This method, CARIC, can help answer key questions in the field of post-transcription of gene recreation by providing a comprehensive census of RNA-binding proteins. The main advantage of CARIC is its ability to capture proteins bound to various kinds of RNAs such as mRNAs and non-coding RNAs. This method can be used in various types and the organism to study the RNA protein interaction at work.
First, culture HeLa cells in supplemented DMEM at 37 degrees Celsius in a five percent CO2 atmosphere. When the cells reach about 80 percent confluence replace the culture medium on each dish with 15 milliliters of prewarmed, fresh medium. Add 15 microliters of 100 millimolar EU to each dish to a final concentration of one millimolar.
For experimental and no UV control samples add 7.5 microliters of 100 millimolar four SU to each dish. Cover the dishes with foil and culture the cells for 16 hours using the previous conditions. After this, add half of the previous amount of EU and/or four SU to the appropriate plates and continue culturing for another two hours.
To begin in vivo cross-linking, remove the culture medium and wash each dish three times with PBS using five milliliters of PBS per wash. Remove the residual PBS as much as possible. For the experimental and no four SU control samples, place the dishes on ice with the lid removed.
Use a UV cross-linker to irradiate the cells with UV light at 365 nanometers and at two jules per centimeter squared. For the no UV control samples, place the dishes on ice and protect them from light. Next, add one milliliter of prelysis buffer to each dish.
Use a rubber cell lifter to scrape the cells and collect the prelysis suspension in a 15 milliliter tube. Add prelysis buffer to a tube containing the suspension from two dishes adjusting the total volume to six milliliters. Then add an equal volume of R lysis buffer.
Pass the cell lysate through a syringe with a narrow needle several times until the lysate is clear and homogeneous. Incubate the lysate at four degrees Celsius with gentle rotation for one hour. After this dilute the lysate in 20 volumes of dilution buffer and divide it into 15 milliliter fractions in 15 milliliter untrafiltration tubes, molecular cutoff of 10 kilodaltons.
Using a swinging bucket rotator, spin the tubes at 4000 times G and four degrees Celsius for about 15 minutes until each fraction is concentrated to a volume less than one milliliter. Add 14 milliliters of dilution buffer to each concentrated lysate fraction and repeat the concentration process. Then combine the fractions and concentrate them to a volume of six milliliters using the previously described concentration process.
First prepare the reaction mix as outlined in the text protocol. Add this reaction mix to the six milliliters of pre-cleared lysate and mix well. Add 162.5 microliters of reducing reagent and mix well.
Incubate at room temperature on an orbital shaker at 800 rpm for two hours. After this quench the reaction by adding five millimolar EDTA and incubating on the orbital shaker at room temperature for five minutes. Next, split the reaction mixture between two 50 milliliter tubes, each containing four volumes of pre-chilled methanol and incubate at negative 30 degrees Celsius for 30 minutes.
Centrifuge at 4000 times G and four degrees Celsius for 15 minutes. Discard the supernatant and add between one and two milliliters of pre-chilled methanol to the pellet. Pipet up and down to break up the pellet making sure that the pellet ends up completely suspended with no visible chunks.
Repeat the centrifugation and resuspension process twice. After this, carefully draw out the residual methanol as much as possible making sure not to disturb the pellet. Add 10 milliliters of reconstitution buffer and pipet up and down to dissolve the pellet.
Centrifuge at 4000 times G and four degrees Celsius for 10 minutes. Then transfer the supernatant to a new tube making sure to collect 20 microliters of the sample for quality control. Transfer the cleaned up and reconstituted sample to the prepared streptavidin agarose beads.
Incubate at four degrees Celsius with gentle rotation overnight. Spin down the beads at 4000 times G for five minutes. Transfer the supernatant to a new tube making sure to collect 20 microliters of the sample for quality control.
Wash the beads with 10 milliliters of wash buffer A.Incubate with gentle rotation at 12 rpm and at room temperature for 10 minutes. Then spin down the beads at 4000 times G for five minutes. Remove the supernatant and repeat the washing and centrifugation process once more.
Repeat this wash process for wash buffer B and wash buffer C as outlined in the text protocol. After this, wash the beads with 10 milliliters of 50 millimolar Tris hydrochloride. Spin the beads down at 4000 times G for five minutes.
Remove the supernatant and split the beads evenly between two 1.5 microcentrifuge tubes. To begin eluding the captured RNPs, add 400 microliters of prepared biotin elution buffer to 400 microliters of washed, settled beads. Incubate on an orbital shaker at 1500 rpm at room temperature for 20 minutes.
Then incubate on an orbital shaker with a heat block at 1500 rpm and 65 degrees Celsius for 10 minutes. Centrifuge the beads at 7800 times G for one minute and collect the eluded RNP. Add 400 microliters of fresh biotin elution buffer to the beads.
Repeat the incubation and centrifugation process to collect a second elute and combine the two elutes in a single 15 milliliter tube. Next, add three volumes of dilution buffer to the combined RNP elutes to decrease the concentration of SDS. Using a 0.5 milliliter ultra filtration tube, concentrate the sample to about 40 microliters as outlined in the text protocol.
Add 0.5 micrograms per microliter RNase A and incubate at 37 degrees Celsius for two hours to release RBPs from cross-linked RNase. Collect two microliters of RBPs for quality control. When optimizing CARIC protocols, quality control steps are critical.
Representative quality control of the click labeled samples is performed via in-gel fluorescence analyses. Only the doubly labeled sample shows a strong smear band at the high molecular weight which represents the signal of cross-linked RNPs. The RNP signal is abolished by omitting either the four SU or EU.It can also be abolished by digestion with RNase A.In some cases a strong smeared band is observed in the no four SU control sample.
This band represents the labeled uncross-linked RNase which will degrade during the heat innaturation in most cases. Quality control of the affinity poll dataficiency is assessed through western blot analysis which shows the biotin signals in the samples both before pulldown and after pulldown. A silver staining analysis of the captured RBPs reveals that for HeLa cells, the general total capture efficiency is between 0.05 and 0.1 percent of input proteins.
While attempting this procedure it's important to remember that the RNase are sensitive to ribonucleases. Keep the experimental environment as clean as possible to reduce degredation of RNase. Following this procedure, protein almake identification using mass spectrometry can be preformed to reveal the iron-bounding proteins.
After its development, the CARIC technique will pave the way for a more comprehensive understanding of the postal transcription OT regulation at work.
