Oct 18, 2011
P75NGFR Target, N35 Pool RNA
Progress, Results, and Discussion:
A round one of practice selection against MBP using N70 RNA pool was completed on October 15, 2011. Due to numerous early setbacks, mostly involving failed reactions and gels which will be discussed later, selection against p75NGFR has not yet begun but will start within a few days. By performing this last practice round of selection, valuable experience and knowledge was gained which will translate to fewer problems on the actual selection against p75NGFR. Practice selection also resulted in desirable data which indicates that selection protocol is understood and properly performed. For example as shown in Figure 1, the following is a cycle course gel of R1 selection against MBP using N70 RNA pool:
Figure 1. Cycle Course Gel of Selection against MBP using N70 RNA Pool: The W0 has a band at 6 cycles, indicating that there was a lot of RNA in the unbound pool. W3 did not have a dark band until 12 cycles, meaning that there was not much RNA in the unbound pool. E1 has a dark band at 9 cycles and large scale PCR was performed with 8 cycles because, this being round one, there were a lot of background binders. The no template negative controls had no bands so there were no artifacts present.
The completed round one of practice selection against MBP using N70 RNA pool had a spec concentration of 564.9 ng/uL. Actual selection against p75NGFR using N34 RNA pool should also produce viable results.
Numerous setbacks were experienced during the practice round of selection. Practice was originally started with a large scale PCR reaction of MBP selected with N59 RNA pool which was accidently left in the -20°C instead of the -80°C over the summer. After two transcription reactions were run through a PAGE gel, no bands were present. A gel was performed on the large scale PCR and no bands were present so practice selection was restarted, this time using N70 RNA pool. The first cycle course gel for this new practice selection showed no bands for E1, indicating that most likely a component was not added to the E1 cycle course PCR reaction. Greater care was taken and the second cycle course gel was successful as shown in Figure 1. Three PAGE gels had to be made because the first two were unsuccessful. The first PAGE gel failed to polymerize correctly, most likely due to the fact that components were not added correctly or that the older APS that was used failed to promote polymerization. New APS was used to eliminate a potential cause of this problem. The second PAGE gel did not fill correctly when the 25 ml of reaction was added, but the transcription reaction sample was loaded anyways and there was an attempt to run the gel. Later, it was realized that the entire top of the gel must be there so that a seal could be made. The loaded transcription reaction was recovered as best as it could be and it was also realized that 25 ml did not fill correctly because incorrect spacers were used. It was previously unknown that there are different spacers and that these require 50 ml of reaction to fill the plates. The correct spacers were used and a functional PAGE gel was run. Although there were many problems in the practice selection which have prevented selection against p75NGFR from starting, valuable lessons were learned which will promote less setbacks in the upcoming selection.
Conclusion and Future Work:
Practice selection against MBP using N70 RNA pool was recently completed and actual selection against p75NGFR using N34 RNA pool will begin soon. This delay was due to the fact that many problems were encountered during the practice selection. Although the protein is large enough (95kDA) to perform filter-based selection, selection will be performed using beads because it involves familiar protocol. The protein needs to be biotinylated so this will be the beginning step of selection. One hundred pmol of p75NGFR will be used per round which is the equivalent of 9.5 ug/round at a cost of $59.85/round. The selection buffer that will be used is PBS because the information that comes with the protein requires resuspension in PBS and PBS is also a good buffer for aptamers with desired applications in the body. Incubation time and temperature for the first round will be 45 minutes at 37°C because more initial binding to the protein is desired and 37°C is body temperature. Three washes using 2 volumes of PBS will be performed and streptavidin beads will be used. N34 RNA pool will be used because it has had no artifact related problems and it is a smaller pool so binding to p75NGFR will be more specific. Two rounds of completed selection are desirable by the next progress report.
I have aliquoted 40ul samples streptavidin and made 3.8% agarose gel.
To see my abstract, click here.