The overall goal of this experiment is to measure changes in RNA synthesis by bromouridine labeling and immunoprecipitation. This method can help answer key questions in the field of RNA metabolism by measuring whether a change in RNA levels is caused by changes in RNA synthesis. The main advantage of this technique is that it can be used on any preferred type of cell culture and the materials used are easily accessible.

To begin the protocol, seed 3.5 times 10 to the sixth HEK293T cells in a 10-centimeter Petri dish in 10 milliliters of growth media to obtain a total of greater than 40 micrograms of RNA upon extraction 48 hours later. Maintain the cells at 37 degrees Celsius and 5%CO2.48 hours after seeding, aspirate eight milliliters of growth media, and leave two milliliters behind to prevent the cells from drying out. Add two millimoles of bromouridine and treatment to the growth media, and remove the residual two milliliters from the cells before reapplying the eight milliliters of bromouridine containing growth media.

Next, incubate the cells for one hour. After incubation, wash the cells in five milliliters of Hank's buffer, and trypsinize the cells with two milliliters of 0.05%trypsin for five minutes. Add eight milliliters of growth media to stop the reaction, transfer the cells to a 15-milliliter tube, and spin the cells for two minutes at 1, 000 times g.

Then, remove the supernatant and extract total RNA from the cell pellet using an RNA isolation kit, and elute the RNA in RNase-free water. This technique is very sensitive, and it's therefore important to work with RNA of a high quality. The RNA extraction is therefore a critical step, but it's also easily performed with today's RNA extraction kits.

Prepare beads in one batch for the number of samples to be investigated plus one extra to account for loss during pipetting and whirl mixing. Resuspend anti-Mouse IgG magnetic beads thoroughly by whirl mixing, and take out 20 microliters of beads per sample in an RNase-free 1.5-milliliter tube. Place the 1.5-milliliter tube in a magnetic stand for 20 seconds for the beads to collect on the side.

Remove the supernatant, take out the tube from the magnetic stand, and resuspend the beads in 40 microliters of one times bromouridine-IP buffer by pipetting. Spin the tube for two seconds in a tabletop centrifuge, place it back in the magnetic stand, and remove the supernatant. Next, block unspecific binding to the beads using heparin.

Resuspend the beads in one milliliter of one times bromouridine-IP plus one milligram per milliliter of heparin buffer, and rotate the tube for 30 minutes. Then, wash the beads one time. Resuspend the beads in one milliliter of one times bromouridine-IP buffer, and add 1.25 micrograms per sample of anti-alpha-bromodeoxyuridine antibody.

Incubate the beads for one hour while rotating. Following incubation, wash the beads three times. Resuspend the washed beads in 50 microliters per sample of one times bromouridine-IP buffer supplemented with one millimolar bromouridine, and leave the beads rotating for 30 minutes.

After rotation, wash the beads three times, and resuspend the beads in 50 microliters per sample of one times bromouridine-IP buffer. Measure RNA concentrations in the RNA extracts using ultraviolet-visible spectrophotometry, and dilute 40 micrograms of RNA from each sample in RNase-free water to a total volume of 200 microliters. After denaturing the RNA for two minutes at 80 degrees Celsius, spin the samples briefly in a tabletop centrifuge.

After spinning, add 200 microliters of two times bromouridine-IP plus BSA, RNase inhibitor. Then, add 50 microliters of the resuspended and prepared beads to each sample, and rotate the samples for one hour. After incubation, wash the beads four times.

Resuspend the beads in 200 microliters of elution buffer to elute the RNA, and quickly add 200 microliters of pH 6.6 phenol to remove any non-RNA molecules from the sample. Whirl the sample to mix, and spin it for three minutes at 25, 000 times g in a tabletop centrifuge. Next, aspirate as much of the upper phase as possible, approximately 190 microliters, and move it to a tube with 200 microliters of pH 6.6 phenol-chloroform.

Make sure to aspirate the same amount across samples. Whirl the sample, and spin it. After the spin, perform chloroform extraction by aspirating the aqueous upper phase, approximately 180 microliters, and transfer it to a tube containing 200 microliters of chloroform.

Whirl and then spin the sample. Aspirate the aqueous upper phase, approximately 170 microliters, and transfer it to a tube containing 17 microliters of three molar pH six sodium acetate to neutralize and precipitate the RNA from the aqueous solution. Next, add two microliters of glycogen, which will precipitate with the RNA and make the pellet more visible.

Add 500 microliters of 96%ethanol to increase binding between the salt ions and the RNA, and mix by inverting the tube a couple of times. Leave the tube overnight at minus 20 degrees Celsius or for 15 minutes on dry ice. Spin the chilled sample, discard supernatant without disturbing the pellet, and wash the pellet in 185 microliters of 75%ethanol to remove any residual salt.

Then, spin the sample at 25, 000 times g at four degrees Celsius for five minutes. Discard the supernatant completely without disturbing the pellet, and leave the pellet to dry for five to 10 minutes with the lid open. Resuspend the pellet in 10 microliters of RNase-free water applied on the side of the tube to avoid disturbing the pellet.

AsPC-1 cells were treated with bromouridine for zero, one, two, and 4.5 hours, followed by total RNA extraction and bromouridine-IP. GAS5 and GAPDH were measured in bromouridine-IP RNA, which demonstrated that one hour of labeling was sufficient to reach nine-and 44-fold change compared to the background. Two millimoles of bromouridine treatment did not induce any morphologic changes in HEK293T cells for either one hour or the longer four-hour treatment.

PLK-2 inhibition caused an increase in alpha-synuclein mRNA in both the input and bromouridine-IP, suggesting that the increase in the steady state RNA is caused by an increase in synthesis of RNA. After watching this video, you should have a good understanding of how to label newly synthesized RNA with bromouridine and perform a subsequent immunoprecipitation of bromouridine and the labeled RNA.