How do we calculate expression level in a qRT-PCR in the case of transgenics (like expressing 'cry genes') where we do not have a calibrator control sample? The calibrator sample (Wild Type: WT or control) in such case does not have the target gene, and thus null expression of the target gene.
Can we calculate relative expression levels using the 2-ΔΔCT method of 'Livaka and Schmittgen 2001'. In the case of the 2-ΔΔCT method, how can we calculate the relative expression levels of transgenic events using WT as the calibrator, because the expression of the gene like 'Cry gene' in WT will be zero.
What can we use in place of calibrator sample?
Can you give a bit more of information?
Did you do a comparision between treated and untreated sample? If yes, you can calculate a fold-change between these samples.
You could use endogenous controls (constitutive expressed transcripts) just as in a regular qRT-PCR experiment and use that to compare expression between your transgenes. Need more details on your experiment to give better advice.
If I understand your experiment correctly...Your setup is simply not suitable for the comparison you suggest. However, you can firmly state that the Cry1Ac dCt value(s) you obtain in the transgene(s) prove that you have expression of your introduced gene and you can give ddCt expression level(s) relative to the endogenous Actin transcript dCt value(s). You can also make comparsisons betwen your transgenic plants to determine which one has the highest expression of the introduced Cry1Ac gene. The non-transgenic should have no detectable amount of the Cry1Ac (as it lacks the gene) and will function conveniently as a negative control in your setup (i.e non-transgenes should not be used as a calibrator in your case). Keep it simple.
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