The overall goal of the following experiment is to isolate translating ribosomes polysome in order to study changes in mRNA translation activity in the cell. This is achieved by adding cyclo hexa to the growth media in order to freeze ribosomes on the mRNA, thereby preventing ribosome runoff as a second step. Ribosomes are extracted from cells and separated based on their density by sucrose density gradient centrifugation to quantify and isolate free ribosomal subunits 40 s and 60 s mono zones a DS, and translating ribosomes polysome.
Next RNAs or proteins can be extracted from sucrose fractions in order to analyze Mr NA and protein abundance respectively in pre and poly somal fractions. The results show quantitative and qualitative changes in mRNA translation, which can be examined on a genome wide level using microarrays or next generation RNA sequencing and analyzed using a NoDa algorithm. The poison profiling method can be used to address major questions when it comes to regulation of gene expression at post transcription levels, such as monitoring changes in mRNA, translation on a genome wide level, as well as the Association of Proteins with ribosome and polys.
Valentina Gand research associate in our lab will demonstrate this technique To begin, prepare 100 milliliters of 60%weight per volume sucrose solution in deionized distilled water, then filter the solution through a 0.22 micrometer filter. Next, dilute the 60%sucrose solution in one X sucrose gradient buffer to make 40 milliliters of 5%and 50%sucrose solutions. Use the marker block provided with the gradient unit to mark the half full point on six poly aamer ultracentrifuge tubes.
Then use the layering device to add 5%sucrose solution to the half full point. Next, add the 50%sucrose solution to the bottom of the tube until the interface of the two solutions reaches the half full point seal tubes with rate zonal caps and place the seal tubes in the tube holder on the gradient maker. Run the gradient maker to obtain a linear gradient from 5%to 50%The gradient maker rotates the tubes at a high tilt angle to form six linear gradients within a few minutes.
On the day of the experiment, cells should be approximately 80%confluent. Treat cells with 100 micrograms per milliliter of cyclo hexa in growth media and incubate for five minutes at 37 degrees Celsius and 5%carbon dioxide Wash cells twice with 10 milliliters of ice cold PBS containing 100 micrograms per milliliter cyclo hexa. Then add an additional five milliliters of the ice cold PBS solution.
Scrape the cells and transfer the cell suspension to a 50 milliliter to centrifuge cells at 200 times G for five minutes at four degrees Celsius. Then discard the supernatant and resuspend cells in 425 microliters of hypotonic lysis buffer. Next, add five microliters of 10 milligram per milliliter cyclo hexa, one microliter of one molar DTT and 100 units of RNAs inhibitor to the cell suspension.
Then vortex the solution for five seconds. Supplement the cell suspension with 25 microliters of 10%tritton X 125 microliters of 10%sodium deoxy coate, then vortex for an additional five seconds. Next, centrifuge the lysates at 16, 000 times G for seven minutes at four degrees Celsius.
Transfer the super natin to a new pre chilled 1.5 milliliter tube and save 10%of the lysate as a control to measure steady state MR.mRNA levels. Measure the OD at 260 nanometers for each lysate sample, and then adjust the to the same OD in hypotonic lysis buffer with a final volume of 500 microliters. Remove 500 microliters from the top of the sucrose gradients, and load the adjusted lysate samples on top of each gradient.
Next, weigh and balance each gradient tube. Place the tubes in an SW 41 TI rotor and put the ultra centrifuge in low break mode. Then centrifuge the samples at 222, 228 times G for two hours at four degrees Celsius.
When ultra centrifugation is finished, carefully remove the gradient tubes from the rotor and place them on ice. First, fill the fraction collector with two milliliter collection tubes. Then turn on the computer pump UV detector and fraction collector half an hour before fractions are collected.
Set the pump to run at three milliliters per minute and then fill the tubing with a chasing solution to remove the air in the system. Run the chasing solution through the tubing until a drop comes out of the needle. Open the analysis software.
Then set the sensitivity to plus or minus 10 milli electron volts and the time base to 100 seconds. Place the ultracentrifuge tube into the UV detector and ensure that the tube is in the orthogonal position. Next, twist the knob below the tube holder to pierce the tube with the needle.
Adjust the pump flow rate to 1.5 milliliters per minute and set the fraction collector to collect fractions every 30 seconds, which is equal to 750 microliters in each fraction. Put the pump in the remote position. Then start the pump and fraction collector and begin recording with the DAQ tracer at the same time.
When the first drop of chasing solution falls into a collection tube, stop the pump and the DAQ tracer. At the same time, add 750 microliters of triol to each collected fraction and flash. Freeze the fractions in liquid nitrogen.
Then follow the manufacturer's instructions to isolate the polysome associated and cytosolic RNA from the samples. Polysome fractionation was used to evaluate the translational activity of C seven breast cancer cells treated with insulin. The proportion of polysome increased with insulin treatment as compared to control cells, and those treated with the mTOR inhibitor, Torin one.
This result indicates that insulin stimulates global translation initiation rates in mc seven cells. The results from four independent trials of the insulin experiment were used to evaluate the reproducibility of this polysome fractionation method. The samples from the same treatment condition and RNA origin are closely positioned on this graph indicating high reproducibility.
After watching this video, you should have a good understanding of how to lys cells, prepare sucrose, gradients, and finally collect fractionated ribosomes. This protocol will allow you to analyze gene expression at the post transcription level.
