Hi,
I have a question on qRT-PCR data analysis. The gene panel has more than 200 genes along with the multiple reference/housekeeping genes. I have 50 paired samples (i.e Before vs After Vitamin supplementation) and 6 pooled controls (3 Pooled controls in one plate and 3 in another plate as interplate controls). In other words, 50 samples in plate 1 (Before) + 3 Pooled Controls and 50 samples in plate 2 (After) + 3 Pooled Controls.
I would like to see the gene expression changes in the Before vs After Vitamin supplementation. Please let me know if the workflow followed looks fine.
Following the calculation of the Delta Ct, does the Delta Delta Ct calculation looks fine? I am bit confused here, if the average of the pooled controls should be considered and subtracted with the individual samples or difference of Before and After samples should be considered individually?
In addition, what values are considered for the statistical analysis like paired t-test, PCA, Scatter Plot and other visualization (Negative Delta Ct) or Delta Delta Ct?
Best Regards,
Toufiq
I'd place before & after samples on the same plate (25 samples on plate#1, 25 on plate #2). If you put before / after on different plates then time effect is perfectly confounded with the plate effect and thus you won't be able to say what the time effect is (unless you can assume that the plate effect is zero - what is quite unlikely).
I also wonder why you would use the geometric mean to average the Ct values of the reference genes. That does not make sense. Should be the arithmetic mean.
The pooled controls could be used to correct for betwee-plate differences.
You would use the (negative) dCt for all analyses. Note: would be smarter to calculate dCt as Ct[ref]-Ct[goi], then the sign is already correct and doe not need to be changed afterwards.
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