The overall goal of this procedure is to capture cytosolic protein that bind to a specific sequence located in RNA molecule in mesothelioma cells. This method can help answer key question in RNA biology field, such as if a predicted RNA binding protein can bind adenosine or uridine-rich sequence in a transcript. The main advantage of this technique is that it complements functional characterization of stabilizing or destabilizing elements assessed by reporter assay.
Demonstrating this technique will be Dr.Jelena Kresoja-Rakic, a postdoc from my laboratory. To begin the protocol, obtain previously prepared ACC-MESO-4 cells with a 80 to 90%confluence. Aspirate the medium, wash the cells with 15 milliliters of 1X phosphate buffered saline, or PBS, and aspirate the PBS.
Next, add three milliliters of 0.25%trypsin-EDTA, and incubate the culture flask at 37 degrees Celsius for three minutes. Add seven milliliters of supplemented RPMI-1640 medium, collect the cells in a 15-milliliter tube, and spin them down at 200 times g for five minutes. Then, aspirate the medium, and resuspend the cell pellet with 1.5 milliliters of 1X PBS.
Then, transfer the cells into a 1.5-milliliter microcentrifuge tube. Spin down the cells again at 500 times g and four degrees Celsius for five minutes. After the spin, discard the supernatant, resuspend the cell pellet with 1.5 milliliters of ice-cold 1X PBS, and spin down the cells at 500 times g and four degrees Celsius for three minutes.
Discard the supernatant, and estimate the cell pellet packed volume by comparing the tube containing the cell pellet with a 1.5-milliliter tube with 10, 20, 50, and 100 microliters of 1X PBS. Add 200 microliters of ice-cold cytoplasmic extraction reagent, or CER I, supplemented with two microliters of 1X proteinase inhibitor. Vigorously vortex the pellet for 15 seconds, and incubate the tube on ice for 10 minutes.
Add 11 microliters of ice-cold cytoplasmic extraction reagent II, or CER II, vigorously vortex for five seconds, and incubate the tube for one minute on ice. Vortex the sample vigorously for five seconds, and centrifuge the tube at 16, 000 times g for five minutes. Transfer the supernatant to the clean, pre-chilled tube on ice.
Resuspend the remaining pellet in 100 microliters of ice-cold nuclear extraction reagent, or NER, supplemented with one microliter of proteinase inhibitor, and vigorously vortex for 15 seconds. Incubate the sample on ice for 40 minutes, and vortex every 10 minutes for 15 seconds. Centrifuge the tube at 16, 000 times g for 10 minutes.
Transfer the supernatant, which is the nuclear protein fraction, to a clean, pre-chilled tube. Immediately measure the protein concentration using the bicinchoninic method. Then, prepare 25 microliters of aliquots of cytosolic and nuclear extracts.
Snap-freeze these aliquots by placing them in liquid nitrogen for five seconds, and store them directly at minus 80 degrees Celsius. Transfer five microliters of 10-micromolar RNA probes into 0.5-milliliter, thin-wall microcentrifuge tubes, and incubate them in a PCR machine at 85 degrees Celsius for five minutes. Place the tubes immediately on ice.
For a single 30-microliter reaction, add 10 microliters of previously prepared mix to the RNA-containing tube. Carefully add 15 microliters of PEG 30%to the reaction, and use a fresh tip to mix the reaction. Incubate the reaction overnight at 16 degrees Celsius.
The next day, prepare the reagents. Add 70 microliters of nuclease-free water to the RNA-labeling reaction tubes. Add 100 microliters of chloroform-isoamyl alcohol, and vortex briefly and spin the sample at 13, 000 times g for three minutes.
Carefully remove only the upper phase. Avoid touching the lower phase. Transfer it to a new nuclease-free, 1.5-milliliter tube.
Add 10 microliters of five-molar sodium chloride, one microliter of glycogen, and 300 microliters of ice-cold 100%ethanol. Place the tube at minus 20 degrees Celsius for two hours. After two hours, centrifuge at 13, 000 times g and four degrees Celsius for 15 minutes.
Carefully discard the supernatant without disturbing the pellet. Add 300 microliters of ice-cold 70%ethanol, and centrifuge again at 13, 000 times g and four degrees Celsius for five minutes. Discard the supernatant completely, and air-dry the pellet for 15 minutes.
Resuspend the pellet in 20 microliters of nuclease-free water. Incubate the RNA at 90 degrees Celsius for two minutes, and place it on ice. Place the labeled RNA on ice during the following step of pre-washing of the streptavidin magnetic beads.
Prewash 50 microliters of streptavidin magnetic beads per 50 picomoles of RNA. Vortex the tube with streptavidin magnetic beads for 15 seconds. After vortexing quickly, remove 200 microliters into a clean, 1.5-milliliter Safe-Lock tube using cut pipette tips.
Next, place the tube on a magnetic stand so that the beads collect at the side of the tube, and wait one minute. Remove the resuspension liquid. To wash the beads, remove the tube from the magnetic stand, add 400 microliters of 0.1-molar sodium hydroxide, 0.05-molar sodium chloride solution, and gently pipette up and down several times.
Place the tube back on the magnetic stand, and wait one minute. Then, collect the supernatant. Wash the beads with 200 microliters of 100-millimolar sodium chloride, and remove the supernatant.
Add 200 microliters of 20-millimolar tris, resuspend the beads by pipetting, and place the tube on the magnetic stand. After one minute, remove the supernatant. Then, remove the tube from the magnetic stand, add 200 microliters of 1X RNA capture buffer, and resuspend streptavidin magnetic beads by briefly vortexing.
Remove 50 microliters of streptavidin magnetic beads, and add it to each labeled RNA tube using a cut pipette tip. Incubate the tubes for 30 minutes at room temperature on a roller. After the tube has been on the magnetic stand for one minute, remove the supernatant.
Add 50 microliters of 20-millimolar tris to the beads, and resuspend them. Then, place the tubes into the magnetic stand. After one minute, remove the supernatant.
Add 100 microliters of 1X protein RNA binding buffer to the beads, and resuspend them. Then, place the tubes back into the magnetic stand. Once the tubes have been in the stand for one minute, collect the supernatant.
Add 100 microliter of master mix B, resuspend by gently pipetting up and down, and avoid creating bubbles. Incubate reaction tubes for 60 minutes at four degrees Celsius on a roller. Move on to the wash and elution steps.
Place the tubes into a magnetic stand, collect the flow through, and transfer it into a new tube. Pipette 100 microliters of 1X wash buffer on the beads, resuspend gently, place them back into the stand, wait one minute, and discard the supernatant. Repeat this step two times.
Add 40 microliters of elution buffer to the magnetic beads, mix by pipetting up and down, and incubate on a tube shaker. Place the tubes into a magnetic stand, wait one minute, and collect eluate sample. Place on ice, and use for downstream analysis.
To demonstrate the purity of the nuclear/cytosolic fractions, proteins were analyzed by Western blot, which showed that alpha-tubulin was only detected in the cytosolic fraction, and PARP protein was detected only in the nuclear fraction. The eluate from the CALB2 three-prime UTR ARE probe demonstrated binding of human R and was absent in the eluate from the mutant probe and the unrelated RNA probe, which binds the iron-responsive protein. To further demonstrate the specificity between the CALB three-prime UTR and human R, the membrane was probed with anti-mesothelin antibody, and all three eluate samples showed no presence of mesothelin, indicating that the stabilizing ARE motif within CALB2 three-prime UTR can specifically bind human R protein.
Coomassie staining of the gel shows that equal amounts of proteins were used to incubate with the three different RNA probes. Following this procedure, additional method protein-centric, like immunoprecipitation of a given protein, can be performed in order to address additional question, like which sequence bind to it. After its development, this technique paved the way for researchers in the field of RNA biology to explore possible interacting partners of a specific RNA.
