The first round of aptamer selection for alpha-synuclein (asynuc) from the n34 RNA pool was completed on October 5, 2010. Despite a few minor setbacks, the first round showed promising results, and the second round of selection is already under way. Being a novice to filter selection, I expected to have some problems at first, but I’m confident that the subsequent rounds will go faster and have fewer problems.
Cycle course PCR was used in order to find the optimum number of cycles for large scale PCR. PCR product was taken out at the end of the certain cycles, and then the products were run down a 3.8% agarose gel to separate them, Figure 1. As you can see, not even the positive control, washes from round 1 (Wr1), showed up in the gel. This indicates that amplification of the DNA during PCR was unsuccessful. This might be due to bad PCR reagents. To test whether or not the reagents used during cycle course PCR were working properly, a positive control with a DNA sample that had worked previously was performed. The control DNA, Wr1, and E1 were put through 9 cycles of PCR with the same reagents used in the previous cycle course PCR and then ran down a gel, Figure 2. The control DNA and Wr1 showed up, but E1 did not. This indicates the PCR reagents are working properly, but E1 hadn’t been amplified enough to show up in the gel yet.
Conclusion and Future Work
For the first round of RNA aptamer selection for asynuc, I got a final concentration of 1399.6 ng/ul of RNA from the n34 pool. I learned a lot about filter selection during this round and I also learned some problem solving techniques for cycle course PCR and ethanol precipitation. My first cycle course PCR yielded an unsuccessful gel. This problem was correct by repeating the cycle course after a control verified that the PCR reagents were working properly. By the next progress report I plan on completing at least 2-3 more rounds of selection.