We have improved the previously published RiboTag method to significantly reduce background. This makes application to complex models like a mutant system simpler from an analysis perspective and much less costly. Aside from experimental animal generation, the RiboTag method is considerably easier and more straightforward than other methods to analyze ribosome associated mRNAs.
Like all RNA experiments, stringent RNase free conditions are fundamental for success. If you have limited experience with RNA, check your conditions by first performing a basic RNA extraction then move on. Demonstrating the procedure will be Lauren Chukrallah, a graduate student from my laboratory.
To prepare homogenization buffer, add 2.5 milliliters of one molar tris at pH 7.4, five milliliters of one molar potassium chloride, 600 microliters of one molar magnesium chloride, and 500 microliters of MP40 to a 50 milliliter tube. Add diethyl pyrocarbonate-treated water to the tube to bring the volume to 50 milliliters and mix until all components are incorporated. To prepare high-salt wash buffer, add 2.5 milliliters of one molar tris at pH 7.4, 15 milliliters of one molar potassium chloride, 600 microliters of one molar magnesium chloride, and 500 microliters of MP40 to a 50 milliliter tube.
Bring it to the volume of 50 milliliters in diethyl pyrocarbonate-treated water and mix until all components are incorporated. Pre-weigh all sample tubes, record the weight, and pre-cool in liquid nitrogen. Pre-cool sterile mortar, pestle, and weighing spatula on dry ice.
Then on dry ice, use the pre-cooled sterile mortar and pestle to break flash frozen tissue samples into large pieces and grind it slowly into a fine powder. Using the pre-cooled spatula, scrape the powder from the mortar into a pre-cooled collection tube taking care to keep on dry ice whenever possible. Weigh the tube with the tissue powder.
Calculate the mass of tissue by subtracting initial weight of the tube from the weight of the tube with the sample in it. Record this value for later calculation of lysis buffer volumes. Prepare lysis buffer by adding into 10 milliliters of homogenization buffer dithiothreitol, RNase inhibitor, Cycloheximide, heparin, and one protease inhibitor tablet.
Mix until all components are incorporated and keep on ice until use. For every 100 milligrams of sample, add one milliliter of lysis buffer. With the same pipette used to add the lysis buffer, carefully pipette the resulting lysate up and down to fragment the cells and mix.
Continue pipetting for generally 25 to 30 strokes until the sample is no longer viscous. Set the samples on ice for 10 minutes to lyse. Then spin the samples in a pre-cooled centrifuge at four degrees Celsius for 10 minutes at 10, 000 times g.
A large loose cloudy pellet forms at the bottom of the tube. Being careful not to disturb the pellet, collect the lysate into a new tube and record the volume for each sample. To prevent sample degradation, ensure that the samples remain cool throughout the remaining protocol storing on ice or at four degrees Celsius whenever possible.
Now, calculate the required volume of magnetic beads coupled to the appropriate antibody binding protein, protein G.For one milliliter of lysate, use 375 microliters of protein G beads to reach 30 milligrams per milliliter. Place the bead solution in a 1.7 milliliter tube on a magnetic tube rack. Remove the bead solvent and add an equal volume of fresh lysis buffer to the beads.
On a benchtop tube rotator set to 20 RPM at four degrees Celsius, rotate the tube five minutes to wash. Place the tube on the magnetic tube rack and remove the wash buffer. After repeating the wash two more times, add lysis buffer at the original volume to equilibrate the beads.
Store at four degrees Celsius or on ice until use. Add 50 microliters of the equilibrated beads for every one milliliter of lysate and rotate at four degrees Celsius for one hour. Then place the tube on the magnet rack and collect the lysate into a fresh tube.
Discard the used beads. To the lysate, add 25 microliters of the equilibrated beads for every one milliliter and rotate at four degrees Celsius for one hour. Store the remaining equilibrated protein G beads at four degrees Celsius overnight.
In the morning, place the tube on the magnet rack and collect the lysate into a fresh tube. Discard the used beads. From the cleared lysate, retain 50 microliters as the sample input control.
Store at minus 80 degrees Celsius. For every one milliliter of the cleared lysate, add five micrograms of anti-HA antibody. To prevent sample loss, seal the tube cap with laboratory film and rotate the samples 16 to 18 hours at four degrees Celsius.
For every one milliliter of the cleared lysate, add 300 microliters of protein G beads. Reseal the tube cap with laboratory film and rotate for two hours at four degrees Celsius. Place the sample tube on the magnet rack to allow the beads to separate from the IPed lysate.
Pipette off flow-through lysate and discard, retaining the beads. Add 800 microliters of high-salt wash buffer to the beads. Place the tube on a rotator for 10 minutes at four degrees Celsius.
Place the sample tube on the magnet rack and allow the beads to separate from the wash. Remove and discard the wash. Add another 800 microliters of high-salt wash buffer to the beads.
Close the tube and allow it to rotate for five minutes at four degrees Celsius. Place the sample tube on the magnet rack and allow the beads to separate from the wash. Remove and discard the wash.
Repeat the wash once more. After the wash, add 3.5 microliters of 14.2 molar beta-mercaptoethanol to the beads and mix by vortexing for 15 seconds. Extract RNA using a commercial RNA purification kit according to the manufacturer's instructions.
Elute the sample in 30 microliters of RNase free water. Store the sample at minus 80 degrees Celsius. In this study, very little RNA was isolated from the samples lacking either Cre or Rpl22HA demonstrating the effectiveness of this protocol to reduce IP background and isolate genuine HA tagged ribosomal RNAs.
A series of antibodies in RNA isolation protocols were tested in Cre and Rpl22HA positive samples. These results demonstrate reagent selection can have a significant impact on IP efficiency. The effectiveness of the RiboTag system to isolate ribosome-associated RNA was examined.
For both wild type input and IP genotypes, the input concentration was significantly higher than the IP concentration indicating that there was more RNA in the input sample. In total RNA pools, RNA integrity number values were expected to be near 10 with a higher RIN correlated to higher inferred sample integrity and quality. While the IP samples had lower RIN than the inputs, the RINs were still within an acceptable range and were not dependent on sample genotype.
Aside from breeding and maintaining RNase free conditions, researchers should ensure they collect and keep each suggested sample. The input RNA especially is key for multiple types of downstream analyses. For our laboratory, we generally perform RNA sequencing after immunoprecipitation.
This allows us to query the entire transcript dome for ribosome association. Alternatives would include microarray analysis or quantitative RT-PCR. For us, this system allows us to identify changes in ribosome-associated RNAs with mutation of specific RNA binding proteins.
