I have came across with some papers where they have queried some mutant strains by qRT-PCR for gene expression study.
However, i am not clear how to do that when they mean “normalized to N2 OP50” for gene transcript level comparison.
I have a real case here,
where there is a drug that could induce gene “B” in N2 on OP50.
I would like to investigate if the compound still can induce gene B in daf-16 mutant background.
Does that mean that,
during Δ (ΔCt) = Δ treatment - Δ control calculation,
in my case it should be,
Δ (ΔCt) = Δ treatment of daf-16 on OP50 - Δ control of N2 on OP50 ?
Thus it means the normalization to N2 on OP50?
If Δ (ΔCt) = Δ treatment of daf-16 on OP50 - Δ control of daf-16 on OP50,
it just simple mean a normalization to own group of daf-16 but didnt give me any info on the transcript level in comparison to N2, right?
Hope if anyone could help to clear my doubts… Thanks…
Merry Christmas to Worm Community members too
As I read the paper (and with the caveat that I’ve not done qPCR, and don’t know how they did these calculations), they normalized within each sample to their preferred housekeeping genes to determine the relative abundance of the gene in question within the sample, and then normalized the resulting relative-abundance value to the relative abundance of the same gene in N2 grown on OP50. Thus, for each gene on which they’ve done qPCR, the sample from N2 on OP50 has a normalized relative-abundance of 1, and the other samples are some fold more or less than the relative abundance of that gene in N2 grown on OP50.
So: I’m pretty sure I understand what they’re showing me, if not necessarily how they’ve done it. But I’ve tried several times to understand your question, and I’m still lost.
you need the following info I guess (if I understand you question correctly…);
First of all you would need to perform an experiment to determine transcript level(s) in your REFERENCE strain (N2) with no treatment, this value would be used as a baseline and set to 1.
a. the transcript level(s) of the gene(s) of interest (corrected for inter-sample variation using invariant housekeeping genes) in N2[no treatment], NORMALISED to 1.
Then you need to determine the transcript level(s) in your mutant under the same conditions,
b. the transcript level(s) of the gene(s) of interest (corrected for inter-sample variation using invariant housekeeping genes) in your mutant [no treatment], RELATIVE to N2.
Then do the experiments with your drug of interest,
c. the transcript level(s) of the gene(s) of interest (corrected for inter-sample variation using invariant housekeeping genes) in N2 [with treatment], RELATIVE to a.
d. the transcript level(s) of the gene(s) of interest (corrected for inter-sample variation using invariant housekeeping genes) in in your mutant [with treatment], RELATIVE to a.
Is this what you meant or did I enjoy too much wine last night?
