Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

The overall goal of this procedure is to identify transcriptional starting points and RNAs cleavage sites in vivo from bacteria using a fluorescent primer extension reaction. This is accomplished by first growing bacteria such as e coli or S aria, and isolating the RNA molecules. Next, the RNA is reverse transcribed using specific fluorescent primers and reverse transcriptase resulting in CD NA fragments.

Then DNA or plasmids are isolated from the bacterial cells, and a fluorescent Sanger sequencing ladder is created from the DNA template using a sequencing kit. Finally, the CDNA fragments and sequencing ladders are separated and simultaneously detected on a poly acrylamide gel using an automated gel sequencer and compared to one another. Ultimately, fluorescent primer extension is used to map the five prime ends of RNA molecules up to a one base resolution.

The main advantage of this technique over other methods such as race or radioactivity based primary extension is rapid identification of the five from ends of RNA molecules as the detection happens simultaneously during electrophoresis. The sensitivity is similar to red activity based primary extensions, but enables laboratory personnel to use this method without the need for rate activity radiation safety training. We have successfully used this fluorescent primary extension technique to investigate the RNAs components of toxin antitoxin systems.

Staphylococci demonstrating the procedure will be Christopher Schuster, a grad student from my laboratory. After preparing a high yield of RNA and designing primers according to the text protocol, carry out the primer extension reaction by first preheating the thermocycler to 95 degrees Celsius. For each RNA sample mix the following compounds in A PCR tube, denature the samples for one minute at 95 degrees Celsius.

Then place the tubes on ice for five minutes to hybridize the primers and the RNA. Next, set the PCR machine to 47 degrees Celsius. Then prepare the reverse transcription master mix as described here.

Add four microliters, a reverse transcription master mix to each hybridized RNA sample and incubate the tubes for one hour at 47 degrees Celsius. To stop the reaction, heat the samples to 95 degrees Celsius for two minutes. Then add six microliters of form amide loading dye and store overnight or up to two weeks in the dark at minus 20 degrees Celsius.

After preparing the genomic DNA or plasmids, according to the text protocol, assemble the Sanger sequencing reaction by mixing 12 microliters of about 10 to 15 micrograms of genomic DNA or 100 to 500 nanograms of plasmid DNA with one microliter of fluorescently labeled primer and one microliter of DMSO pipette. One microliter of each sequencing mix into separate pre-labeled PCR tubes. Then add three microliters of the D-N-A-D-M-S-O primer mix to each tube.

Set the PCR machine to the following program and run it after placing the samples in the machine. After the run, add six microliters of loading dye to the samples and then on ice. If using right away or at minus 20 degrees Celsius long-term using clean dust-free 25 centimeter plates, place 0.25 millimeter spacers on the rear glass plate and lower the notched glass plate on the top with the notched end of the glass plates and the rail entry pilots both facing upwards.

Attach the gel rails to both sides of the glass plates and lightly tighten the knobs To cast the gel. Combine the compounds listed here in a beaker with a stir bar and place on a magnetic stir plate. Immediately after adding a PS and teamed, use a 50 milliliter syringe to take up the acrylamide solution and place a 0.45 nanometer filter on the tip.

Place the sandwiched plates at a 10 to 20 degree angle and slowly dispense the gel solution between the glass plates while continuously moving the syringe tip from one side to the other. Stop dispensing the gel solution once it meets the bottom end of the glass plates. Using a bubble hook, remove any bubbles that may have formed.

Then slide the gel pocket spacer between the glass plates at the notched end, submerging it into the gel solution, and fixing it by attaching the casting plate lightly fasten the upper rail screws and allow the gel to set for one to two hours. Before removing the casting plate and pocket spacer, use distilled deionized water to clean the pocket of salt and gel residues to run the gel. Slide the buffer tank holder into the gel rails on the front glass plates and tighten the knobs.

Place the gel into the lower gel tank of the automated gel imager against the heating plate and fix by sliding the rail entry pilot into the apparatus brackets. Use one XTBE buffer to fill the upper and lower gel buffer chambers. Then close the lower buffer chamber and use the power cord to connect the upper buffer chamber to the power.

If salt is present in the gel pocket, use buffer to repeatedly pipette into the pocket to clean it. Then use the top buffer lid to close the top buffer tank chamber. After turning on the imager and computer and starting the base image IR data collection software according to the text protocol, use the settings listed here to set up the scanner control.

Next, pre-run the empty gel for 20 minutes by selecting voltage on and pressing enter. In the meantime, using a PCR machine, heat the sequencing ladder and the primer extension products to 90 degrees Celsius for two minutes Before cooling on ice, stop the electrophoresis, open the automated gel sequencer and remove the upper buffer tank lid. Then insert the shark tooth comb in between the glass plates and with the shark teeth.

Slightly pierce the gel pipette one to two microliters of the primer extension products or sequencing ladder reactions into each gel pocket. For any empty pockets. Use loading dye to fill them to prevent inconsistent running behavior, close the buffer tank and the door of the gel sequencer.

Then start the electrophoresis by selecting voltage on for the laser select scan status on and press enter. Once the region of interest has passed the laser, stop the electrophoresis as shown here. A primer extension reaction can be used to determine the transcriptional starting points or TSP of transcripts of interest and can help to deduce promoter regions.

In this example, the TSP of MPE mRNA was identified as G consistent with earlier published data. The minus 35 and minus 10 promoter elements were deduced to be T-T-G-T-A-A and T-A-G-A-C-T, and these motifs differ only to bases from each of their consensus sequences using primer extension to identify mRNA cleavage products. The RNA mapping of cleavage close to the START co on by the RNA Ybi SEC two is shown here when the RNAs is not present or inactive, no primer extension products are formed close to the start co on as a result of RNAs activity.

However, two strong bands appear just downstream of the start coat on this figure shows a failed primer extension experiment due to excess RNA in the reverse transcription reaction that prevents the identification of individual bands. This demonstrates that the total amount of template RNA must be adjusted to the abundance of the RNA of interest. After watching this video, you should have a good understanding on how to elucidate transcriptional starting points and RNAs cleavage sites in vivo using fluorescent primers and an automated gel sequencer with some practice.

This method is quick and easy to perform. Good luck with your experiments.