The psiCheck-2 vector contains two luciferase genes, renilla as the experimental reporter while firefly the control reporter. Renilla is the experimental reporter as you can clone in regulatory sequences in the 3' or even 5' UTR (via available R.E. sites); and the resulting luciferase activity values will be normalized to the ones obtained from firefly. Subsequently, the renilla/firefly values can be compared between the different experiment conditions. To understand if the changes/regulation observed is transcriptional or post transcriptional, we could do qPCR to look at the luciferase transcript levels.
For my case, I dont intend to clone in any regulatory sequences into the renilla luciferase. I would just like to use the psiCheck-2 vector as a reporter for the translational status of the cell, in response to a certain stimuli. qPCR would be done to check the luciferase transcript levels, if there are no changes in the transcript levels but there are changes in the luciferase activity (renilla/firefly stimuli vs renilla/firefly no stimuli), this would suggest a post transcriptional effect.
I have carried out the experiment and I observed that when I analyse the results, I see a drop in the translation status when I compare the renilla/firefly activity (qPCR transcript levels renilla/firefly no change). However, since I did not make any changes to either the renilla or firefly genes, technically I can also do the reverse by taking firefly/renilla values;meaning using the renilla as the control reporter. But when I do this, I get the opposite effect (an increase in translation status). What i interpret this as is the stimuli has an inhibitory effect on the translation of both renilla and firefly, but the effect is greater on renilla. thus renilla/firefly show decrease whereas firefly/renilla show increase. When I look at the individual raw values, I do see this between stimuli vs no stimuli. However, this might be subjective as without any normalization, it is hard to compare.
So my question is for the psiCheck-2 vector, is there anything unique about the firefly luciferase gene that it should always be used as the control? From the protocol, this is stated:
This firefly reporter cassette has been specifically designed to be an intraplasmid transfection normalization reporter; thus when using the psiCHECK™-2 Vector, the Renilla luciferase signal can be normalized to the firefly luciferase signal.
Any other advice to set this experiment up? Thanks.
Hi Jit,
Generally I do get higher luminescence values for Rluc as compared to firefly, roughly one to two folds higher. Example values for firefly ~1000 vs Rluc ~2000-3000.
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