If I'm doing a qPCR experiment where I need to determine the relative decrease in gene expression from RNAi lines that were generated by T-DNA insertion do I need to run my reference primers on each independent line? Or can I use the wild-type sample for all normalization? For example I will have 30 independent lines of tomato that each potentially have a different T-DNA insertion for our gene of interest and I will be comparing their expression to a pooled sample of wild-type tomato.
The reference gene/s is/are used as a loading control, accounting for differences in the amount of biological material, RNA extraction and RT efficiency. If you can make sure (by whatever means) that these stept all wore identically between your samples and lines then you won't need any loading control.
If not, you first need to find reference gene/s that is/are expressed constantly and at an indentical level in all your lines. This can be a very difficult task.
If you have such a reference gene(set) then you will need to use it as a loading control for each individual sample.
The reference gene/s is/are used as a loading control, accounting for differences in the amount of biological material, RNA extraction and RT efficiency. If you can make sure (by whatever means) that these stept all wore identically between your samples and lines then you won't need any loading control.
If not, you first need to find reference gene/s that is/are expressed constantly and at an indentical level in all your lines. This can be a very difficult task.
If you have such a reference gene(set) then you will need to use it as a loading control for each individual sample.
Jochen, that was what my gut was telling me. Thanks for the confirmation!
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