RNA Aptamer Selection for Alpha-Synuclein: Progress Report #2

Progress, Results, and Discussion

Three rounds of selection for alpha-synuclein from theN34 RNA pool have been completed. These rounds were done under the same conditions using filter selection, HEPES SELEX as the selection buffer, and a starting pool to target ratio of 400pmol:400pmol. The binding reactions were washed three times with 100ul of binding buffer and incubated for 30 minutes at room temperature. Negative selection was started during round 3 and will continue to be used for subsequent rounds.

Round 2 took two weeks to complete. The cycle course gel (Fig 1) was successful and showed the best amplification for 9 and 12 cycles of PCR. For large scale PCR, 10 cycles were used. The DNA was transcribed into RNA and ran down a PAGE gel. The RNA successfully showed up on the PAGE gel (Fig 2), but ethanol precipitation of the elution had some problems. The gel was eluted with 3ml of 1X TE and .3M NaOAc so the ethanol precipitation reaction had to be split into eight 1.7 ml tubes. Since each of these small tubes only had a small amount of RNA, the pellets formed were very small and difficult to see. Also, while combing the samples, some RNA was probably left in the tubes and pipette filter. To prevent unnecessary loss of product, the ethanol precipitation was not split into different tubes for during the third round.

Round 3 also took about two weeks to complete. At the beginning of this round, a negative selection was performed in which RNA from round 2 and binding buffer were pulled through the filter without the target present. This procedure selects for RNA that binds to the filter instead of the actual target. This RNA stays with the filter, and the RNA and buffer that made it through the filter are used for the binding reaction with the target on a clean filter. The cycle course gel (Fig 1) showed the best amplification at 9 cycles of PCR, so 9 cycles were used for large scale PCR. As previously stated, the PAGE eluant was not split into separate, small tubes during ethanol precipitation during round 3. The bench top centrifuged had to be utilized to spin the reaction in a 15ml conical. Since this is a large volume to pull RNA through, it was difficult to get a pellet to form. The reaction was frozen longer, centrifuged repeatedly for longer periods of time, and more ethanol was added. These techniques were used until a pellet was finally produced. For round 4, the PAGE gel will be eluted with 1ml instead of 3ml. Hopefully, this will prevent the ethanol precipitation problems faced in the previous three rounds by increasing the RNA concentration.

Problems Encountered
The major, reoccurring problem in this project has been the ethanol precipitation of the RNA eluant from the PAGE gel. This issue, along with problem solving techniques used and changes made in each round, is discussed at length in the previous section and in the first progress report. Also, the UV light camera used to photograph agarose gels wasn’t working properly during round two. This is why the round 2 cycle course gel picture (Fig 1) is blurry. The camera and software used to analyze the image were fiddled around with until they finally worked. The 2X blue dye used to visual the RNA crashed out of solution. It is now being kept at room temperature instead of the freezer to prevent this.

Conclusion and Future Work
The final concentrations of RNA at the end of each round were 989.8 and 1124.7 ng/ul for rounds 2 and 3 respectively (Fig 3). Ten cycles of PCR were used for round 2, and 9 cycles were used for round 3. To prevent future ethanol precipitation problems, the PAGE gel chunk will be eluted with 1ml instead of 3ml. Round 4 is already under way and the results will be included in the final manuscript. By that time, I plan on completing at least one or two more rounds and possibly a binding assay.
`
Round Large Scale PCR Cycles Absorbance Quantity of NA Used Quantity of NA Recovered
1 20 16.245 400 pmol 41988 ng
2 10 11.962 400 pmol 29694 ng
3 9 13.031 400 pmol 33741 ng
4 20 Not completed 400 pmol Not completed
Figure 3. Data from each round of selection for asynuc from the N34 pool.