Sds software version 1

Genomic DNA (gDNA) was prepared from 200 uL of blood collected in EDTA from 37 patients and extracted using a QIAamp DNA blood mini kit (QIAgen, Hilden, Germany) in a volume of 100 ul of elution buffer. Genomic DNA from HMC1.2 cells^{10 (link)} was used as the KIT D816V mutation positive control. Genomic DNA from peripheral blood of a HV was used as a negative control. The concentration of each DNA sample was determined using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Both mutation-specific and control real-time qPCR assays for KIT D816V were performed for each sample in the same plate using the TaqMan Universal PCR Master Mix with AmpErase UNG on the 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) as described.^{11 (link)} Each real-time qPCR reaction was performed in triplicate with 50 ng of gDNA in a total volume of 25 ul. Results were analyzed using SDS software version 1.3.1 (Applied Biosystems, Grand Island, NY). Samples with two of three or three of three analyses generating a threshold cycle (Ct) value below 41 were defined as mutation positive. Mutation negative samples tested negative with zero of three reactions producing a Ct value below 44.^{12 (link)} The percentage of the cells carrying the KIT D816V allele among the total number of nucleated blood cells was then calculated as described.^{11 (link)}

RNA was isolated from each hippocampal region (n=5–6 per region of each age and OVX duration group) using the RNeasy Lipid Tissue Mini kit (Qiagen, catalog number: 74804) and DNase digestion was performed with the RNase-Free DNase Set (Qiagen, catalog number: 79254). Following isolation, the concentration was measured using the NanoDrop 2000 spectrophotometer (Thermo Scientific). Reverse transcription was performed using the QuantiTect Reverse Transcription kit (Qiagen, catalog number: 205311) and quantitative polymerase chain reaction (qPCR) was completed using the TaqMan Gene Expression Assays (Esr1: Rn01640372_m1, Gapdh: Rn01775763_g1) in a 7300 Real-Time PCR system with SDS software version 1.3.1 (Applied Biosystems). The ΔΔCT method (Livak and Schmittgen 2001 (link)) was used to determine the relative cDNA levels and the CA1 region from young short-term rats were used as the calibrator samples.