3. 15ml锥形管(Corning,Falcon ®,目录号:352096)
    4. 1.5ml硅化G管(Bio Plas,目录号:4165SL)

    的 组织
    1. 50ml锥形管(Corning,Falcon ®,目录号:352098)
    2. 5 ml离心管(Eppendorf,目录号:0030119401)
    3. 1.5ml硅化G管(Bio Plas,目录号:4165SL)

    试剂
    1. 液氮(LN 2 )
    2. 适当的培养基(例如,用于酿酒酵母的YPD; DMEM,FBS和哺乳动物细胞系的添加剂)
    3. 1x磷酸缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
    4. 萤光素酶RNA(Promega,目录号:L4561)
      注意:在-80°C下储存100ng /μl储存液。
    5. HEPES(Sigma-Aldrich,目录号:H4034)
    6. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:68475)
    7. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
    8. 肝素(Sigma-Aldrich,目录号:H3149)
      注意:在4°C下储存10 mg / ml储液。
    9. Triton X-100溶液(Sigma-Aldrich,目录号:93443)
    10. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D9779)
      注意:将过滤后的等分量的1M储备溶液储存在-20°C。
    11. 环己酰亚胺(AMRESCO,目录号:94271)
    12. Superase-IN(Thermo Fisher Scientific,Invitrogen TM,目录号:AM2696)
      注意:将过滤的等分试样的100毫克/毫升储备溶液储存在-20℃。
    13. cOmplete,mini,EDTA-free蛋白酶抑制剂鸡尾酒(Roche Diagnostics,目录号:11836170001)
    14. 蔗糖(Sigma-Aldrich,目录号:S5016)
      注意:存储解决方案在4°C,请参阅食谱部分。
    15. 裂解缓冲液(见配方)
    16. 10%蔗糖溶液(见食谱)
    17. 50%蔗糖溶液(参见食谱)

  2. 阶段2:蔗糖梯度分馏
    材料
    1. 50ml SteriFlip(EMD Millipore,目录号:SCGP00525)
    2. 打开顶部多清洁离心管(Seton Scientific,目录号:7030)
    3. SW41标记块(包含分馏器)
    4. 60 ml注射器(BD,目录号:309653)
    5. 不锈钢注射器针,不带点,〜10英寸,〜12号(Sigma-Aldrich,Cadence Science,目录号:Z116971)
    6. 短帽(Biocomp,目录号:105-514-6)
    7. 管架(Beckman Coulter,目录号:331313)
    8. 2毫升管,带螺帽(USA Scientific,目录号:1420-8700)
    9. 贴膜或石蜡膜

  3. 阶段3:mRNA分析
    材料
    1. qPCR板(RPI,目录号:141328)
    2. qPCR膜(Bio-Rad Laboratories,目录号:MSB1001)
    3. PCR管

    试剂
    1. Cell-to-Ct试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:AM1728)
    2. Superase-In(Thermo Fisher Scientific,Invitrogen TM,目录号:AM2696)
    3. XenoRNA(Thermo Fisher Scientific,Invitrogen TM,目录号:4386995,对照试剂盒的一部分)
      注意:以-80°C的小等分存储。
    4. TaqMan基因表达主混合物(Thermo Fisher Scientific,Applied Biosystems TM,目录号:4369016)
    5. 用于感兴趣的基因的引物/探针qPCR测定(Thermo Fisher Scientific,Ac00010014_a1(XenoRNA)和Mr03987587_mr(萤光素酶)))

设备

  1. 移液器(例如,,P1000,P10)
  2. 250ml带挡板的烧瓶(Corning,PYREX ,目录号:4444-250)
  3. 2升挡板(Corning,PYREX ,目录号:4444-2L)
  4. 过滤系统(Restek,目录号:KT676001-4035)
  5. Coors瓷砂浆和杵(Sigma-Aldrich,目录号:Z247472和Z247510)
    制造商:CoorsTek,目录号:60316和60317。
  6. Dounce,组织研磨机(DWK Life Sciences,WHEATON,目录号:357538)[可选]
  7. 台式冷离心机(Eppendorf,型号:5424 R)
  8. SW41 Ti转子(Beckman Coulter,型号:SW 41 Ti,目录号:331362)
  9. 超速离心机(Beckman Coulter,型号:L8-80M)
  10. 梯度台(Biocomp,目录号:153-001)
  11. 馏分收集器(Gilson,目录号:FC 203B)
  12. BIORAD EM-1 Econo UV监视器(Bio-Rad实验室,型号:EM-1 Econo TM )
  13. NanoDrop 2000分光光度计(Thermo Fisher Scientific,Thermo Scientific,型号:NanoDrop TM,目录号:ND-2000)
  14. CFX96 Touch Real-Time PCR检测系统(Bio-Rad Laboratories,型号:CFX96 Touch TM,目录号:1855195)

软件

  1. Gradient Profiler V2'软件

程序

协议分为三个阶段(见图1):
阶段1裂解液制备
1A。细胞的生长和收获(a。酵母,b。细胞系,c。组织)
1B。样品制备
阶段2蔗糖梯度分馏
2A。梯度准备
2B。超速离心
2C。分馏
第3阶段mRNA分析
3A。 DNase治疗和控制RNA飙升
3B。逆转录
3C。实时PCR
推荐的工作流程时间是:

注意:阶段2A需要30-60分钟的孵化,并在阶段1B之前进行以优化时间。


图1.来自多种细胞类型的蔗糖多聚体梯度方案的工作流程示意图这说明了分析多核糖体梯度上的RNA分布的过程的总体步骤。

  1. 阶段1:裂解物制备
    1. 第1A部分。细胞的生长和收获
      的 酵母
      1. 第0天
        1. 将细胞从板上接种到50ml的YPD中,在250ml带挡板的烧瓶中
        2. 成长过夜饱和。
      2. 第1天
        1. 在2L带挡板的烧瓶中,将600ml OD值的0.05稀释到400ml YPD中。
          注意:如果培养多种文化,那么稍微延长文化差距(第一种和最后一种文化之间30分钟的差距就足够了)。
        2. 当培养物达到0.5-0.6的OD 600时,通过快速过滤收获细胞:
          1)在倾倒文化之前,将YPD倒入过滤器。
          2)将整个文化倾倒在过滤容器的侧面,注意避免倾倒将聚集在过滤器顶部的泡沫。
          3)随着最后的液体流出,快速取出过滤器的夹具和顶部,快速地从过滤器上刮掉细胞,但轻轻地使用电池升降器,并浸入含锥形的液氮中。流过过滤器的最后液体和浸没在液氮中的电池之间的总时间不应超过5秒。
          注意:细胞刮刀应在液氮中预冷。
        3. 将锥形圆锥形放在-80°C冷冻箱中,并允许液氮沸腾。
          注意:将帽子松开收紧。
        4. 用研钵和杵研磨裂纹细胞 1)将预冷砂浆和杵用液氮(〜2-3分钟)放入冰桶中。
          2)从砂浆中倒出任何残留的LN 2 。
          3)将细胞沉淀添加到砂浆中。
          4)将细胞沉淀物轻轻倒入〜1ml至1ml的LN 2 注意:添加太多LN 2 将显着增加处理时间。 br />5)用研杵打碎细胞,直到所有LN 2 沸腾,然后再烘干干粉〜1-2分钟。 注意:在砂浆中所有液氮蒸发后,颗粒快速达到粉末状稠度1-2分钟。从进一步研磨中没有获得好处。从经验来看,研磨时间不会太长。
          6)将细胞粉末悬浮在液氮中,小心倒入原来的锥形管中
        5. 将锥形管放置在-80°C冷冻箱中,并允许液氮沸腾。
          注意:将帽子松开收紧。
        6. 暂停点。
        7. 继续到2A阶段。

        8. 哺乳动物细胞
          1. 第0天
            1. 实验所需的平板细胞 注意:该程序已成功应用于在6孔板,10厘米培养皿和15厘米培养皿中培养的各种哺乳动物细胞系,收获范围为10 -6至10 7 单元格。通过控制电镀密度来避免收获时的融合,考虑细胞操作对翻译的影响是重要的。过度融合,耗尽的营养物或血清,或培养基的变化可引起快速的翻译反应。推荐使用稳定的细胞系进行瞬时转染的细胞,以确保重复性。
            2. 在最佳生长条件下孵育细胞。
          2. 第1天
            1. 向终浓度为100μg/ ml的培养基中加入放线菌酮(CHX),在37℃下孵育10分钟。可以省略此步骤。
              注意:虽然生长培养基中的CHX预处理是可选的,但我们建议将CHX加入PBS和裂解缓冲液,以防止在收获过程中核糖体流失。
            2. 在冰上预冷PBS和裂解缓冲液并添加添加剂(参见食谱)。
            3. 将组织培养皿转移到冰桶。
            4. 吸气媒体。
            5. 用10ml冰冷的PBS清洗两次。
            6. 在5毫升冰冷的PBS中彻底清洗细胞。
            7. 将细胞悬浮液转移到15ml锥形管中。
            8. 在4℃,500×g离心5分钟,弃去上清液
            9. 闪存冷冻细胞沉淀并储存在-80°C。
            10. 继续到第2A阶段。

          哺乳动物组织
          1. 第0天
            1. 解剖整个组织样本。
            2. 用冰冷的PBS清洗组织,然后在液氮中冷冻。
          2. 第1天
            1. 用研钵和杵研磨碎裂并撕开组织。
              1)将预冷砂浆和杵用液氮(〜2-3分钟)放入冰桶中。
              2)从砂浆中倒出残留的LN2。
              3)将冻结的纸巾加入砂浆中。
              4)在冷冻的组织上倒入〜1ml至1ml的LN2 &nbsp;&nbsp;&nbsp; &nbsp;注意:添加太多LN2将显着增加处理时间。
              5)用研杵打碎细胞,直到所有LN2沸腾,然后再磨干粉〜1-2分钟。
              注意:在砂浆中所有液氮蒸发后,颗粒快速达到粉末状稠度1-2分钟。从进一步研磨中没有获得好处。从经验来看,研磨时间不会太长。
              6)将细胞粉末悬浮在液氮中,倒入原来的锥形管中
            2. 将锥形管放置在-80°C冷冻箱中,并允许液氮沸腾。
              注意:将帽子松开收紧。
            3. 暂停点
            4. 继续进行第2A阶段

            5. 哺乳动物细胞
              1. 第0天
                1. 实验所需的平板细胞 注意:该程序已成功应用于在6孔板,10厘米培养皿和15厘米培养皿中培养的各种哺乳动物细胞系,收获范围为10 -6至10 7 单元格。通过控制电镀密度来避免收获时的融合,考虑细胞操作对翻译的影响是重要的。过度融合,耗尽的营养物或血清,或培养基的变化可引起快速的翻译反应。推荐使用稳定的细胞系进行瞬时转染的细胞,以确保重复性。
                2. 在最佳生长条件下孵育细胞
              2. 第1天
                1. 向终浓度为100μg/ ml的培养基中加入放线菌酮(CHX),在37℃下孵育10分钟。可以省略此步骤。
                  注意:虽然生长培养基中的CHX预处理是可选的,但我们建议将CHX加入PBS和裂解缓冲液,以防止在收获过程中核糖体流失。
                2. 在冰上预冷PBS和裂解缓冲液并添加添加剂(参见食谱)。
                3. 将组织培养皿转移到冰桶。
                4. 吸气媒体。
                5. 用10ml冰冷的PBS清洗两次。
                6. 在5毫升冰冷的PBS中彻底清洗细胞。
                7. 将细胞悬浮液转移到15ml锥形管中。
                8. 在4℃,500×g离心5分钟,弃去上清液
                9. 闪存冷冻细胞沉淀并储存在-80°C。
                10. 继续到2A阶段。

              哺乳动物组织
              1. 第0天
                1. 解剖整个组织样本。
                2. 用冰冷的PBS清洗组织,然后在液氮中冷冻。
              2. 第1天
                1. 用研钵和杵研磨碎裂并撕开组织。
                  1)将预冷砂浆和杵用液氮(〜2-3分钟)放入冰桶中。
                  2)从砂浆中倒出残留的LN2。
                  3)将冻结的纸巾加入砂浆中。
                  4)在冷冻的组织上倒入〜1ml至1ml的LN2 &nbsp;&nbsp; &nbsp;&nbsp; 注意:添加太多LN2会大大增加处理时间。
                  5)用研杵打碎细胞,直到所有LN2沸腾,然后再磨干粉〜1-2分钟。
                  注意:在砂浆中所有液氮蒸发后,颗粒快速达到粉末状稠度1-2分钟。从进一步研磨中没有获得好处。从经验来看,研磨时间不会太长。
                  6)将细胞粉末悬浮在液氮中,倒入原来的锥形管中
                2. 将锥形管放置在-80°C冷冻箱中,并允许液氮沸腾。
                  注意:将帽子松开收紧。
                3. 暂停点
                4. 继续进行第2A阶段
                5. 第1B部分:样品制备(第2天)
                  注意:所有以下步骤均在冰块或4°C冷藏室进行。
                  酵母程序
                  1. 在冰上解冻粉碎5分钟。
                    过早加入裂解缓冲液可能导致冻结。
                  2. 同时,每个样品预先标注四个硅化微量离心管:
                    再悬浮粉末
                    澄清未稀释的裂解物 澄清稀释的裂解物 等分裂解物(多管)
                    注意:您还将需要三个0.6 ml管,每个样品含有90μlddH2O。
                  3. 在每个锥形细胞中加入1 ml裂解缓冲液至细胞粉末
                  4. 旋转每个管轻轻混合,然后使用P1000上下移动完全重新悬挂。
                  5. 将整个管内容物转移到预先标记的"再悬浮"1.7ml管中
                  6. 旋转10分钟,1,300 x g,4°C。
                  7. 将澄清的裂解物(〜800μl)转移到"澄清的未稀释"标记的1.7 ml管中。澄清的裂解物应具有白色/黄色色调的半透明外观。
                  8. 将10μl转移到90μl水中,以指定NanoDrop的RNA浓度,作为总裂解物浓度的代用品。
                    注意:
                    1. Triton X-100干扰阅读,因此需要稀释。
                    2. 空白将具有10μl裂解缓冲液+90μlddH 2 O。
                  9. 用裂解缓冲液稀释所有裂解物至25 OD 260 U / ml(1μg/μlRNA)。
                  10. 规定稀释的裂解物,以确保所有样品在彼此的约5%以内
                  11. 加入外源未封端的萤光素酶RNA(Promega)至终浓度为100ng / ml
                  12. 将等分试样150μl加入1.7ml管中。
                  13. 在-80°C储存裂解液不需要立即进行实验。
                  14. 继续到2B阶段。

                  哺乳动物细胞程序
                  1. 在冰上解冻细胞沉淀。
                  2. 将细胞沉淀物重悬于100μl溶胞缓冲液/10μl细胞中
                  3. 将裂解液转移至1.7 ml管
                  4. 在冰上孵育10分钟,通过上下移动混合。
                    注意:最佳溶解时间和洗涤剂浓度可能因细胞类型而异。在裂解期间在不同时间的相位对比显微镜下检查细胞裂解。 Triton可以用其他洗涤剂如NP-40或使用均质器进行机械裂解。
                  5. 在4℃,12,000×g离心10分钟。
                  6. 将澄清的裂解液转移到"澄清的未稀释"标记的1.7ml管中。&nbsp;
                  7. 将10μl裂解物稀释至90μl水中,以指定NanoDrop的RNA浓度,作为总裂解物浓度的代用品(见附注8)。
                  8. 通过加入适量的裂解缓冲液将所有样品稀释至相同的浓度 注意:我们建议稀释至〜20-100μg/ ml。调整浓度并按照规格进行测量,以确保所有样品在5%以内。
                  9. 将外源未封端的萤光素酶RNA刺激至终浓度为100ng / ml。&nbsp;
                  10. 将等份200-500μl分装到1.7ml管中,将剩余的裂解物(输入)储存在-80℃。
                  11. 继续到2B阶段。

                  组织过程
                  1. 将50毫克(一勺)冷冻粉末称取至2升冷冻的5毫升Eppendorf管中。
                    1. 快速行动以避免管道升温。
                    2. 将管浸入LN 2,并摇匀以分散粉末(主要为50ml圆锥形)。
                  2. 在加入裂解缓冲液(LB)之前,让它在冰中"解冻"至4°C
                  3. 加入每毫克粉末50-100μl裂解缓冲液。
                  4. 上下移动混合,大力旋转,放在冰上。
                    1. 使Triton X-100在旋转之前加入LB后,将脂质裂解5-10分钟。
                    2. 再次旋转。
                  5. 在4℃下旋转750℃×10分钟10分钟。
                  6. 将上清液分离成新管。&nbsp;
                    注意:全组织样本:必须仔细准备富含脂质的样品以避免脂质污染。因此,我们建议在离心后将中间的75%澄清的裂解物吸收,以避免干扰上层脂质层或底部不溶物质。
                  7. 在4°C下旋转12,000 x g 10分钟。
                  8. 将上清液分离成新管。
                    注意:从中间抽取75%的液体。
                  9. 将10μl上清液转移到90μl水中以规范NanoDrop的RNA浓度,其作为总裂解物浓度的代谢(见注8)。
                  10. 将等分试样250μl加入1.7ml管中,并将剩余的裂解物(输入)储存在-80℃下
                  11. 继续到2B阶段。

                6. 阶段2:蔗糖梯度分馏(第2天)
                  1. 第2A部分。梯度准备
                    建立应在室温下和裂解液制备的第二步之前进行。当梯度冷却至4℃时,准备并澄清裂解物。
                    1. 准备蔗糖溶液
                      1. 将40ml预过滤的蔗糖溶液(储存于4℃,参见食谱部分)分装成锥形管,并使其温热至室温。
                      2. 将DTT,放线菌酮和Superase-IN加入蔗糖溶液中,然后轻轻旋转混合。
                    2. 准备裂解缓冲液,放上冰冷至4℃
                    3. 使用SW41 Ti标记块标记Polyclear离心管,在顶部标记块线上的每根管上绘制一条线。
                    4. 用50%蔗糖溶液(见食谱)慢慢填充50ml注射器(以避免气泡)。通过将注射器垂直握住(套管朝上)来安装插管并排出任何空气。
                    5. 保持管使得标记的线处于眼睛水平,快速和垂直地将插管插入管的底部(避免50%蔗糖溶液泄漏到10%溶液中)。
                    6. 慢慢地排出50%的蔗糖溶液,同时保持插管的底部〜弯液面以下5毫米。
                    7. 每个管盖(注意避免任何气囊)。
                    8. 使用P1000,移出通过盖帽孔推出的任何残留的蔗糖。
                    9. 将管放入梯度制管器支架(已经使用制造商提供的级别进行预平衡)。
                    10. 在准备裂解物时,将管转移到冷藏室(但不要除去帽子)。

                    11. 此时,打开超离心机将其预冷至4°C。
                  2. 第2B部分。超速
                    1. 轻轻取出10-50%蔗糖梯度的帽子。&nbsp;
                    2. 将蔗糖梯度管滑入转子桶。
                    3. 从每个梯度的顶部去除(X + 100)μl,其中X是您将加载的裂解物的量(通常为100μl,但最多可达600μl)。
                      注意:本协议的下游RNA分析步骤对于使用&lt; 100μg裂解物(基于A260单位)。对于较高的裂解物负载量,可能需要一些放大和优化协议的下游步骤。
                    4. 在梯度上缓缓层层100-600μl裂解物。裂解物应形成可见和整齐的层。&nbsp;
                      注意:保存至少10μl裂解物作为下游qRT-PCR分析的"输入"分数。
                    5. 称量每个梯度管的平衡,并仔细调整每个管的重量,如果需要,通过添加裂解缓冲液。平衡在转子上彼此面对的铲斗对:1-4,2-5和3-6。&nbsp;
                    6. 盖上桶。
                    7. 将铲斗连接到SW41 Ti转子。
                    8. 将转子轻轻放入离心机中,用手轻轻旋转转子,确保所有水桶均正确连接。
                    9. 输入离心设置:
                      真空打开
                      温度-4°C
                      Speed-36,000 rpm(160,000 x g )
                      时间2.5小时&nbsp;
                      加速-1
                      去加速-7
                    10. 启动离心机并确保达到所需的速度。离心机可能会以3,000 rpm的速度暂停加速,直到真空完全接合。
                      注意:在SW41 Ti转子上,36,000 rpm对应于rav的160,000 x g。如果您使用不同的转子,请参阅手册以使用正确的速度。
                  3. 第2C部分。分馏
                    阅读并遵循制造商的说明。我们建议您联系当地的Biocomp代表进行高级教程。
                    1. 在旋转过程中,打开Bio-Rad Econo UV显示器进行预热,并标记并冷却螺旋盖管。
                      注意:
                      1. 允许Bio-Rad Econo UV Monitor预热至少2小时,然后再设置零。
                      2. 在离心过程中:每个梯度预先标记16个螺旋盖管(USA Scientific),用保鲜膜或Parafilm覆盖,以防止灰尘/ RNase污染并在4°C储存。
                    2. 打开Gilson馏分收集器,Biocomp梯度计,计算机和打开的"Gradient Profiler V2"软件。
                    3. 用干净的水Bio-Rad Econo UV显示器设置零UV读数。&nbsp;
                    4. 确保UV读数稳定,不变动
                    5. 从离心机上取下转子,将转子管放在机架上,放在冷藏室中 注意:除非需要进行分馏,否则请勿卸下螺帽。
                    6. 使用以下设置将分馏梯度分成2ml螺旋盖管:
                      注意:如果在任何时候Econo-UV监视器指示灯变为红色,请拉起活塞,释放空气阀,并用水重新调零。
                    7. 在冷室中储存级分,直到整套样品分馏。
                    8. 快速冻结所有管并储存在-80°C。

                    9. 阶段3:mRNA分析(第3天)
                      1. 第3A部分。 DNA酶处理和对照RNA加入
                        1. 解冻分数管,输入管和细胞到Ct停止溶液。&nbsp;
                        2. 通过加入6μl至174μl无RNase的水稀释输入样品30倍,然后放入冰上。
                        3. 准备含有以下(每个样品)的裂解液的主混合物:
                          细胞到Ct裂解缓冲液
                        4. 每个梯度,准备16个PCR管(用于15个分数加上输入),含有10.1μl裂解液主混合物。
                        5. 将每个级分(或输入)1μl直接加入裂解主混合物(即,不是管壁),然后上下移动2-3次。&nbsp;
                        6. 反转管数次轻轻混合,然后短暂旋转。
                        7. 在室温下孵育5分钟,然后放在冰上(在孵化过程中,可以将分馏管放回-80°C冰箱)。
                        8. 吸管1μl细胞到Ct将溶液直接停止到每个PCR管(即,不是管壁)中。
                        9. 反转管数次轻轻混合,然后短暂旋转。
                        10. 在室温下孵育2分钟,然后放在冰上。
                        11. 第3B部分。反向转
                          1. 准备包含以下(每个样本)的RT主混合:
                          2. 使用P10将5.5μlRT主混合物分配到PCR管。
                          3. 使用多通道P10加入4.5μl裂解物
                          4. 使用以下程序在热循环仪中进行RT反应:37℃1小时,95℃5分钟,4℃永久。
                          5. 通过加入50μl水稀释每个RT反应并彻底混合。
                          6. 储存在-20°C或直接进行PCR。
                          7. 第3C部分定量实时PCR方案
                            用每个探针的qPCR技术重复分析每个部分。
                            1. TaqMan测定程序仪器:
                              1. 探针用FAM染料和非荧光猝灭剂标记
                              2. 循环条件:50℃2分钟(UDG孵育),95℃10分钟(酶活化),40×[95℃15秒+60℃1分钟](PCR)。
                            2. 通过旋转瓶混合2x TaqMan基因表达主混合物,通过短暂涡旋混合20x测定并离心;将所有解决方案都保留在冰上。
                            3. 对于每个感兴趣的基因(包括Xeno和萤光素酶对照),制备含有(用于每个qPCR反应)的5μl2x TaqMan基因表达主MIX +0.5μl20x TaqMan测定(基因特异性)的TaqMan PCR Cocktail。 >
                            4. 使用P10将5.5μlPCR混合液在室温下分配到实时PCR板中
                            5. 使用多通道P10为每个qPCR反应添加4.5μlRT反应,通过移液混合
                            6. 小心地盖上板,短暂离心(〜800 x g 几秒钟)。
                            7. 在实时PCR仪器中放置反应并开始运行。
                          8. 反转管数次轻轻混合,然后短暂旋转
                          9. 在室温下孵育2分钟,然后放在冰上。
                          10. 第3B部分。反向转
                            1. 准备包含以下(每个样本)的RT主混合:
                              2x Cells-to-Ct RT缓冲区
                              20x Cell-to-Ct RT酶混合物
                            2. 使用P10将5.5μlRT主混合物分配到PCR管。
                            3. 使用多通道P10加入4.5μl裂解物
                            4. 使用以下程序在热循环仪中进行RT反应:37℃1小时,95℃5分钟,4℃永久。
                            5. 通过加入50μl水稀释每个RT反应并彻底混合。
                            6. 储存在-20℃或直接进行PCR。
                            7. 第3C部分定量实时PCR方案
                              用每个探针的定量PCR技术重复分析每个部分。
                              1. TaqMan测定程序仪器:
                                1. 探针用FAM染料和非荧光猝灭剂标记
                                2. 循环条件:50℃2分钟(UDG孵育),95℃10分钟(酶活化),40×[95℃15秒+ 60℃1分钟](PCR)。
                              2. 通过旋转瓶混合2x TaqMan基因表达主混合物,通过短暂涡旋混合20x测定并离心;将所有解决方案都保留在冰上。
                              3. 对于每个感兴趣的基因(包括Xeno和萤光素酶对照),制备含有(用于每个qPCR反应)的5μl2xTaqMan基因表达主MIX +0.5μl20xTaqMan测定(基因特异性)的TaqMan PCR Cocktail。 >
                              4. 使用P10将5.5μlPCR混合液在室温下分配到实时PCR板中
                              5. 使用多通道P10为每个定量PCR反应添加4.5μlRT反应,通过移液混合
                              6. 小心地盖上板,短暂离心(~800 x g 几秒钟)。
                              7. 在实时PCR仪器中放置反应并开始运行。
                      2. 食谱

                        1. 裂解缓冲液(每次新鲜)
                          20mM HEPES-KOH(pH 7.4)
                          5mM MgCl 2
                          100 mM KCl
                          200μg/ ml肝素 1%Triton X-100
                          2 mM DTT
                          100μg/ ml放线菌酮
                          20 U / ml Superase-IN
                          完全不含EDTA的蛋白酶抑制剂鸡尾酒(每10ml溶液1片)
                        2. 10%蔗糖溶液
                          碱:20mM HEPES-KOH(pH7.4),5mM MgCl 2,100mM KCl,10%蔗糖 过滤灭菌在4℃下储存&gt; 2周
                          添加剂每次添加新鲜(终浓度):2mM DTT,100μg/ ml放线菌酮,20U / ml Superase-IN
                        3. 50%蔗糖溶液
                          碱:20mM HEPES-KOH(pH7.4),5mM MgCl 2,100mM KCl,50%蔗糖 过滤灭菌在4℃下储存&gt; 2周
                          添加剂每次添加新鲜(终浓度):2mM DTT,100μg/ ml放线菌酮,20U / ml Superase-IN
                          体积:1个梯度= 12ml总体积(〜6ml 10%蔗糖溶液,〜6ml 50%蔗糖溶液)对于6个梯度(可在SW41Ti转子中同时旋转),将40ml的每种蔗糖溶液充分

                      致谢

                      我们承认Jonathan Weissman,Raul Andino,Keith Yamamoto和Alan Frankel慷慨分享设备。这项工作得到了UCSF突破性生物医学研究计划的支持(由Sandler基金会资助)和NIH主任早期独立奖(DP5OD017895)。

                      参考

                      1. 阿拉瓦, Y.,Wang,Y.,Storey,J.D.,Liu,C.L。,Brown,P.O。和Herschlag,D。(2003)。 mRNA翻译简档的基因组范围分析 在酿酒酵母中 Natl Acad Sci U S A 100(7):3889-3894。
                      2. 杰维尔, T.E.and Green,R。(2012)。 延伸,终止和回收阶段的翻译真核生物 Spring Harb Perspect Biol 4(7):a013706。
                      3. 迪 Santo,R.,Aboulhouda,S.and Weinberg,D.E。(2016)。 故障安全机制 未切割的HAC1 mRNA的转录后沉默。 5。
                      4. 地板, S.N.and Doudna,J.A。(2016)。 可调蛋白质合成 人类细胞中的转录本同种型。 5。
                      5. Hinnebusch, A.G.和Lorsch,J.R。(2012)。 真核生物的机制 翻译开始:新的见解和挑战 Spring Harb Perspect Biol 4(10)。
                      6. Ingolia, N.T.,Ghaemmaghami,S.,Newman,J.R。和Weissman,J.S。(2009)。 基因组范围 分析体内的翻译 核糖体分析的核苷酸分辨率。科学 324(5924):218-223。
                      7. 马塞克, T.,Valasek,L。和Pospisek,M。(2011)。 Polysome analysis and RNA 从蔗糖梯度纯化 Mol Biol 703:293-309。
                      8. Ødegaard公司, J.I.,Lee,M.W.,Sogawa,Y.,Bertholet,A.M.,Locksley,R.M.,Weinberg,D. E.,Kirichok,Y.,Deo,R.C。和Chawla,A。(2016)。 围产期许可证 IL-33和ST2的产热。细胞 166(4):841-854。
                      9. Pfaffl, M.W.(2001)。 新的数学模型 实时RT-PCR中的相对定量 Acids Res 29(9):e45。
                      10. 华纳, J.R.,Knopf,P.M.and Rich,A。(1963)。 多重核糖体结构 在蛋白质合成中。"Proc Natl Acad Sci U S A"49:122-129。
                      11. 温伯格 D.E.,Shah,P.,Eichhorn,S.W.Hussmann,J.A.,Plotkin,J.B。和Bartel, D.P。(2016)。 改进的核糖体足迹 和mRNA测量提供了对酵母的动力学和调节的见解 翻译。 Cell Rep 14(7): 1787年至1799年。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Aboulhouda et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Aboulhouda, S., Di Santo, R., Therizols, G. and Weinberg, D. E. (2017). Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation. Bio-protocol 7(19): e2573. DOI: 10.21769/BioProtoc.2573.
  2. Di Santo, R., Aboulhouda, S. and Weinberg, D. E. (2016). The fail-safe mechanism of post-transcriptional silencing of unspliced HAC1 mRNA. Elife 5.
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