Progress Report 2 CXCL-1

Final Manuscript is available here

PROGRESS, RESULTS, DISCUSSION

Initial Conditions:

Target: CXCL-1

Pool: N34

Beads: Nickle-NTA

Wash volume: 3 washes, 2 volumes

Buffer: Tris SELEX

Incubation time and temperature: 25 mins at 37 degrees.

Ratio: 200:400 pmol

Round 1 cycle course determined the large scale to be run f or 9 cycles. From this cycle course, it was determined that stringency was to increase. Negative selection began in Round 2. The protein beads were incubated at 37˚ for thirty minutes, while the reaction containing the RNA incubated for twenty minutes. As well, the wash volume in Round 2 was doubled to 400µl.

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Problems were encountered during reverse transcription which were most likely the factor to the odd results from the cycle course. As a result, the cycle was restarted using all of the same changes as before.

Round 3 changes to stringency included increasing the individual RNA reaction incubation time by five minutes to twenty five minutes total. As well, the incubation of the RNA and protein combined decreased by six minutes to total 19. The last change to Round 3 included increasing the time between washes from two to four minutes, as per protocol. The cycle course for round 3 is shown below.

All the final results are shown below in the table:

	Cycle Course	Absorbance	Quantity of NA used	Quantity of NA recovered
Round 1	Cycle 9	19.729	400pmol	1728.1 ng/µL
Round 2	Cycle 9	13.314	400pmol	1232.7 ng/µL
Round 3	Cycle 12	20.174	400pmol	1807.4 ng/µL

PROBLEMS ENCOUNTERED

Several problems were encountered, most of which were due to human error. During round 2 the initial cycle course PCR amplified significantly later than the first round. This can be seen above in the first cycle course for round 2. This was most likely due to a problem encountered during the reverse transcription.All of the first set of components were added to reverse transcription reaction. However after the RNA denaturing step, when the Super Script II was about to be added, it became apparent there was only 1 µl remaining. The 1µl was added to the E1 reaction, while the W0 and W3 were left on ice for approximately 40 minutes before being able to react in the thermocycler. The 3.8% agarose gel illustrated amplification at cycle 20, 11 cycles later than the previous round. Although stringency was changed from round 1 to round 2, the difference was too significant. The round was restarted and yielded expected results.

FUTURE WORK

The purpose of this study was to select RNA against CXCL-1, in pursuit of creating a suitable aptamer through selection. Up until this point, only three rounds of selection have been completed, but have gone as per protocol. Conclusive results cannot be determined until more rounds of selection are performed. A binding assay will be the next step, once a sufficient amount of rounds of selection have been performed. This will determine how well the RNA has bound to this specific target.