October 18, 2011
Pool N35, RNA, Selection Against Her2
After completing a successful practice round of selection against MBP using the N58 pool, a filter based nucleic acid aptamer selection against Her2 was commenced. The target to RNA was mixed in a ratio of 100pmol Her2 to 400pmol N35 RNA. The target was incubated at 25°C for 25 minutes, and 1X PBS selection buffer was used for the selection process. During round one, the filter ripped during selection. The filter rip was not noticed until elution of the bound species. Though the filter had ripped and thus prevented more bound species from being eluted, the selection process proceeded. Ethanol precipitation was preformed, and the RNA pellets were re-suspended in 10uL diH20.
Reverse transcription was performed by adding 8.5 ul of re-suspended RNA pellet, 20uM reverse primer, and 500uM dNTPs. The reaction was heat denatured at 65°C for 5 minutes and let cool for 5 additional minutes. The reaction was completed by adding 4 ul 5X first strand buffer, 10mM DTT, and 1ul SuperScript II reverse transcription enzyme. The reaction was incubated for 50 minutes at 42°C and heated to 70°C for 15 minutes. The PCR reactions were made using 2 ul DNA from reverse transcription, 10 ul 10X PCR buffer, 200uM dNTPs, 400nM N35 reverse primer and forward primer, 2.5U Taq DNA polymerase and 78.5uL sterile diH20. The samples were collected after 6, 9, 12, 15, and 18 cycles of PCR. The conditions for PCR cycle course are as follows:
#1 Initial: 94°C for 2 minutes
#2 Denature: 92°C for 45 seconds
#3 Anneal: 54°C for 45 seconds
#4 Elongate: 72 °C for 60 seconds
Repeat steps #2-#4 17 times and chill to 4°C forever.
After cycle course PCR, the agarose gel showed clear amplification of an elution bands as well as the W0 bands. No bands were visible for W3, implying that the number of washed preformed were likely sufficient. Upon examination of the gel, 16 cycles of PCR were determined to be the proper amount for optimal amplification.
Figure 1: Figure one shows the round 1 samples from the N35 pool selection against Her2. E1, W0, and W3 were run after cycle course PCR in a 3.8% agarose gel.
Large scale PCR was run for 16 cycles followed by, ethanol precipitation and transcription. Transcription was performed by incubating 2 uL 10X transcription buffer, 10mM DTT, 7.5mM of ATP, CTP, UTP, GTP, 5uL of dsDNA from large scale PCR, 3 uL diH20, and 2 ul T7 enzyme solution overnight. One ul DNase solution was added to the transcription reaction for 15 minutes and the reaction was quenched by adding 1 volume (21ul) of 2X blue denaturing dye. The sample was incubated at 65°C for 3 minutes and run through a PAGE gel. The RNA eluted from the PAGE gel had a concentration of 2765.7 ng/uL. The large concentration indicates that the conditions should be more stringent, and a negative selection should be performed in the next round.
In round two, the target to RNA ratio was 100pmol Her2 to 100pmol N35 RNA. A negative selection was performed by running the buffer, H2O, and RNA solution through a filter 3 times before incubation. The round 2 cycle course gel produced a faint band for the elution, but a dark band for cycle 20 of W4. The presence of the band indicates that more washes should be preformed. During large scale PCR 18 cycles of PCR will be run in attempt to optimally amplify the RNA.
Figure 2: Figure 2 shows the E1, W1, and W4 samples from round 2 of selection. The strong band present in W4 after 20 cycles of PCR indicates that more washes should be preformed.
During the selection and elution process the filters were quite troublesome. The filters kept ripping when placed into the apparatus. The ripping of the filters adversely affects the selection by breaking the integrity of the filter. When the filter is not fully intact, the bound species cannot properly bind to the filter and will not be eluted. This results in a decreased number of quality binders in the final RNA product.
Conclusion and Future Work
Thus far, the N35 pool selection against Her2 has been reasonably successful. All gels run for the selection have worked and the first round yielded a high concentration of RNA. A negative selection will be performed for each future round. More washes will be performed for each round to wash away as many unbound species as possible. Ideally, the gels will ultimately have no bands or only faint bands visible for the sample of the last wash. For all future rounds, 100pmol of nucleic acid will be added to the binding reaction. The processes will be more stringent in attempts to keep as many strong binders as possible.
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