This method can help us answer key questions in the field of mRNA biology such as, whether in silico predicted mRNA target can directly interact with a particular cellular microRNA. The main advantage of this method is that it utilizes biotinylated mimics to pull down the target mRNA. And these biotinylated mimics have locked nucleic acids or LNA based mimic chemistry which helps in formation of very stable oligonucleotides with a very high affinity that can enhance the specificity of the reaction.
The implications of this methodology is that it can extrain from understanding the biology of the microRNA to microRNA-based therapeutics. This is mainly because cause we all know microRNAs play critical roles in every aspect of cellular biology. So by identifying the targets of the microRNAs, those are the mRNAs, we can certainly provide critical knowledge that would be important for discovery and therapeutics.
Demonstrating the protocol will be Sabyasachi Dash, a graduate student from our laboratory. To coat the magnetic beads with streptavidin, first, vortex the beads thoroughly to resuspend. Then, transfer 30 microliters of the resuspended beads in a two millimeter nuclease-free microfuge tube.
Leave the tube filled with the magnetic bead suspension on the magnet for two minutes. After two minutes, remove the supernatant with the help of a micropipet when the beads are drawn to the side of the tube in contact with the magnet. Then, add 100 microliters of wash buffer to the beads.
After adding the wash buffer, remove the tube from the magnet. Then, vortex the beads for 15 seconds to wash them. Next, transfer the tube containing the magnetic beads on the magnet for two minutes.
After the incubation is over, pipet out the supernatant and discard it. Then, add 100 microliters of RNase freeing solution to the beads. Vortex the beads for 15 seconds to mix the RNase solution.
Then, incubate the beads at room temperature for five minutes. After five minutes of incubation, transfer the tube containing the magnetic beads on the magnet for two minutes. After the incubation, pipet out the supernatant and discard it.
Next, add 100 microliters of resuspension solution to the beads, and vortex for 15 seconds. After vortexing, incubate the tube at room temperature for five minutes. After five minutes, transfer the tube with the magnetic beads on the magnet for two minutes.
After two minutes, pipet out the supernatant and discard it. Next, add 200 microliters of bead blocking solution to the beads. To resuspend the beads in the blocking solution, vortex it thoroughly at room temperature for 15 seconds.
Then, leave the tube on a multi tube rotator at four degrees Celsius for 16 hours. To prepare the lysate, use a cell scraper and scrape the transfected HEK293T cells under the laminar hood. Then, transfer the cell suspension to a two milliliter microfuge tube.
Next, subject the cell suspension to centrifugation at 1, 500g for five minutes. Post-centrifugation, dissolve the cell pellet in 1x phosphate buffered saline maintained at pH 7.2. Repeat the process of centrifugation, to obtain a residual medium-free pellet.
After the centrifugation is over, quickly transfer the pellet on ice. Next, prepare fresh cell lysis buffer. After preparing the buffer, add 260 microliters of complete cell lysis buffer to the sample in the microfuge tube.
Pipet the cell pellet several times for homogenous suspension. To lyse the cells, incubate the tubes at 80 degrees Celsius for 10 to 15 minutes. Then, leave the cells on ice.
Repeat the centrifugation process at 16, 000g for five minutes in a benchtop centrifuge at four degrees Celsius. After the centrifugation, transfer the supernatant containing lysate in a sterile 1.7 milliliter microfuge tube on ice and expel the pellet. To the supernatant, add five molar sodium chloride to achieve a final concentration of one molar.
First, prepare the pull-down wash buffer. After overnight incubation post bead blocking, transfer the tube containing the magnetic beads on the magnet for two minutes. Discard the supernatant after the incubation period.
To the beads, add 150 microliters of ice-cold pull-down wash buffer. Then, vortex the beads at room temperature for 15 seconds. And immediately incubate at room temperature for 30 to 60 seconds.
After a minute, transfer the tube containing the magnetic beads on the magnet for two minutes. Discard the supernatant after the incubation period. Then, resuspend the beads in 300 microliters of complete pull-down wash buffer.
After adding sodium chloride, transfer 300 microliters of the cell lysate to a microfuge tube filled with 300 microliters of beads. Then, leave the mixture on the nutating mixer for an hour at room temperature. After an hour, transfer the tube on the magnet for five minutes.
Discard the supernatant after the incubation period. Next, add 300 microliters of ice-cold complete pull-down wash buffer to the beads. After the buffer is added, vortex the beads for 15 seconds at room temperature.
Then, place the tube on the magnet for five minutes. Discard the supernatant after the incubation period. Then, dissolve the beads in 100 microliters of nuclease-free water, and store on ice.
Isolate the total RNA from the beads. Then, dissolve the RNA isolated in 25 microliters of nuclease-free water. Next, measure the concentration of RNA in the spectrophotometer.
Finally, prepare the working stock solution of RNA at 50 nanograms per microliter. Next, use 50 nanograms of the total isolated RNA to synthesize complimentary DNA following the manufacturer's instructions. Then, add oligo primers to a final volume of 20 microliters.
Place the PCR tube on the thermocycler and start the run. After the qPCR is complete, transfer nine microliters of the PCR reaction mix in each well of the plate. To each well, add one microliter of complimentary DNA to achieve a final volume of 10 microliters.
Next, use heat resistant PCR plate sealer to seal the PCR plate and load on the thermocycler. Then, start the program on the thermocycler. In order to validate the targets of miR-125b, the expression levels of target mRNAs of PARP-1 and p53 is studied.
Interestingly, the expression levels of PARP-1 and p53 mRNAs are comparatively higher than the Actin mRNA, which is the negative control. Other negative controls used in the experiment are no template, and three prime biotinylated scrambled sequences. Additionally, the three prime untranslated region of PARP-1 and p53 quantified, also show varying levels of amplification.
This is in comparison to the negative controls, such as Actin, no template, and three prime biotinylated scrambled sequences. This amplification of PARP-1 and p53 indicates that these are strong targets of cellular miR-125b. Once mastered, this technique starting with the plating of cells, to transfection of the biotin-tagged LNA mimics and concluding with the PCR analysis can be done within a week, if performed properly.
After watching this video you should have a good understanding of how to validate the putative mRNA targets in mammalian cells. This can be achieved with high specificity to low noise ratio by introducing the three prime biotin-tagged locked nucleic acid mimics based transfection in the mammalian cells. Don't forget that working with RNA, especially small RNA, such as micro RNAs, can be highly sensitive and prone to degradation.
Therefore, necessary precautions should be taken while performing this assay, such as usage of nuclease-free water, nuclease-free microfuge tubes, and sterile buffers, and freshly prepared buffers and reagents should be considered while performing this assay to get better sensitivity and better results.
