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Supplementary Materials
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Methods
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Theoretical base for comparison of the mRNA levels between different enzymes
and receptors
We used the method of comparison in order to compare mRNA levels of different
enzymes/receptors, which were obtained by using different primers, as described in
(Kimoto et al., 2010). The following consideration is necessary. As an example, we
compare AR (Ar) in CC at 24 m and that of P450arom (Cyp19a1) in the hippocampus
(Hi) at 3 m. Since we use P450arom in 3m Hi as a standard, we obtain
NAr CC / NCyp19a1 Hi
(1)
where ‘N’ is the number of mRNA molecules and we assumed that we obtain the ng i
molecules of cDNA from Ng i molecules of mRNA via RT with an equal efficacy in the
same sample. Therefore, in the process of RT, we have the following relationship
nAr CC / NAr CC = nGapdh CC / NGapdh CC
(2)
nCyp19a1 Hi / NCyp19a1 Hi = nGapdh Hi / NGapdh Hi
(3)
where Gapdh is a house-keeping standard gene which is assumed to express at equal
level in these organs (NGapdh CC = NGapdh Hi).
In Fig.1, we obtained nGapdh CC = nGapdh Hy = nGapdh CL = nGapdh Hi, indicating that Gapdh is
a good internal standard gene. Finally we obtain
NAr CC / NCyp19a1 Hi = (nAr CC / nGapdh CC) / (nCyp19a1 Hi / nGapdh Hi) (4)
Then we need to obtain ‘nAr CC / nGapdh CC’ from the EtBr band intensity ‘I’.
The number of cDNA molecules, ‘n’, is exponentially amplified by PCR, and its PCR
product contains ‘n × L ×(1+e)c ’ molecules, where ‘c’ is a PCR cycle number, ‘L’ is a
product length, and ‘e’ is an amplification efficacy obtained from the PCR amplification
curves in the exponential amplification phase (linear phase with semi-log plot). The
band intensity of the PCR product ‘I’ is proportional to the molecule numbers.
Therefore, we can obtain ‘n’ from the next equation:
I = A× n × L × (1+e)c
(5)
where ‘A’ is a proportionality coefficient between ‘I’ and ‘n × L ×(1+e)c’.
From amplification curves, we choose the exponential amplification phase of PCR
cycles which satisfy the equation (5). The PCR cycle in the exponential phase was
24-30, the product length (LAr CC) was 536 for 17β-HSD type 3 in the CC (Table S1) and
we chose cAr CC = 26 and determined eAr CC = 1.0850, by fitting the data of ‘c’ and ‘I’ to
the equation (5) (I = 0.87 × 2.0850c, R2 = 0.9037). For the Gapdh bands, the same
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calculation was performed, and then we obtained cGapdh CC = 18 and eGapdh CC = 0.9310 (I
= 17.53 × 1.9310c, R2 = 0.9295). From these values we can calculate ‘n’ from the band
intensity ‘I’.
nArCC / nGapdh CC
= (IArCC / IGapdh CC) × (LGapdh CC / LArCC) × (1+0.9310)18 / (1+1.0850)26 (6)
By substitution eq. (6) into eq. (4), we can compare the mRNA levels for different
enzymes and receptors.