Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The overall goal of this procedure is to study the role of erythrocyte microRNAs in post transcriptional gene regulation of plasmodium falciparum transcripts. This method can help answer key questions in the malaria field, such as how RNA splicing events with host or parasite small RNAs can affect the translational potential of fusion mRNAs. The main advantage of this technique is the ability to capture total and small RNAs together in one pool, demonstrating the presence of small RNAs and fusion RNAs in one cell.

Additionally, this procedure utilizes polysome profiling to show how those small RNAs affect translation of their fusion mRNA products. To begin set up the transfection by centif 300 microliters of red blood cells in complete malaria medium at 800 times G for five minutes. Wash the erythrocytes twice with RPMI medium resuspend the cells in complete cyto mix at 50%hematocrit.

Next, transfer the cells to an electroporation vete and add 10 micrograms of DYS th biotin conjugated micro RNA or an unconjugated negative control micro RNA. To electroporated the cells, place the vete in an electroporated and deliver a single pulse. Then plate the cells and infect them with plasmodium falciparum after four hours as described in the text protocol.

Then add 50 microliters of packed, stripped avid and beads to 10 micrograms of parasite RNA in a microcenter tube, and incubate the tube with rotation for one hour at four degrees Celsius. Centrifuge the strip davan and beads at 800 times G for 30 seconds, and then wash the pellet with 500 microliters of RNP buffer containing a one to 1000 dilution of RNA inhibitor. Next elute, the RNA from the beads by Resus, suspending them in 200 microliters of RNA.

Capture elution buffer containing excess biotin. Incubate the beads overnight at four degrees Celsius with rotation. Then centrifuge the beads at 800 times G for 30 seconds and collect the supernatant RNA.

It is critical to elute with an excess of biotin and to use the minimum amount of strep avid and beads to ensure proper specific elution. This reduces the background RNA enrichment. Finally, determine the degree of enrichment of P falciparum transcripts and micro RNA enrichment by quantitative R-T-P-C-R as described in the text protocol to begin polysome separation.

First place five milliliters of a 50%sucrose solution into an ultracentrifuge tube. Then carefully layer five milliliters of a 15%sucrose solution over the 50%solution. Next, seal the gradient tube with perfil and carefully tilt the tube until it lies horizontally on the benchtop.

Make sure the tube is in a stable position to prevent rolling. Keep the tube in this position for at least two hours to allow the gradient to form. In the meantime, collect approximately 100 milliliters of an asynchronous culture of plasmodium infected blood at three to 5%persit.

Then add 10 milliliters of culture medium containing two millimolar cyclo, heide, and incubate at 37 degrees Celsius for 10 minutes. Next, centrifuge the cells at 500 times G for seven minutes and then wash them twice with 80 milliliters of PBS containing 200 micromolar cyclo heide resus. Suspend the pellet in PBS with cycloheximide and place it on ice.

Pellet the cells and remove the supernatant to estimate the pellet volume. Then lyce the cells in a final volume of 4.25 milliliters of lysis, buffer and incubate for 10 minutes at four degrees Celsius with rotation. Previous attempts at polysome profiling and malaria causing species revealed mostly monos because ponent lysis leads to the breakdown of polysome into mono zones.

Here we utilize a lysis buffer that lysis the erythrocyte and parasites simultaneously to preserve the polysome pattern of plasmodium falciparum, Transfer the lysate to micro centrifuge tubes and then spin at 16, 000 times G at four degrees Celsius for 10 minutes. In a separate pre chilled five milliliter ultracentrifuge tube, add 1.25 milliliters of cold 0.5 molar sucrose cushion solution using a syringe with a 27 gauge needle. Carefully layer 3.75 milliliters of the lysate supernatant above the sucrose cushion.

Centrifuge the sample at 366, 000 times G at four degrees Celsius for 146 minutes. During this time, slowly return the sucrose slayed ultracentrifuge tube to a vertical position and let it stand on ice for at least 15 minutes. After removing the lysate from the ultracentrifuge, carefully collect the supernatant in a 15 milliliter conical two.

Store the supernatant at minus 80 degrees Celsius to preserve the RNA fraction unbound by the ribosomes. Next, resuspend the ribosome pellet in 500 microliters of Resus suspension, buffer by pipetting for five minutes to mix centrifuge the sample at 16, 000 times G at four degrees Celsius for 10 minutes. To remove insoluble material carefully remove the param seal on the gradient tube to avoid disturbing the gradient, and then layer the ribosomal suspension on top with a syringe and a 27 gauge needle.

After centrifuging the tube at 200, 000 times G at four degrees Celsius for 180 minutes, store the gradients at four degrees Celsius until ready to load onto the fractionator. Place an empty ultracentrifuge tube into the gradient fractionator and wash the system with RNA free water for five minutes. During the wash, set the sensitivity of the UV absorbance detector at 254 nanometers to 0.2.

Although this may need to be adjusted depending on the signal. Next, set the baseline signal to zero as water flows through the detector. After washing the collector, reverse the fluid flow through the fractionator to empty the lines and then remove the empty ultracentrifuge tube.

Run a 60%sucrose solution through the FRACTIONATOR until it exits the needle apparatus. Then place the loaded gradient tube at the top of the loading chamber and tighten the seal. Pierce the bottom of the tube with the needle.

Then reset the flow speed of the fractionator to 12.5 by 10 and collect 18 second fractions in micro centrifuge tubes. Start the forward flow of the 60%sucrose solution to elute the fractions before the first drop of the gradient solution enters a micro centrifuge tube. Begin both the collection and the live recording of the absorbance at 254 nanometer signal.

When the absorbent signal drops sharply at the interface of 50%and 60%sucrose solution, stop the flow and the recording. Store the gradient fractions at minus 80 degrees Celsius. Then reverse the fluid flow until the 60%solution empties out of the ultracentrifuge tube.

Remove the tube and begin analysis of the next gradient. Transfection of red blood cells with microRNA 4 5 1, followed by recovery of the biotinylated micro NA mRNA hybrids revealed enrichment of PKAR fusion transcripts. Mock, or unrelated micro RNA transfection did not enrich the PKAR transcript.

The data show the elucian peaks of the 40 s and 60 s ribosomal subunits, the a DS ribosome and the polysome fractions containing the indicated number of ribosomes. The ribosomes were associated with the transcripts isolated from PCI residing inside red blood cells. Northern blood analysis demonstrated that the ribosomes and the polysome were associated with the 18 S and the 28 SRNA.

Along with this procedure, other methods like R nase H digestion, ribonuclease protection assays, and northern blotting can be performed in order to additionally validate the presence of microRNA MR.NA fusions. After watching this video, you should have a good understanding of how to transfect microRNAs into erythrocytes and subsequently capture small RNAs with total RNA, including chimeric transcripts. You should also be able to recover polysome to determine the ribosomal occupancy and hence the translational potential of these fusion transcripts.