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).
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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
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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.
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