What Does Sucrose Gradient Fractionation Do Again

• Journal List
• Bio Protoc
• v.7(19); 2017 Oct 5
• PMC5697790

Bio Protoc. 2017 October 5; 7(19): e2573.

Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation

Soufiane Aboulhouda

¹Department of Molecular and Cellular Pharmacology, Academy of California, San Francisco, CA, USA

Rachael Di Santo

¹Department of Molecular and Cellular Pharmacology, Academy of California, San Francisco, CA, USA

Gabriel Therizols

¹Department of Molecular and Cellular Pharmacology, University of California, San Francisco, CA, United states of america

David Weinberg

¹Section of Molecular and Cellular Pharmacology, Academy of California, San Francisco, CA, USA

²Sandler Kinesthesia Fellows Program, University of California, San Francisco, CA, USA

Received 2017 Jul 13; Revised 2017 Aug 22; Accepted 2017 Sep 5.

Abstruse

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 past non-specific mRNA–ribosome interactions. Moreover, our directly-from-fraction qRT-PCR protocol eliminates the need for RNA purification from gradient fractions, which greatly reduces the corporeality of easily-on time required and facilitates parallel analysis of multiple weather condition or gene targets. Additionally, no phenol waste is generated during the process. We initially developed the protocol to investigate the translationally repressed land of the HAC1 mRNA in S. cerevisiae, but nosotros also particular adjusted procedures for mammalian jail cell lines and tissues.

Keywords: Translation, Gene regulation, Ribosome, Polysome analysis, Sucrose gradient fractionation, Reproducibility

Groundwork

The translation of mRNA into protein is a highly regulated procedure that can occur at unlike rates depending on the cistron, cellular context, or environment. Each stride 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 fourth dimension betwixt sequent initiation events is usually shorter than the time required for elongation, most mRNAs are associated with more than ane 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 country of mRNA. Traditionally, this counting has been achieved past sucrose gradient fractionation (likewise sometimes called polysome assay), in which mRNAs are separated by ultracentrifugation based on their size/shape and and so quantified ( Mašek et al., 2011 ). The detection of mRNAs in gradient fractions can either be done for individual mRNAs past RNA blotting or qRT-PCR, or for the entire transcriptome past microarrays or loftier-throughput RNA sequencing ( Arava et al., 2003 ; Flooring and Doudna, 2016). In this manner, the absolute number of ribosomes associated with individual mRNA molecules tin 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 past ribosomes are captured and subjected to high-throughput sequencing ( Ingolia et al., 2009 ). When combined with full RNA sequencing to determine mRNA abundances, ribosome-profiling data can mensurate the translational efficiencies of mRNAs in a genome-wide manner. Nonetheless, 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 merely a pocket-size number of mRNAs are of involvement. For these reasons, sucrose gradient fractionation remains an of import 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 grooming, sucrose gradient fractionation, and RNA-abundance assay. Our protocol was initially developed to streamline the analysis of multiple RNAs in parallel, just in the procedure of protocol development nosotros likewise advisedly optimized each step to ensure that the assay provided an accurate and reproducible measure of ribosome clan. We developed the protocol for the budding yeast S. cerevisiae (Di Santo et al., 2016 ) only since so have as well applied it to a broad variety of human and mouse jail 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 not-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 assay pace we adapted a qRT-PCR kit previously used for cell lysates to work straight with slope 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 only also eliminates generation of phenol waste. Finally, to control for variations in RT-PCR efficiencies amid fractions (which differ in sucrose concentration and macromolecular composition), we spike in an equal amount of artificial RNA to each fraction simply later collection to serve as a normalization reference. In summary, our protocol–presented in detail beneath–contains a collection of improvements and internal controls that together provide an accurate, streamlined assay for polysome analysis.

Materials and Reagents

1. Phase one: Lysate grooming

   Materials

   Yeast

   1. Inoculation loop (Fisher Scientific, itemize number: 22-363-604)

   2. 50 ml conical tube (Corning, Falcon^®, catalog number: 352098)

   3. one.5 ml siliconized G-tube (Bio Plas, itemize number: 4165SL)

   4. 0.45 micron filters (Mantle, catalog number: 60206)

   5. Cell lifter (Corning, catalog number: 3008)

   Mammalian cells

   1. six-well plate (Corning, itemize number: 3516) or 10-cm cell culture dish (Corning, catalog number: 353803)

   2. Cell lifter (Corning, itemize 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. fifty ml conical tubes (Corning, Falcon^®, catalog number: 352098)

   2. 5 ml centrifuge tubes (Eppendorf, catalog number: 0030119401)

   3. 1.five ml siliconized G-tubes (Bio Plas, itemize number: 4165SL)

   Reagents

   1. Liquid nitrogen (LN_two)

   2. Appropriate culturing media (e.g., YPD for Due south. cerevisiae; DMEM, FBS and additives for mammalian cell lines)

   3. 1x phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10010023)

   4. Luciferase RNA (Promega, itemize number: L4561)

      Annotation: Store aliquotted 100 ng/μl stock at -80 °C.

   5. HEPES (Sigma-Aldrich, catalog number: H4034)

   6. Magnesium chloride (MgCl_two) (Sigma-Aldrich, itemize number: 68475)

   7. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541)

   8. Heparin (Sigma-Aldrich, catalog number: H3149)

      Note: Store ten 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 -xx °C.

   11. Cycloheximide (AMRESCO, catalog number: 94271)

   12. Superase-IN (Thermo Fisher Scientific, Invitrogen^TM, itemize number: AM2696)

       Note: Store filtered and aliquotted 100 mg/ml stock solution at -20 °C.

   13. cOmplete, mini, EDTA-costless Protease Inhibitor Cocktail (Roche Diagnostics, catalog number: 11836170001)

   14. Sucrose (Sigma-Aldrich, catalog number: S5016)

       Note: Store solution at four °C, run across Recipes section.

   15. Lysis buffer (see Recipes)

   16. 10% sucrose solution (see Recipes)

   17. 50% sucrose solution (encounter Recipes)

2. STAGE ii: 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. threescore ml syringe (BD, catalog number: 309653)

   5. Stainless steel syringe needle, noncoring point, ~ten inches, ~12 gauge (Sigma-Aldrich, Cadence Science, catalog number: Z116971)

   6. Curt caps (Biocomp, itemize number: 105-514-six)

   7. Tube rack (Beckman Coulter, itemize number: 331313)

   8. 2 ml tubes w/screw caps (The states Scientific, itemize number: 1420-8700)

   9. Cling movie or Parafilm

3. STAGE 3: mRNA analysis

   Materials

   1. qPCR plates (RPI, itemize number: 141328)

   2. qPCR film (Bio-Rad Laboratories, catalog number: MSB1001)

   3. PCR tubes

   Reagents

   1. Cells-to-Ct kit (Thermo Fisher Scientific, Invitrogen^TM, catalog number: AM1728)

   2. Superase-In (Thermo Fisher Scientific, Invitrogen^TM, catalog number: AM2696)

   3. XenoRNA (Thermo Fisher Scientific, Invitrogen^TM, itemize number: 4386995, part of command kit)

      Note: Store in minor aliquots at -80 °C.

   4. TaqMan Factor Expression Principal mix (Thermo Fisher Scientific, Applied Biosystems^TM, 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.1000., P1000, P10)

2. 250 ml baffled flask (Corning, PYREX^®, catalog number: 4444-250)

3. 2 Fifty baffled flask (Corning, PYREX^®, catalog number: 4444-2L)

4. Filtration system (Restek, itemize number: KT676001-4035)

5. Coors porcelain mortar and pestle (Sigma-Aldrich, catalog numbers: Z247472 and Z247510)

   Manufacturer: CoorsTek, itemize 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, itemize number: 331362)

9. Ultracentrifuge (Beckman Coulter, model: L8-80M)

10. Slope 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 Econo^TM)

13. NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Thermo Scientific, model: NanoDrop^TM 2000, catalog number: ND-2000)

14. CFX96 Touch Real-Time PCR detection system (Bio-Rad Laboratories, model: CFX96 Touch^TM, itemize number: 1855195)

Software

1. Gradient Profiler V2' software

Process

The protocol is divided into 3 stages (see Effigy i):

Workflow schematic of the sucrose polysome slope protocol from multiple cell types.

This illustrates the overall steps of the procedure to analyze RNA distribution across a polysome gradient.

Stage 1 Lysate Preparation

1A. Growth and harvesting of cells (a. yeast, b. cells lines, c. tissues)

1B. Sample training

Phase 2 Sucrose Gradient Fractionation

2A. Slope preparation

2B. Ultracentrifugation

2C. Fractionation

Stage iii mRNA Assay

3A. DNase handling and control RNA spike-in

3B. Opposite transcription

3C. Real-time PCR

The recommended workflow timing is:

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

1. STAGE 1: Lysate Training

   1. Function 1A. Growth and harvesting of cells

      Yeast

      1. Day 0

         1. Inoculate cells from plate into fifty 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 ii L baffled flask.

            Note: If growing multiple cultures, stagger civilization growth slightly (30 min difference between first and concluding cultures is sufficient).

         2. When culture reaches an OD₆₀₀ of 0.five-0.6, harvest cells by rapid filtration:

            1. Pour YPD on filter prior to pouring culture.

            2. Cascade unabridged civilisation downwardly the side of the filtration vessel, taking care to avert pouring foam that will collect on top of the filter.

            3. As the final liquid pours through, quickly remove the clamp and top of the filter unit, scrape cells from the filter speedily only gently using a prison cell lifter, and submerge into conical containing liquid nitrogen. The total time betwixt the final liquid flowing through the filter and the cells existence 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-arctic mortar and pestle with liquid nitrogen (~2-3 min) in an ice bucket.

            2. Pour out whatever residual LN_two 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 besides much LN₂ will significantly increment processing times.

            5. Grind with a pestle to break autonomously cells until all LN₂ boils off, so grind the dry powder for an additional ~i-ii min.

               Notation: After evaporation of all the liquid nitrogen in the mortar, the pellet reaches a powder-like consistency quickly, 1-ii min. No benefits are gained from further grinding. From feel, there are no adverse effects of grinding for too long.

            6. Re-append the prison cell powder in liquid nitrogen and carefully pour back into the original conical tube.

         5. Place the conical tube in a -eighty °C freezer and permit liquid nitrogen to boil off.

            Note: Leave cap loosely tightened.

         6. Pause indicate.

         7. Go along to Stage 2A.

      Mammalian cells

      1. Solar day 0

         1. Plate cells every bit required by experiment.

            Note: The procedure has been successfully applied to diverse mammalian prison cell lines cultured in half-dozen-well plates, x-cm dishes, and fifteen-cm dishes, with a harvested range of 10⁶ to x⁷ 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 tin induce quick translational responses. Using a stable prison cell line is recommended over transiently transfected cells to ensure reproducibility.

         2. Incubate cells under optimal growth atmospheric condition.

      2. Twenty-four hour period one

         1. Add cycloheximide (CHX) to media at a final concentration of 100 μg/ml, incubate for 10 min at 37 °C. This footstep can be omitted.

            Note: While CHX pre-treatment in growth media is optional, nosotros recommend adding CHX to the PBS and lysis buffer to forestall ribosome run-off during harvesting.

         2. Pre-arctic 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 x ml ice-cold PBS.

         6. Scrape cells thoroughly and quickly in 5 ml of ice-cold PBS.

         7. Transfer cell interruption to a 15 ml conical tube.

         8. Centrifuge for 5 min at 4 °C at 500 × g, discard supernatant.

         9. Flash freeze jail cell pellet and store at -80 °C.

         10. Proceed to Phase 2A.

      Mammalian tissue

      1. 24-hour interval 0

         1. Dissect out a whole tissue sample.

         2. Launder tissue with ice-cold PBS prior to freezing in liquid nitrogen.

      2. Day 1

         1. Break autonomously 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 saucepan.

            2. Pour out any residual LN₂ from the mortar.

            3. Add the frozen tissue to the mortar.

            4. Cascade ~1-5 ml of LN₂ on the frozen tissue.

               Note: Calculation also much LN_ii will significantly increase processing times.

            5. Grind with a pestle to break apart cells until all LN₂ boils off, then grind the dry pulverisation for an additional ~one-2 min.

               Note: After evaporation of all the liquid nitrogen in the mortar, the pellet reaches a powder-similar consistency apace, i-2 min. No benefits are gained from further grinding. From feel, there are no agin effects of grinding for too long.

            6. Re-suspend the cell powder in liquid nitrogen and cascade back into the original conical tube.

         2. Identify the conical tube in a -80 °C freezer and allow liquid nitrogen to boil off.

            Note: Leave cap loosely tightened.

         3. Pause betoken.

         4. Continue to Phase 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 process

      1. Thaw grinded powder on ice for 5 min.

         Prematurely adding lysis buffer can cause information technology to freeze.

      2. In the meantime, pre-label iv siliconized microcentrifuge tubes per sample:

         Re-suspended pulverisation

         Clarified undiluted lysate

         Clarified diluted lysate

         Aliquoted lysate (multiple tubes)

         Note: You will also need three 0.half dozen ml tubes per sample containing 90 μl ddH_twoO.

      3. Add one 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' one.vii ml tubes.

      6. Spin for 10 min at 1,300 × g at iv °C.

      7. Transfer antiseptic lysate (~800 μl) into 'clarified undiluted' labeled ane.7 ml tubes. Clarified lysate should have a translucent appearance with a white/yellow hue.

      8. Transfer ten μl into 90 μl water to spec on NanoDrop for RNA concentration, which serves equally a proxy for total lysate concentration.

         Notes:

         1. Triton X-100 interferes with reading so dilution is needed.

         2. Bare will have ten μl lysis buffer + 90 μl ddH_twoO.

      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 inside ~5% of each other.

      11. Add together exogenous uncapped Luciferase RNA (Promega) to a final concentration of 100 ng/ml.

      12. Aliquot 150 μl into 1.7 ml tubes.

      13. Shop lysates not immediately needed for experiment at -80 °C.

      14. Go on to Stage 2B.

      Mammalian cells procedure

      1. Thaw prison cell pellet on ice.

      2. Resuspend cell pellet in 100 μl lysis buffer per ten⁶ cells.

      3. Transfer lysate to a i.vii ml tube.

      4. Incubate for 10 min on ice, mix by pipetting up and downwardly.

         Note: Optimal lysis time and detergent concentration may vary depending on the cell blazon. Check cell lysis under a microscope with stage contrast at different times during lysis. Triton tin exist substituted by other detergents such every bit NP-40 or mechanical lysis using a dounce homogenizer.

      5. Centrifuge for 10 min at 4 °C at 12,000 × 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 Notation 8).

      8. Dilute all samples to the same concentration by adding an advisable amount of lysis buffer.

         Note: We recommend diluting to ~twenty-100 μg/ml. Adjust concentrations and measure out past spec to ensure all samples are within v% of each other.

      9. Fasten in exogenous uncapped Luciferase RNA to a final concentration of 100 ng/ml.

      10. Aliquot 200-500 μl into i.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 LN_ii chilled v ml Eppendorf tubes.

         1. Act quickly to avert tube warming up.

         2. Dip tubes into LN_ii and shake to separate powder frequently (chief l ml conical).

      2. Let to 'thaw' to iv °C in ice earlier adding lysis buffer (LB).

      3. Add 50-100 μl of lysis buffer per mg of powder.

      4. Pipette upward and down to mix, vortex vigorously, and let sit on water ice.

         1. Permit Triton X-100 to lyse lipids for 5-ten min subsequently adding LB before spinning.

         2. Vortex again.

      5. Spin 750 × 1000 for x min at 4 °C.

      6. Divide supernatant into a new tube.

         Note: Whole tissue samples: Lipid-rich samples must exist carefully prepared to avert lipid contamination. As such, we recommend taking the heart 75% of the antiseptic lysate after centrifugation to avert disturbing the top lipid layer or bottom insoluble material.

      7. Spin 12,000 × thou for 10 min at four °C.

      8. Separate supernatant into a new tube.

         Annotation: Take 75% liquid from the center.

      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 (run into 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, so vortex to mix.

      12. Aliquot 250 μl into i.vii ml tubes and store the remaining lysate (input) at -lxxx °C.

      13. Go along to Stage 2B.

2. STAGE 2: Sucrose Slope Fractionation (Solar day 2)

   1. PART 2A. Slope preparation

      The prepare upwards should be done at room temperature and prior to the 2d step of lysate preparation. While gradients are cooling to 4 °C, prepare and clarify the lysates.

      1. Fix sucrose solutions

         1. Aliquot 40 ml of pre-filtered sucrose solutions (stored at iv °C, see Recipes section) into a conical tube, and permit warm to room temperature.

         2. Add DTT, cycloheximide, and Superase-IN to sucrose solutions, then mix by gentle rotation.

      2. Set up lysis buffer and put on water ice to absurd to 4 °C.

      3. Mark Polyclear centrifuge tubes using the SW41 Ti marker cake by drawing a line on each tube at the top marker block line.

      4. Using a stripette, make full centrifuge tubes with 10% sucrose solution (see Recipes) upwards to ~2 mm higher up the marked line.

      5. Make full upwards a 50 ml syringe with the l% sucrose solution (come across Recipes) slowly (to avoid bubbles). Attach the cannula and expel whatever air by belongings the syringe vertically (with the cannula pointing up).

      6. Belongings the tube such that the marked line is at eye level, chop-chop 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 chop-chop pull out the cannula.

      8. Cap each tube (taking care to avoid any air pockets).

      9. Using a P1000, pipette out whatever 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 brusk 10-l%' program (encounter Notation 1 for program data).

      12. Transfer the tubes to the cold room (but do not remove caps yet) while y'all 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 ten-l% sucrose gradients.

      2. Slide sucrose-gradient tubes into rotor buckets.

      3. Remove (X + 100) μl from the top of each gradient, where 10 is the amount of lysate you lot volition load (typically 100 μl only 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 superlative of the gradient. The lysate should course a visible and not bad layer.

         Note: Save at to the lowest degree 10 μl of lysate equally the 'Input' fraction for downstream qRT-PCR assay.

      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-six.

      6. Cap the buckets.

      7. Adhere buckets to the SW41 Ti rotor.

      8. Gently lower the rotor into the centrifuge, and lightly spin the rotor past manus to ensure that all buckets are connected properly.

      9. Enter centrifugation settings:

         Vacuum–ON

         Temp–iv °C

         Speed–36,000 rpm (160,000 × g)

         Time–2.5 h

         Acceleration–1

         De-acceleration–vii

      10. Kickoff 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 thousand at r_av. If you lot are using a dissimilar rotor, delight refer to the manual to use the correct speed.

   3. PART 2C. Fractionation

      Read and follow manufacturer'southward instructions. Nosotros 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 sixteen screw cap tubes (United states of america 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 figurer and open 'Gradient Profiler V2' software.

      3. Set up the zero UV reading with make 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 post-obit settings:

         Notation: If at any point the Econo-UV monitor calorie-free turns red, pull upward 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

         Altitude/fraction: 5.00 mm

         Volume/fraction: 0.71 ml

      7. Store fractions in the common cold room until the entire set of samples take been fractionated.

      8. Flash freeze all tubes and shop at -80 °C.

3. Stage 3: mRNA Analysis (Day 3)

   1. Function 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 past adding 6 μl to 174 μl RNase-free water, then put on water 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- DNase

         0.ane μl XenoRNA

      4. Per gradient, prepare 16 PCR tubes (to be used for xv fractions plus the input) containing ten.ane 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), so pipette upwardly and down ii-3 times.

      6. Capsize 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 downwardly.

      10. Incubate at room temperature for ii min, and so put on water ice.

   2. PART 3B. Reverse transcription protocol

      1. Prepare RT Principal mix containing the post-obit (per sample):

         5 μl 2x Cells-to-Ct RT buffer

         0.5 μl 20x Cells-to-Ct RT enzyme mix

      2. Utilise P10 to distribute v.5 μl RT master mix to PCR tubes.

      3. Use multichannel P10 to add 4.v μl of lysate.

      4. Perform RT reaction in a thermocycler with the following programme: 37 °C for 1 h, 95 °C for 5 min, 4 °C forever.

      5. Dilute each RT reaction past adding fifty μl h2o and mixing thoroughly.

      6. Shop 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. Plan musical 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 + lx °C for i min] (PCR).

      2. Mix 2x TaqMan Gene Expression Master mix past swirling the bottle, mix 20x assays by vortexing briefly and centrifuging; keep all solutions on ice.

      3. For each cistron-of-interest (including the Xeno and Luciferase controls), fix a TaqMan PCR Cocktail containing (for each qPCR reaction) five μl 2x TaqMan Cistron Expression Master MIX + 0.5 μl 20x TaqMan analysis (cistron specific).

      4. Use a P10 to distribute v.5 μl of PCR cocktail into a real-time PCR plate at room temperature.

      5. Utilise a multichannel P10 to add iv.5 μl of RT reaction for each qPCR reaction, mix by pipetting.

      6. Comprehend the plate carefully and briefly centrifuge (~800 × g for a few seconds).

      7. Place reactions in a real-fourth dimension PCR instrument and start the run.

Data analysis

1. Per sample, gather raw Cq data from the qPCR machine for:

   1. Xeno

   2. Luciferase

   3. Actin (or other well-translated gene)

   4. Boosted genes of interest

2. Assemble values by fraction numerical order.

3. Boilerplate Cq values from technical duplicates. Also calculate the difference between replicates and echo qPCR reactions for any samples a difference greater than 0.5 Cq units (run into Note 2).

4. Summate mRNA affluence in each fraction relative to the input (Pfaffl, 2001) taking into business relationship differences in qRT-PCR efficiency calculated by normalizing to XenoRNA Cq values:

   T a r g east t a b u north d a north c e i n f r a c t i o n _ X = two C q t a r chiliad e t , i northward p u t - C q t a r thou eastward t , f r a c t i o northward _ 10 2 C q X e north o , i n p u t - C q Ten east n o , f r a c t i o n _ X

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

   P e r c e n t i north f r a c t i o north _ X = T a r g e t a b u n d a northward c e i due north f r a c t i o n _ X ∑ Y = i 15 T a r g e t a b u n d a n c e i northward f r a c t i o n _ Y

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

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

Representative information generated from this polysome gradient analysis protocol.

The pinnacle panel shows the A₂₆₀ absorbance trace of a fractionated yeast lysate with the species of ribosome associated with each superlative annotated. The bottom panel shows a representative plot of the relative distribution of RNA associated with each fraction of the gradient every bit analyzed past qRT-PCR. Represented is a translationally repressed mRNA (orange), a well-translated mRNA (bluish) and the uncapped luciferase RNA (greenish), which serves as a control for non-specific interactions. The well-translated mRNA is mainly polysomic and sediment deep in the slope toward the bottom of the tube. Both the translationally repressed mRNA and the exogenous command RNA are not associated to ribosomes and remain in the acme fractions.

Notes

1. Fractionation plan setup:

   Gradient Chief 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/twenty

   Sequence of steps: abcbdbabcbdbef

2. Ideally the averaged Cq values for XenoRNA volition exist roughly the same in all fractions and input (since equal amounts of XenoRNA were added to samples earlier qRT-PCR). In exercise, 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'due south Cq values, there was likely a trouble introduced at the RT step. If a fraction is significantly different in ane probe's Cq values, there was likely a trouble introduced at the qPCR step.

Recipes

1. Lysis buffer (made fresh each fourth dimension)

   xx mM HEPES-KOH (pH 7.4)

   5 mM MgCl₂

   100 mM KCl

   200 μg/ml Heparin

   ane% Triton X-100

   2 mM DTT

   100 μg/ml cycloheximide

   20 U/ml Superase-IN

   cOmplete mini EDTA-gratuitous Protease Inhibitor Cocktail (i tablet per 10 ml solution)

2. 10% sucrose solution

   Base of operations: 20 mM HEPES-KOH (pH 7.4), 5 mM MgCl₂, 100 mM KCl, 10% sucrose

   Filter sterilize. Shop at 4 °C for > ii weeks

   Additives added fresh each time (last concentration): ii mM DTT, 100 μg/ml cycloheximide, 20 U/ml Superase-IN

3. 50% sucrose solution

   Base of operations: 20 mM HEPES-KOH (pH 7.4), 5 mM MgCl_two, 100 mM KCl, fifty% sucrose

   Filter sterilize. Store at 4 °C for > ii weeks

   Additives added fresh each time (final concentration): 2 mM DTT, 100 μg/ml cycloheximide, 20 U/ml Superase-IN

   Volumes: i gradient = 12 ml total book (~6 ml x% 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 past an NIH Director's Early Independence Award (DP5OD017895).

Commendation

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5697790/

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