For a clear understanding of the purpose for this selection, please reference the abstract and proposal, found here.
Here is my final report, detailing the progress of my selection for this semester.
One round of N35 RNA bead-based selection has been performed against Tau, a microtubule-associated protein linked to symptoms unique to Alzheimer’s disease.
Round One Conditions:
Target: Tau Microtubule Associated Protein (6X-His expressed protein)
Beads: Nickel NTA
Pool: N35 RNA*
Pool:Target Ratio: 200:200 pmol
Buffer: 10X PBS (pH 7.4)
Incubation Time/ Temperature: 25 degrees Celsius, 25 minutes
Wash Number/Volume: 3 washes, 1 volume (100 ul)
*Note that N34 RNA was used for the first attempt at selection, but due to a positive reading for the no-template control during Cycle Course PCR, the starting Round was repeated using the N35 pool to avoid artifact interference. See Figure 1 and Problems Encountered for more detail.
Progress, Results, and Discussion
A Nickel bead-based selection was performed against Tau protein because of its classification as a 6X-His expressed protein. The protein was previously stored in 10X PBS buffer such that it was already sequestered into 200 pmol aliquots. In preparation for the start of selection, Ni-NTA beads were washed, and incubated with Tau protein target for thirty minutes at 25 degrees Celsius in 400 ul 1X PBS solution. The supernatant was decanted, and the beads were washed three times with 400 ul of the 1X PBS Solution. While the protein was incubating, N35 RNA was denatured at 65 degrees Celsius for five minutes, and cooled down to 25 degrees Celsius, thereby removing tertiary structures. The beads were exposed to the RNA pool, where Tau protein, Nickel beads, and the N35 RNA pool was incubated for 25 minutes at 25 degrees Celsius as described initially in the round conditions above. Supernatant was removed to represent the wash 0 binders. The beads were then washed in 100 ul 1X Binding Buffer for three more times, and the supernatant was removed in separate quantities. The bound species was then eluted from the beads using 400 ul of 80 degrees Celsius diH2O. This step was repeated two more times, each time removing supernatant to be used later. Water was added to each wash volume to reach 400 ul, and ethanol precipitation was performed to concentrate the RNA. Wash 0, Wash 3, and the Eluted reactions were precipitated and re-suspended in 10 ul diH2O.
Reverse transcription was carried out with 7 ul of the resuspended RNA, 20 uM of N35 reverse primer, and 500 uM of dNTPs. After heat denaturing the reaction for 5 minutes at 65°C, 1X First Strand Buffer, 10 mM DTT, and 1 ul of SuperScript II Reverse Transcriptase enzyme was added. The final solution was subjected to 42°C for 50 minutes, and 70°C for 15 minutes, providing optimal conditions for Reverse transcription.
PCR reactions allowed for the amplification of the N35 pool. Cycle course PCR reactions were prepared, using 1X PCR buffer (lab stock), 200 uM dNTP, 400 nM N35 T7 forward primer, 400 nM of N35 reverse primer, 2 ul of the ssDNA from reverse transcription, and 2.5 U of Taq DNA polymerase. As Figure Two exemplifies, the optimal number of cycles for PCR was 15 cycles. Recall the temperature conditions per cycle of PCR involves initial exposure at 94°C for two minutes, denaturing at 92°C for 45 seconds, annealing at 54 degrees Celsius for 45 seconds, and elongation at 72°C for 60 seconds. A large scale PCR reaction was carried out for 15 cycles with 1X PCR buffer (lab stock), 200 uM dNTP, 500 nM N35 T7 forward primer, 500 nM N35 reverse primer, 2 ul of the ssDNA obtained from reverse transcription, and 2.5 U of Taq DNA polymerase. Ethanol precipitation concentrated the PCR product. A transcription reaction was prepared using 1X Ampliscribe Transcription Buffer, 10 mM DTT, 7.5 mM of each NTP, 5 ul of dsDNA from lsPCR, and 2 ul of T7 enzyme solution. The reaction was incubated at 42°C for approximately 3 hours before adding 1 ul DNase I at 37°C. After pouring, loading, and visualizing the PAGE gel with the RNA product from Transcription, RNA was visualized and eluted from the gel using the RNA Crashing Method.
Problems Enountered
As mentioned earlier, the original Cycle Course PCR gel showed a positive “No Template Control”. This implies that there is an artifact within one of the reagents, possibly 10X PCR Buffer, or one of the primers specific to the N34 pool. It was decided to restart the round using the N35 RNA pool instead to prevent the risk of contamination.
Additional Work
The RNA crashing method was used to recover the RNA from the PAGE gel and elute it in a concentrated solution suitable for starting the next round of selection. This new technique was introduced during the summer and mastered this semester. In addition, I learned how to create fresh 3.8% Agarose gel for the benefit of the entire stream. A significant amount of time was spent making personal aliquots of primers, deionized water, and a stock solution of 10X TE.
Conclusion and Future Steps
As shown below in Table 1, the initial concentration of RNA was 3679.4 ng/ul. Such a large concentration indicates the presence of background binders interfering with the beads of the tube’s surface. To confirm that this concentration was valid, the concentration of diluted RNA was recorded. This value (280.6 ng/ul) was used to calculate a final concentration of 109.9 uM, confirming that conditions for the next round will need to be altered to allow for more stringent binding. For Round 2 of selection, the wash volume will be doubled to remove any non-competitive binders . If successful, then the concentration of RNA yielded from the previous round will be significantly higher, proving the background binders were eliminated.
| Table 1: Current Progress for Tau Aptamer Bead Based Selection with N35 Pool | |||||
| Round | LsPCR Cycles | Quantity of NA used | Wash # and volume | Concentration (ng/ul) | Concentration (recovered in uM) |
| 1 | 12 | 200pmol | 3w/1v | 3679.4 ng/ul | 109.9 uM |
| 2 | Pending | 400pmol | 3w/2v | Pending | Pending |
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