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R50 Selection Against CD105/Endoglin - Progress Report #1 (10/18/11)
Progress, Results and Discussion
Dry protein CD105/Endoglin(1600pmol) was resuspended in PBS buffer to achieve desired working concentration of around 20uM. Aliquots consisted of 3X200pmol (10ul) and 10X100pmol (5ul). Binding reaction (100ul) was made with 400pmol R50 pool and 200pmol target protein. Filter-based selection was designated optimum partioning method for size of protein (61.2kDa).
Millipore nitrocellulose membrane filter (0.45um) was pre-washed with 100ul 1X PBS SELEX buffer and five total washes executed after addition of binding reaction. RNA was eluted with 400ul elution buffer (5M urea and 25mM EDTA buffer). Ethanol Precipitation of RNA was performed with 1/10th volume 3M NaOAc, 3ul glycoblue, 2.5 volumes 100% ethanol. Reverse transcription achieved with following conditions: incubate at 42°C for 50 minutes and heat inactivate the enzyme at 70°C for 15 minutes. The correct R50 T-7 Forward primer and R50 reverse primer were used to prepare the 100ul PCR reaction with 2ul ssDNA from reverse transcription which was performed on W0, W5, and E1. Reactions were cycled for 20 cycles under following conditions: initial at 94°C for 2 minutes, denature at 92°C for 45 seconds, anneal at 54°C for 45 seconds, elongate at 72°C for 60 seconds. Five ul aliquots were extracted during end of cycles as shown in Figure 1 in orange.
As seen in figure 1, 11 cycles was chosen to be performed during large scale PCR. Ethanol precipitation of PCR yielded dsDNA for subsequent transcription reaction. Five ul precipitated dsDNA was transcribed using the Ampliscribe Transcription Kit from Epicentre and incubated at 37°C for 16 hours. RNA was purified with 8% Polyacrylamide Gel Electrophoresis, 10% APS and TEMED. Denaturing bromophenol blue (2X) dye was used in a 1X TE TBE rig. Visualization of RNA shadow in gel through UV illuminator was very small and faint. Crush soak elution and precipitation was continued with the ssDNA PAGE band. Nanodrop specing revealed 78ng/ul or a concentration of 2.33uM. This concentration was too low to continue on to the next round and steps to address this issue are discussed in following section.
Initially, 400pmol of target protein was to be added to 400pmol (20ul) pool to begin selection. However the pool mixture RNA was not denatured before protein was added and the process had to be repeated. With only ~1400pmol left of protein, 200pmol (10ul) was used in consideration of minimal remaining amount. While adding protein (W0), no resistance against filter was observed unlike in pre-wash. The filter was then assumed to contain a hole, but selection was continued and stringency decrease noted. A no-template negative control failed to be performed, but will be in future gels.
Concentration of RNA after one round deemed insufficient to proceed. Possible reasons for low amounts were mainly attributed to small RNA shadow observed in PAGE. Failure during transcription posed as leading cause for low detection. Therefore an agarose gel was run to validate this hypothesis as seen in figure 2.
As seen in Figure 2, the dsDNA band was found to be around equal length to the 100bp. Therefore the problem occurred after large scale PCR and transcription needed to be duplicated with DNA from PCR. Also, the smudging around what should be a clearly defined band was noted and could indicate possible over amplification. Transcription was then repeated with careful precaution to use of reagents and alteration of Ampliscribe Kit then that of the previous used. It was then realized that 0.1M DTT had been used instead of 100mM thus explaining reason for low amplification during first transcription reaction. This new transcription reaction that used 100mM DTT was incubated for 16 hours. When placed in incubator no precipitate or evidence of a nucleotide crashing out was evident however when retrieved the next morning clear evidence of white precipitant was observed. DNA template was still removed and preparation of PAGE in hopes of a still successful transcription reaction.
Conclusion and Future Work
As of October 18th, 2011 one round of selection has been executed using the R50 pool against Endoglin/CD105. However, this round was not completely successful due to insufficient RNA concentration amount of 2.33uM. Error in amount of DTT was concluded to be reason for transcription failure and correct amounts were used in new reaction. PAGE on second transcription reaction will hopefully reveal successful concentrations of RNA to continue on to second round where a negative selection will also be performed. In the next reporting period I hope to accomplish a couple more rounds of selection.
Here is a link to my proposal: https://docs.google.com/document/d/1kBQJ-g00D5QQ3sC3XXkGz-pPoDAdWXKPS34uYDIXR3A/edit?hl=en_US
Link to abstract: http://aptamerstream.blogspot.com/2011/08/nucleic-acid-aptamer-selection-against_9456.html#links
Final Manuscript: https://docs.google.com/document/d/1DRo2m--Hr9V4lYq8MnboE8rvMq-Y5Hyni1zN0zEZ2GU/edit