This method is significant because no other protocol allows newly synthesized RNA to be analyzed in such a short time scale, and allow pulse-chase experiments with such a short pulse. This protocol works well with protein depletion, such as the Best AID 4U protocol. The main advantage is that the background is low.
This allows the short labeling times, by removing almost all the nonthiolated RNA. The written protocol also details many different types of experiment that could be performed and how to integrate them into this technique. To begin this procedure, grow yeast in YMM medium to an OD 600 between 0.6 and 0.8.
In a fume hood, add a methanol equivalent to 30 to 50%of the intended sample volume to a 50 milliliter tube. Close the tubes tightly, label them, and place them on dry ice to chill. Next, label two milliliter tubes for long term storage of the samples and place on ice to cool.
Add zirconia beads to the two milliliter tubes. Cool at least one milliliter of water per sample on ice. If an S.pombe spike is to be added to the culture, place an aliquot of thiolated S.pombe cells on ice to thaw.
Vortex the thawed aliquot and add it to the culture. Then add 4tU to the culture to a concentration of 10 micromolar and mix vigorously. Thio-label for a length of time between 15 seconds and five minutes.
Take samples of the culture at regular intervals to the end of the time course. Add each sample to one of the prepared centrifuge tubes containing methanol. Seal each tube, shake to mix thoroughly, and place on dry ice.
Once all the samples have been taken, place them all on ice. Ensure that none of the samples have frozen. If any have frozen, warm them gently in the hand while inverting constantly.
Centrifuge the cells at 3, 000 times g and at four degrees Celsius for two minutes to pellet the cells. Pour off the liquid, and resuspend the pellet in at least one milliliter of ice cold water by vigorously pipetting up and down. Transfer the suspension to the prepared screw cap tube and place the tube on ice.
Centrifuge the samples briefly at a speed greater than 13, 000 times g to re-pellet the cells. Then place them back on ice, and remove the liquid. First, add in 400 microliters of AE buffer, 40 microliters of SDS, and 800 microliters of phenol at a low pH.
Resuspend by vortexing for 10 seconds. Then lyse the cells in a homogenizer for three two minute bursts at the lowest power setting. Leave the samples on ice for two minutes between homogenization pulses.
Place the lysed cells on dry ice for five minutes until it solidifies. Next, use a microcentrifuge to spin the cells at a speed over 13, 000 times g for five minutes at room temperature. After this, perform phenol chloroform extraction and chloroform extraction as outlined in the text protocol.
Add between 1/3 to 1/2 of the volume of 10 molar lithium chloride and mix to precipitate the RNA. Centrifuge at a speed over 13, 000 times g for five minutes. Remove the fluid, and briefly re-spin the sample to remove the dregs.
Then, wash the pellet with 300 to 500 microliters of 70%ethanol. Centrifuge the washed pellet briefly, and remove any remaining ethanol. Re-dissolve the RNA pellet in 90 microliters of TE at a pH 7.0 by heating at 65 degrees Celsius while shaking.
After checking for full RNA solubilization, transfer the suspension to a 0.2 milliliter tube. To begin, biotinylate by adding 10 microliters of HPDP-biotin solution to the RNA and mix thoroughly. Incubate in the dark at 65 degrees Celsius for 15 to 30 minutes.
Next, prepare a small resin volume, size exclusion column to exclude the unincorporated biotin. Remove the bottom tag of the column, loosen the cap, and place the column into a two milliliter centrifuge tube. Centrifuge at 1, 500 times g for one minute to flush out the buffer and discard the flow-through.
Then gently add 0.3 milliliters of TE to the top of the column and spin again using the same conditions. Transfer the washed column to a fresh 1.5 milliliter tube. When the sample incubation is complete, add the sample to the top of the column.
Centrifuge at 1, 500 times g for two minutes, leaving the biotinylated RNA sample at the bottom of the tube. Discard the column. And add 1/3 to 1/2 volume of 10 molar lithium chloride to the tube containing the sample.
Mix to re-precipitate the RNA. First, re-dissolve the RNA in 200 microliters of DEPC-treated water. Measure the RNA concentration, and calculate volumes for each sample that will give equal amounts of RNA for all samples.
Add this amount of RNA to a fresh tube, and add DEPC-treated water back up to a total volume of 200 microliters. When a sample is at room temperature, add 25 microliters of 10 x NaTM buffer, 25 microliters of one molar sodium phosphate, and 2.5 microliters of 10%SDS. Mix thoroughly, and centrifuge gently at approximately 100 times g for less than 30 seconds.
Prepare two milliliters of bead buffer for each sample with one x NaTM buffer, 0.1 molar sodium phosphate, and 0.1%SDS. Add 50 microliters of streptavidin beads to a low retention 1.5 milliliter tube. Place the tube on the magnetic rack and wait for the beads to settle.
Then, remove the fluid by aspiration. To wash the beads, add 200 milliliters of bead buffer and vortex until the bead pellet is fully resuspended. Centrifuge the tube at approximately 100 times g for a maximum of five seconds.
Place the tube in the magnetic rack to allow the beads to be captured by the magnet. Remove the fluid by aspiration if there are a small number of samples. Pour off the fluid and then aspirate if there are many samples.
Block with 200 microliters of bead buffer, 10 microliters of glycogen, and 2.5 microliters of tRNA. Incubate at room temperature for 20 minutes while rotating end over end at moderate speed. After this, remove the fluid and wash again, as outlined in the text protocol.
Resuspend the beads in the sample, and incubate at room temperature for 30 minutes while rotating. During this incubation, prepare a fresh 1.5 milliliter tube for each sample. Add 1/10 volume of three molar sodium acetate at pH 5.3 and 20 micrograms of glycogen.
Centrifuge at approximately 100 times g for three seconds. Remove the unbound RNA from the beads, and wash them as outlined in the text protocol. To elute the RNA, add 50 microliters of freshly prepared 0.7 molar beta mercaptoethanol to the beads.
After vortexing and centrifugation, place the slurry in the magnetic rack. Pipette the RNA containing solution into the prepared 1.5 milliliter centrifuge tubes. Elute once more as previously described.
Then, place the sample back in the magnetic rack to remove residual beads from the eluted RNA and transfer the fluid to a fresh, low binding 0.5 milliliter centrifuge tube. Mix the sample. And then add 2.5 volumes of ethanol to precipitate nsRNA.
After mixing and incubating, centrifuge at a speed of at least 13, 000 times g and at four degrees Celsius for 20 minutes. Typical yields for snRNA recovered using this protocol are shown here. Note that RNA recovery at time point zero is a very small portion of that recovered from longer time points with only 0.3 micrograms of RNA being recovered from approximately 30 billion cells.
After just 30 seconds of labeling, however, over twice as much RNA is collected from the same number of cells. In the bioanalyzer trace, our RNA precursors can be seen as a peak near 1, 000 nucleotides and a doublet of peaks at 1, 700 to 1, 800 nucleotides. The abundance of these intermediates increases as thiolation continues.
Thiolation is then performed with samples being taken at 15 second intervals from the start of thio-labeling and the processing of ACT1 RNA transcript is monitored. As can be seen pre-mRNA and lariats are generated even after just 15 seconds of labeling. After about 45 seconds to one minute, the amount of lariats and pre-mRNA reach equilibrium with as much of these RNA species being created by transcription as are processed away by splicing.
The most difficult steps are those involving the beads, washing, and blocking. Be careful not to lose the beads, or to let them dry out. Once you have purified newly synthesized RNA, you can use any traditional RNA analysis procedure.
It is recommended that you run the RNA on a bioanalyzer or similar. After that, we normally use QPCR and RNA-Seq to analyze the RNA. This method proved ideal for analyzing the kinetics of RNA processing.
The most hazardous steps are those using phenol and chloroform in the RNA extraction. Any time you use these chemicals in an unsealed container, do so in a fume hood, and wear appropriate protective clothing, a lab coat, and gloves. Take particular care that there are no zirconia beads in the thread of the screw cap tube, as that will lead to a leak.
