The overall goal of this RNA immunoprecipitation technique is to selectively isolate and characterize endogenous RNA protein complexes formed in eukaryotic cells via a specific oligonucleotide directed enrichment. This method can help answer key questions in the post-transcriptional control field, such as the composition of RNA binding proteins that define the five prime UTR of a retroviral transcript to regulate its gene expression. The main advantage of this technique is that it allows a researcher to selectively isolate and characterize a specific RNA protein complex of interest while maintaining the possibility to conduct a more global analysis of the RNP downstream.
Well the implications of this technique extend toward fundamental understanding of viral infections and numerous cellular diseases. And provides significant insight into the mechanisms of post-transcriptional control of gene expression. That could mean new viable drug targets.
After culturing cells and carrying out immunoprecipitation of a FLAG-tagged protein according to the text protocol, transfer 60 microliters per IP of pro-TG magnetic bead slurry to a 1.7 milliliter microcentrifuge tube. Place the tube on a magnetic rack and remove the storage solution by carefully pipetting. Then remove the tube from the rack.
Add 600 microliters of 1x wash buffer. And place the tube on an end over end rotator at room temperature for three minutes, to wash and equilibrate the beads. Place the tube on the magnetic rack and remove the wash buffer.
Then add 10 volumes of fresh 1x wash buffer. Add immunoprecipitating FLAG antibody to the equilibrated pro-TG magnetic beads. And then rotate the tube at room temperature for at least 30 minutes, to conjugate the immunoprecipitating antibody.
Place the tube on the magnet and remove the supernatant. Then remove the tube from the magnet and add 1x wash buffer to the beads and rotate at room temperature for three minutes. Repeat the wash step twice and remove the final buffer.
Remove the culture medium from the cells by aspiration and use 1.5 milliliters of ice-cold PBS to wash the cells twice. With a cell scraper dislodge the wet, adherent cells. Pipette the cell suspension into fresh tubes and then centrifuge the culture at 226 times G in four degrees celsius, for four minutes.
Add 375 microliters of ice cold low salt buffer to the cell pellet, and incubate the sample on ice for five minutes to allow swelling. It is important to take care when adding and re-suspending the cell pellet in the low salt lysis buffer, to ensure complete submersion and swelling without premature mechanical disruption. To collect the cytoplasmic cell lysate, add 125 microliters of ice cold lysis buffer, and then use a pre-chilled Dounce homogenizer to perform ten strokes.
Dounce homogenize with enough force to enable plasma membrane lysis and contaminants of the released cytoplasmic lysate. But take care to not overextend the mechanical disruption and risk nuclear contaminations, disruption of RNA protein complex, or loss of released Spin the homogenate in a microfuge at top speed for one minute. Then transfer the supernatant to a new 1.7 milliliter tube that has been on ice.
Determine the total protein concentration. Reserve 10%for western blot analysis, to be used as input control. To carry out immunoprecipitation, add the desired volume of harvested cell lysate to the target antibody conjugated beads.
Using 1x wash buffer, bring the total volume up to 600 microliters and rotate the sample at room temperature. After 90 minutes of incubation, place the IP tube on the magnet rack to collect the beads. Then collect the supernatant and reserve it as flow through.
Next, add 10 volumes of ice cold NETN 150 wash buffer to the RNP bound, antibody conjugated beads, and rotate the sample at room temperature for three minutes. After re-suspending the immobilized RNP complexes in 1x binding buffer according to the text protocol, adjust the remaining volume in 100 microliters of ice cold 1x binding buffer. And heat the tube at 70 degrees Celsius for three minutes.
To isolate cognate RNA protein complexes, add an approximately 30 nucleotide DNA oligo, complementary to the three prime sequence boundary of the RNA of interest. And incubate for 30 minutes at room temperature with gentle rocking. Add five to ten units or RNase H to the tube and incubate the sample at room temperature for one hour to cleave the RNA from the RNA DNA hybrid.
Then transfer the supernatant containing the captured RNP complexes of interest to a sterile 1.7 milliliter microcentrifuge tube. To collect the immunoprecipitated RNA, re-suspend half of the total sample of captured RNA complexes in 750 microliters of acid guanidinium thiocyanate reagent. Add 200 microliters of chloroform to the tube, shake vigorously for 10 seconds an incubate at room temperature for three minutes.
After centrifugation, collect the aqueous phase in a fresh tube, and add an equal volume of isopropanol. Mix well and incubate the sample at room temperature for at least ten minutes. Add one microliter of glycol blue to the sample and store it at negative 20 degrees celsius for efficient precipitation or processing at a future date.
Centrifuge the tube at 16, 000 times G in four degrees Celsius for ten minutes. The use a P200 micropipette to carefully collect and discard the supernatant so as not to disturb the RNA pellet. Add 500 microliters of 75%ethanol to each tube.
Vortex and centrifuge the tubes for five minutes. Carefully collect and discard the supernatant as just demonstrated. Air dry the pellet for two to three minutes and use 100 microliters of RNase free water to re-suspend it.
Apply 100 microliters of the RNA sample to an RNA clean up column. And process it according to the manufacturers protocol. Finally, elute the RNA in 30 microliters of RNase free water and store it at negative 80 degrees Celsius for up to three months.
To isolate post-transcriptional control elements, or PCE, GAG, RNA, RHA ribonucleoprotein complexes formed in proliferating cells. FLAG-MS2 RNP immunoprecipitation was carried out in transfected HECK293 cell lysates. The results show efficient precipitation of the FLAG-MS2 protein.
As DHX9/RHA is found in the RNP complex and not within the IgG isotope control, nor within the negative control. Shown here are western blots from RNase H digested RNA DNA complexes. RNase H cleaved PCE GAG RNA, releasing the RNP complex specifically bound to the five prime UTR.
The DHX9/RHA associated RNPs were enriched in the eluents. Importantly, the FLAG-MS2 bound RNPs remained with the antibody conjugated beads. In this experiment, HO immunoprecipitation of the MS2 stem loop, containing retroviral PCE GAG RNA in cells and in eluents, was validated by RT-PCR and QRT-PCR.
The results confirmed a select association between DHX9/RHA and the retroviral five prime UTR, which has been highlighted as a unique RNP, important for targeted translation control. Once mastered, this technique can be done in approximately five hours, if it is performed properly. While attempting this procedure, it's important to remember to perform all steps efficiently and with care, to ensure maintenance of intact RNP complexes and the isolation of the specific RNP complex of interest.
Following this procedure, other methods like mass spectrometry and RNA-Seq, can be performed in order to answer additional questions, like global protein association with the target RNA. And region or complete analysis of all transcripts engaged within a particular RNP. After its development, this technique paved the way for researcher in the field of post-transcriptional gene control to explore the distinct RNA protein complexes governing pathogenic viral gene expiration and general mechanism of cellular gene expression control.
After watching this video, you should have a good understanding of how to harvest intact cellular RNP complexes and determine their composition using selective oligonucleotide directed enrichment. Do not forget too, working with pathogenic viruses and biological samples and hazardous chemicals like acid or phenol, can be extremely hazardous. All precautions, such as appropriate personal protective equipment should always be taken while performing this procedure.
