The overall goal of this procedure is to image single RNA transcripts in living cells in real time. This is accomplished by first hybridizing two oligonucleotides that were designed to form a hairpin forming probe known as a ratio metric by molecular beacon. The second step is to purify ratio metric by molecular beacons by size exclusion chromatography.
Next, the ratio metric by molecular beacons are introduced into cells expressing the engineered RNA target via micro porion. The final step is to see the cells into a microwell plate suitable for live cell microscopy. Ultimately, fluorescence microscopy is used to monitor the movements of individual RNA transcripts visualized as bright fluorescent spots within single cells.
The main advantage of this technique is that it utilizes synthetic probes that are bright photo stable, and exhibit a high signal to background, which opens up the possibility of imaging any endogenous RNA Begin the preparation of the ratio metric by molecular beacons or RBM BS by mixing the following reagents in a tube. 20 microliters of Rbm B one 30 microliters of Rbm B two six microliters of 10 x phosphate buffer, and four microliters of DNAs and RNAs free water. This is typically sufficient for approximately 50 studies.
Incubate the mixture at room temperature for 30 minutes. Meanwhile, prepare a liquid chromatography column for removing any UN hybridized RBM B two oligonucleotides. Use 75 prep grade super decks in an eight milliliter liquid chromatography column with an about four milliliter bed volume using a syringe pump running at a flow rate of 0.6 milliliters per minute.
Wash and equilibrate the SUEx with about 50 milliliters of one x phosphate buffer before all of the fluid enters the bed volume. Stop the syringe pump, detach it, and let the remaining liquid go through the column by gravity. Next, load the RBMB mixture onto the chromatography column.
After the RBMB sample has completely entered the bed slowly add another 250 microliters of one x phosphate buffer to the top of the bed to ensure that the entire sample has completely entered the column. Fill the column to the rim with one x phosphate buffer. Close the top and start the syringe pump.
The syringe is filled with about 50 milliliters of one XPBS and runs at a flow rate of 0.6 milliliters per minute. Typically, the RBMB sample can be readily visualized due to the color of the dye incorporated into the probe. Once the RBMB nears the bottom of the column, collect the flow through at two drops per micro centrifuge tube.
Stop collecting the sample. Once the color of the sample within the micro centrifuge tubes becomes clear, combine the tubes containing the colored RBMB sample, typically five to six tubes and load into a centrifugal filter device. Centrifuge the sample at 10, 000 RCF for 20 minutes or until the desired volume.
This speed and time will typically yield a final volume of about 30 microliters. In this protocol, a human fibro sarcoma cell line was engineered to express Q-F-P-R-N-A with 96 tandem repeats of the RBMB target sequence in the three prime UTR, the control cell line was engineered to express wild type Q-F-P-R-N-A one day before probe delivery plate cells in a T 25 flask at 40 to 50%co fluency culture. The cells with DMEM media supplemented with 1%pen strep and 10%fetal bovine serum and incubate at 37 degrees Celsius with 5%carbon dioxide on the following day.
Turn on the micro por and set up the micro portration parameters optimized for HT 10 80 cells. Fill the micro porion tube with four milliliters of electrolytic buffer and place it on the micro preparation station. Defrost a stock sample of purified RBMB and dilute several microliters to a final concentration of 12 micromolar with one x phosphate buffer.
One microliter is needed for each micro porion. For each micro porion reaction Pipette one milliliter of culture medium with FBS, but without antibiotics into a micro centrifuge tube. Set it aside near the micro porion device.
It will be used later to suspend the cells immediately after micro preparation. Antibiotics are not used because they would decrease the cell viability after micro preparation. The engineered HT 10 80 cells plated the previous day should now be 60 to 80%confluent.
Remove the cell culture media and wash the cells once with one milliliter of calcium and magnesium free DPBS incubate with one milliliter of trypsin for one to two minutes. Stop the trypsin by adding one milliliter of DMEM media supplemented with 10%fetal bovine serum without antibiotics and phenol red and transfer the cells into two 1.5 milliliter micro centrifuge tubes. Spin down the cells in the micro centrifuge tube at 200 times G for five minutes.
Remove the supernatant resuspend and combine the cell pellets in a final volume of one milliliter DPBS. Remove 10 microliters from the well mixed cell suspension to count the cells, pipette the cells up and down several times to make sure they are well dispersed and transfer 300, 000 cells with DPBS into a new 1.5 milliliter micro centrifuge tube. Spin down at 200 times G for five minutes.
Remove the supernatant being careful not to disturb the cell pellet. Resuspend the pellet in 11 microliters of Resus suspension, buffer and pipette up and down several times to ensure that the cells are well dispersed. Be sure not to generate any air bubbles.
Add one microliter of the diluted RBMB and mix well by pipetting up and down several times. Again, be careful not to generate any air bubbles. It's critical to avoid introducing any air bubble into the RBMB sales suspension because air bubbles will cause a spark during micro operation and result in poor RBMB delivery and Cell Deaths.
Using the micro preparation pipette aspirate 10 microliters of the RBMB cell mixture and insert the pipette into the micro preparation tube. Push the start button to start the micro corporation. There should be no visible air bubbles in the tip when the screen of the micro por shows completion.
Remove the pipette from the station and expel the 10 microliter mixture into the previously prepared micro centrifuge tube containing one milliliter of culture medium with FBS, but without antibiotics mixed gently by rocking the tube side to side several times. Spin the cells at 200 times G for five minutes. Wash the cells twice in one milliliter of phenol red free culture medium with FBS, but without antibiotics to remove any rbm BS that were not delivered into the cells.
After the second wash. Resus suspend the cells in 400 microliters of phenol red free culture medium with FBS, but without antibiotics. Plate the micro prorated cells into a previously prepared poly de lysine coated eight well chambered cover glass at 200 microliters per well or at the desired co fluency.
Place the chambered cover glass into a cell culture incubator. Imaging can be performed as soon as 30 minutes post micro preparation. To begin this procedure, turn on the live cell stage top incubation system and equilibrate it until it reaches 37 degrees Celsius, 5%carbon dioxide and 75%humidity.
Turn on the microscope and the fluorescent light source and open the metamorph image acquisition software. Apply immersal oil to the objective. Transfer the chambered cover glass with micro pored cells to the live cell stage.
Top incubation system. Incubate the stage top system until the temperature and carbon dioxide levels are stabilized. Open the acquire tab in the metamorph software.
Click the show live button to find the field. Adjust the focus under white light and click the stop live button. Under the acquire tab.
Click and open the stream acquisition popup button and set up the desired movie acquisition parameters. Acquire 150 frames using the SCI five CF six 40 R filter. Save the images to the hard drive.
Shown here are representative images of HT 10 80 cells stably expressing RNA with 96 tandem repeats, or four tandem repeats of the RBMB binding site in the three prime UTR. Following the intracellular delivery of mbs. The presence of 96 tandem repeats allows individual RNA transcripts to appear as bright fluorescent spots.
In contrast, RNA containing only four tandem repeats appear as very dim fluorescent spots and are often only detectable in areas of exceptionally low background. The acquisition of streaming images allows individual RNA transcripts to be imaged in real time. Individual RNA transcripts can readily be seen within the cytoplasm and nucleus of the cells undergoing brownian or sub diffusive movements.
In rare cases, an RNA undergoing directed transport will be observed as shown here. RNA transcripts undergoing directed transport typically move rapidly along a straight path. In this montage, the trajectory of the RNA transcript is shown overlaid over a single frame of the time series.
The directed RNA movements are consistent with the transport of RNA along microtubules and micro filaments. After watching this video, you should have a good understanding of how to prepare and purify racial metric by molecular beacons. Deliver racial metric by molecular beacons into cells via micro parion and image single RNA transcripts by fluorescent microscope.
