Supplementary Materials 2: Methods—SERT mRNA levels were measured in brain and lymphoblast RNA samples by real-time PCR using an ABI 7000 DNA sequence detection system (Applied Biosystems, Foster City, CA). Complementary DNA (cDNA) was amplified, using heat-activated Taq DNA polymerase, dNTPs, buffer, SYBR Green, plus a reference dye (Applied Biosystems, Foster City, CA). PCR primers and reaction conditions were the same as those used for the SERT AEI assays (Materials and Methods). PCR amplified reaction mixtures were analyzed by their melting curves on the ABI 7000, to confirm the homogeneity of PCR products (not shown). Standard curve for quantification of SERT mRNA—To determine the number of SERT mRNA molecules in our samples, we established a standard curve by plotting the log of the number of DNA templates (N0) added to the reaction mixture versus the measured cycle-threshold (CT), i.e., the cycle at which fluorescence associated with the PCR products reached a fixed value (Fig. S2a). The number of PCR products at the cyclethreshold (NT) is related to the initial number of DNA templates (N0) by the equation NT = N0 CT), where is the efficiency of the PCR amplification. Taking the log of both sides and rearranging yields the equation: CT = [-1/log(1 + ] logN0 + log NT/ log(1 + ]The equation CT = -3.52 logN0 + 35.43, was determined to be a good fit for the data from linear regression analysis; R2 = 0.9854; R = correlation coefficient). We calculated the efficiency of the PCR amplification from the slope [-1/log(1 + = -3.52] of this line: = 10-1/slope -1 = 0.92). 1 Fig. S2-1. Logarithmic plots of real-time PCR amplifications with increasing amounts of template DNA performed on the ABI Prism® 7000 Sequence Detection System using SYBR Green I fluorescence to detect the PCR products. CT (cycle threshold) is the fractional cycle number at which the total fluorescence passes a fixed threshold (T). A. PCR amplification of 1, 5, 10, 20, 30 and 50 ng template DNA, corresponding to approximately 300, 1500, 3000, 6000, 9000 and 15000 molecules, respectively. These traces were used to determine the CT for each amount of template DNA. B. The log (number of DNA molecules) was plotted versus CT as determined in A. The data points were fitted to the equation: CT = (slope) x log10(number of DNA molecules) + (Yintercept); R2 = 0.985; ( 10-1/slope – 1). Quantification of SERT mRNA expression in pons samples—Fig. S2-2 plots CT values for SERT mRNA for pons sections in our sample. As indicated, CTs ranged from approximately 25 to 18.5 cycles, corresponding to approximately 1,000 to 64,000 mRNA per 50 ng of pons cellular RNA. We have previously determined that the reproducibility of allelic mRNA ratio measurements declines with decreasing amounts 2 of mRNA in the samples. Reliable results are usually obtained with RNA samples yielding CTs of 25 or lower. Fig. S2-2. Assay of SERT mRNA levels in pons RNA samples using quantitative realtime PCR. Number of samples examined: (l/l) = 12, (l/s) = 15 and (s/s) = 2. N0 = estimated initial number of SERT mRNA molecules in each sample. The wide range of measured SERT mRNA levels probably reflects differences in the quality of tissue samples. This is an obvious confounding factor that makes meaningful comparisons between individuals extremely difficult. Measuring allelic mRNA ratios using RNA isolated from the same tissue sections allows each allele to serve as a control for the other, equalizing artifacts associated with the condition of the tissue. There were no statistically significant differences among the cycle thresholds (CTs) among the l/l, l/s and s/s SERT genotypes (one-way ANOVA, p = 0.222). To address the question whether mRNA levels in the pons tissue sections reflect specific expression in serotonergic neurons, rather than nonspecific background expression, we also applied real-time PCR to other brain regions. As shown in Fig. S2- 3 4, the average CT for cycle thresholds for SERT mRNA in non-pons regions of brain was 29.6, which is equivalent to approximately 50 molecules of SERT mRNA per 50 ng cellular RNA. In contrast, the average CT SERT mRNA in pons was 19, corresponding to approximately 46,000 molecules of SERT mRNA per 50 ng cellular RNA. The considerably higher SERT mRNA levels in pons tissue sections suggest that this SERT mRNA derives from the serotonergic neurons of the raphe nuclei in these sections. Fig. S2-3. Assay of SERT mRNA levels in RNA of non-pons brain regions (n = 5) and pons (n = 26) using quantitative real-time PCR. cDNA was prepared from 1 g of total RNA isolated from pons, cerebellum or the occipital, frontal, parietal and temporal lobes of the cortex and 1/20 of this cDNA was used as a template for quantitative real-time PCR. SERT mRNA levels were significantly higher in pons compared to other brain regions (One-way ANOVA, p = 1.36E-17). N0 = initial number of SERT mRNA molecules in each sample. Relative mRNA expression of SERT mRNA in lymphoblasts—Fig. S2-4 shows the results of quantitative RT-PCT measurements of SERT mRNA levels in 33 Epstein-Barr virus4 transformed lymphoblast cell lines. As for the pons samples, ANOVA revealed no statistically significant differences in mRNA expression levels among the l/l, l/s and s/s genotypes. Fig. S2-4 SERT mRNA expression in Epstein-Barr virus transformed lymphoblasts was determined by quantitative real-time PCR. One g of total cellular RNA was used for cDNA synthesis and 1/20 of this cDNA was used as a template for quantitative realtime PCR. Number of samples examined: (l/l) = 11, (l/s) = 9 and (s/s) = 7. There was no statistical difference in cycle threshold (CTs) among the three SERT genotypes (Oneway ANOVA, p=0.980). N0 = initial number of SERT mRNA molecules in each sample. 5