If you recall, on my last post I had said that none of my experiments had really worked. Well, as of Friday the 13th, all of that changed...
In my final week in Dr. Dolan's lab, which also coincided with finals week (which wasn't a big deal since I only had one take home final to work on), I decided that it would be worthwhile to repeat my knockdown experiment one last time, except with a few changes: 1) shorter time point of 6 hours vs. 24 hours, 2) added the siRNA to the nucleofection 96-well plate before adding the cells+NFS mixture, 3) allowed the cells to rest for 10 minutes at room temperature in the hood before adding pre-warmed media after the nucleofection, and 4) pooled four wells per sample instead of two.
On top of changes to the protocol, my technique was solid on the RNA isolation, RT-PCR, and real-time PCR. My RNA yield was the best it'd been all quarter, and my real-time results were not only beautiful but more importantly were believable because the standard curves (for the standards and samples) had a slope of about -3.4 which is much closer to the ideal -3.3 that's expected for totally pure cDNA.
So what was the final result? At the highest concentrations tested (40 and 60 pmol), I saw an almost 80% decrease in CYP1B1 RNA for both cell lines after 6 hours of transfection. These results are very exciting since it shows that transfection is possible with these LCLs (otherwise considered a very difficult system in which to transfect siRNA) and that the vector system we've chosen (96-well, the particular program and NFS) are workable, if not necessarily optimal, choices.
The next step is to carefully note any and all the changes to the protocol I made (some of which were really just reverting back to what Amaxa, the supplier of the transfection stuff, recommended) and then explain those to others in the lab. Woohoo!
In my final week in Dr. Dolan's lab, which also coincided with finals week (which wasn't a big deal since I only had one take home final to work on), I decided that it would be worthwhile to repeat my knockdown experiment one last time, except with a few changes: 1) shorter time point of 6 hours vs. 24 hours, 2) added the siRNA to the nucleofection 96-well plate before adding the cells+NFS mixture, 3) allowed the cells to rest for 10 minutes at room temperature in the hood before adding pre-warmed media after the nucleofection, and 4) pooled four wells per sample instead of two.
On top of changes to the protocol, my technique was solid on the RNA isolation, RT-PCR, and real-time PCR. My RNA yield was the best it'd been all quarter, and my real-time results were not only beautiful but more importantly were believable because the standard curves (for the standards and samples) had a slope of about -3.4 which is much closer to the ideal -3.3 that's expected for totally pure cDNA.
So what was the final result? At the highest concentrations tested (40 and 60 pmol), I saw an almost 80% decrease in CYP1B1 RNA for both cell lines after 6 hours of transfection. These results are very exciting since it shows that transfection is possible with these LCLs (otherwise considered a very difficult system in which to transfect siRNA) and that the vector system we've chosen (96-well, the particular program and NFS) are workable, if not necessarily optimal, choices.
The next step is to carefully note any and all the changes to the protocol I made (some of which were really just reverting back to what Amaxa, the supplier of the transfection stuff, recommended) and then explain those to others in the lab. Woohoo!
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1 comment:
So, Friday the 13th has been very lucky for you!!! Good for you.
love mom
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