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how to minimize the variation among the replicas


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4 replies to this topic

#1 rosepolaris

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Posted 09 November 2010 - 11:14 AM

I am new to this field :)

I have done transfection and luciferase assay a couple of times. Sometimes, I got huge variations among the replicas (three replicas). Although I may use Renilla to "solve" this problem. I really think I should improve my technique!

Would you share your tricks??



Thanks,
Xun

#2 microRNAboy

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Posted 22 November 2010 - 04:04 AM

Are you co-transfecting your reporter vector with an internal control (Renilla or Bgal) plasmid? You mention Renilla, but it is not clear if you are actually using it.

#3 rosepolaris

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Posted 23 November 2010 - 12:55 PM

Are you co-transfecting your reporter vector with an internal control (Renilla or Bgal) plasmid? You mention Renilla, but it is not clear if you are actually using it.



I know Renilla can normalize the data. But I want to do without it, in other words, to improve my technique. Sometimes I got about 100 fold difference within replicas. I don't understand how that would happen. I did everything extremely carefully.

#4 microRNAboy

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Posted 24 November 2010 - 12:08 AM


Are you co-transfecting your reporter vector with an internal control (Renilla or Bgal) plasmid? You mention Renilla, but it is not clear if you are actually using it.



I know Renilla can normalize the data. But I want to do without it, in other words, to improve my technique. Sometimes I got about 100 fold difference within replicas. I don't understand how that would happen. I did everything extremely carefully.


There probably isn't anything wrong with your technique. In my experience with reporter assays you need to use an internal control (Renilla or B-Gal plasmid). The reason you're getting such a big difference is due to different transfection efficiencies between the replicates. This will be due to how evenly the cells are plating down, which will have a knock-on effect to how many survive the transfection etc etc. The internal control will normalise this because it will correct for transfection efficiency. This is very important if you are planning to do experiments where the reporter expression will be altered. How will you know what is a real change compared to a change due to error?

I have done a lot of reporter assays and without using an internal control pretty much all of the data would have been useless!

#5 rosepolaris

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Posted 16 December 2010 - 08:33 PM

Thank you very much !




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