Progress Report Number 1
October 18, 2011
Aptamer Stream Fall 2011
Pool N34, Glucagon
Progress, Results and Discussion
Glucagon is found in the -80 degree Celsius freezer prior to my research proposal. There are plenty of 100 ul aliquots of Glucagon in the freezer and that leaves this experiment with much room for error given conservative and strict practices. The proposal for this research experiment can be found here along with a link to the abstract here .
Practice rounds are complete and hence selection for Glucagon can begin. October 10, 2011 marks the beginning of Glucagon selection via RNA pool N34. This day began with the much awaited creation of HEPES buffer solution at 10x and then the dilution of this buffer to 1x. Diluting solutions was good to learn again because the pH of HEPES at 7.4 may need to be increased to pH 7.6 via findings in the proposal for Glucagon use. This experiment will continue to use HEPES at pH 7.4 until three rounds produce some sort of significant result. Many conditions in the original bead based selection protocol were changed in order to accommodate Glucagon. Glucagon was immobilized using strep beads within the target immobilization. During target immobilization, 1xPBS was also created to fulfill a total of three bead washes.
Moving on, binding and selection was a strong step moving forward because the incubation times for the target was changed to 45 minutes instead of 25 minutes. Since this is ultimately an experimental design, many incubation times where doubled in order to assure specific binding. Figure 1 shows the current design of this experiment with respect to the initial round being label round 0 all the way to the 3rd and final round. Respectively, W1, W2, and W3 were created after the washes and all three were eluted immediately with RNA N34. During precipitation W0, W3, and E1 were dried with glycol-blue in or
der to make sure that the pellets were not only noticeable but also not harmed when removing supernatant. Reverse Transcription went well and the protocol for this s
tep was kept the same as in the bead based selection protocol.
Figure 1: This figure indicates the requirements for
each round of selection. These parameters and conditions will be used for the first rounds of selection. Once data is recovered and analyzed via the yield from each round, these parameters are willing to change and be modified.
During the cycle course two gels were made in order to accommodate the amount of aliquots that were being used per gel. Figure 2 shows the results of the gels that were made during the cycle course. Also to combat the idea that N34 RNA and its primers are contaminated, a negative control was set up for each cycle set after the first six cycles.
Figure2: This figure is combined from two separate gels that were made in order to leave space between each cycle set (ex. 6, 9, 12, 15, 20) and not cause confusion. There was not a Negative control for cycle 6 but all other cycle sets included a negative control. The Cycle course shows that the Negative controls did not show up any bands during the PRC. This indicates that the N34 Primers that were used were in fact not contaminated. As seen by the gel, there is a weak sign of amplification at cycle 9 W0 . There is also considerable amplification at cycle set 20 for W0, W3, and E1-1.
The N34 RNA pool has been a suspect to contamination via artifacts that are appearing in lab. Since this is the RNA pool that has been used for the first round of selection in this experiment, this causes concerns to future use of this RNA pool. No strong bands of any of the negative selections used for N34 have shown up therefore it will be continued to be used until otherwise told to do so. Due to a working environment that is shared with other labs, there have been considerable pauses in between my protocol steps that may have been too long. For example, waiting in line for a Thermocycler or needing a specific pipettor. Referring to pipettors, a great way to eliminate time gaps is by using a larger pipettor but this can cause imprecision. This refers to using an SL-10 instead of an SL-2 when trying to measure 1ul of RNA or some other substance that is required. Although this is considered a problem it is not significant enough to distinguish any work done so far because this only happens sometimes. These issues are indicated in my lab notebook when they occur. Also problems have occurred in relation to a faulty or wrong program used on the Thermocycler. This refers to running a cycle course for 20 cycles that only was programmed to go 11 cycles. This is a personal error that occurred when I did not check the specifics of a written program prior to running a cycle course. Last but not least, contamination is always a factor to worry about in lab and with the Flu season upon us, there is no doubt that sneezing in the freezer or on any other area in the lab is helpful towards keeping a clean lab environment. Therefore sneezing in a shoulder or covering your mouth in any general way should be practiced enthusiastically.
Conclusion and Future Work
Round 1 of selection for Glucagon is not complete therefore the most essential gateway procedure for future work is to finish the round and obtain results. Without a doubt, our lab’s protocol for magnetic bead based selection was not written for Glucagon selection and this creates a lot of personal caution when moving on to other steps. Time is not currently a major factor within this research. Slowly calculating buffer concentrations and other mixes within this experiment has proven successful so far but a major concern is precision and accuracy in order to go about this experiment with a true “plus or minus” trail of error conclusion. This experiment is constantly being revised and updated via new findings about the characteristics and physical makeup of Glucagon. Therefore personal lab work is done with much concern towards fulfilling goals that are set before entering lab.
The future of this research will not be limited to bead based selection due to the lack of information there is regarding Glucagon based aptamers. Currently a trial and error methodology is being used to find out how to manipulate Glucagon into some sort of future application with aptamers. After results from three rounds of selection, a Glucagon binding assay is required for comparison via the many structures Glucagon takes up when used in different environments. The next goal of this experiment is to formulate a specific binding assay protocol for use with Glucagon. The Proposal and abstract also needs much updating via new findings and changes that were made during the evaluation period of this experiment.