For gene expression analysis, relative expression values were calculated according to the formula: relative expression gene x = 2^ − (Ct gene x − Ct internal reference) and the mean expression and standard deviation for each triplicate was calculated.
Dinoflagellate gene-coding percentages were estimated based on this formula: (total gene number x average gene length/genome size)×100%, where average gene length was approximated as 1.346 kbp, a value previously found highly conserved in eukaryots [18].
Finally, the mRNA expression ratio of cancerous (C) to noncancerous (N) tissues was calculated using the following formula: R=log{target gene (C /GAPDH (C }, R=log{target gene (N /GAPDH (N)}.
We weighted the genes that were selected in six cancer-specific EMT signatures using the formula: for gene g, the weight of the gene is given by: where D is the total number of diseases (D = 6 in this case), fc gd and q gd are the fold-change and q-value% of the gene, g, of disease, d, as computed by SAM.
Finally, the mRNA expression ratio of cancerous (C) to noncancerous (N) tissues was calculated from the following formula: R={target gene (C /GAPDH (C)}/{target gene (N /GAPDH (N)}.
The expression levels for the tumour samples were calculated by the formula: CT gene expression level=2(c−d), c=beta-actin Ct obtained from the equivalent of 50 ng of tumour RNA, d=CT gene Ct obtained from the equivalent of 50 ng of tumour RNA.
The calculation formula for gene expression specificity is presented in Materials and Methods [ 22].
The ΔCt for each sample was then calculated according to the formula Cttarget gene - Ctactin; ΔΔCt values then were obtained by subtracting the ΔCt of a reference sample from the ΔCt of the studied samples.
The level of expression of each GPCR "X" was normalized to the geometric mean of the expression levels of the selected reference genes, R1 to R3, in the same PCR plate according to the formula: Reference genes were selected according to the GeNorm procedure [ 18].
