Total RNA (750 ng) was subjected to reverse transcription reaction to synthetize cDNA using oligo dT, RNase Out and M-MLV RT enzyme (Life Technologies, ThermoFisher Scientific) according to the manufacturer’s instructions.
Real time qPCR experiments were carried out with the 7500 Fast real-Time PCR system (Applied Biosystems, ThermoFisher Scientific), using TaqMan® Fast Universal PCR Master Mix (2X) and the following TaqMan® Gene Expression Assays: Oxt (Mm00726655_s1), Avp (Mm00437761_g1), Lcn2 (Mm01324470_m1), Ptgs2 (Mm00478374_m1), Esr1 (Mm00433149_m1), Igf1 (Mm00439560_m1), Gapdh (Mm99999915_g1). Experiments used 7.5 ng of previously prepared cDNA and samples were run in triplicates on six different biological samples for each group. Relative expression levels were determined according to the ΔΔCt method where the expression level of the mRNA of interest is given by 2-ΔΔCT where ΔΔCT = ΔCT target mRNA − ΔCT reference mRNA (Gapdh) in the same sample as previously described [4 (link), 128 (link)].
Real time qPCR experiments were carried out with the 7500 Fast real-Time PCR system (Applied Biosystems, ThermoFisher Scientific), using TaqMan® Fast Universal PCR Master Mix (2X) and the following TaqMan® Gene Expression Assays: Oxt (Mm00726655_s1), Avp (Mm00437761_g1), Lcn2 (Mm01324470_m1), Ptgs2 (Mm00478374_m1), Esr1 (Mm00433149_m1), Igf1 (Mm00439560_m1), Gapdh (Mm99999915_g1). Experiments used 7.5 ng of previously prepared cDNA and samples were run in triplicates on six different biological samples for each group. Relative expression levels were determined according to the ΔΔCt method where the expression level of the mRNA of interest is given by 2-ΔΔCT where ΔΔCT = ΔCT target mRNA − ΔCT reference mRNA (Gapdh) in the same sample as previously described [4 (link), 128 (link)].
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