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.
- Calculation of Delta Ct = Difference between the Gene of interest and Geometric Mean of Multiple housekeeping genes
- Calculation of Delta Delta Ct = Difference between the samples (before and after vitamin supplementation) and average of pooled control samples
- Calculation of the 2 to the power of (negative Delta Delta Ct) to evaluate fold gene expression levels
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?