Quantifying Gene Expression in Mouse Conceptuses

Total RNA was isolated from GD/ED 10 mouse conceptuses using the RNeasy Plus Mini Kit (Qiagen; Germantown, MD) following homogenization in lysis buffer using the TissueMiser (Fisher Scientific; Waltham, MA) or Bullet Blender Gold (Next Advance; Troy, NY). A minimum of 3 conceptuses were pooled per dam. Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Foster City, CA). Relative transcript abundance was measured using Power SYBR® Green Supermix (Applied Biosystems; Foster City, CA) with the C1000 Touch Thermal Cycler (CFX96 Real Time Systems; Bio-Rad, Hercules, CA). Each sample was assayed in duplicate for all target and housekeeping genes. Average threshold cycle (Ct) values were normalized to average Ubc^{78 (link),79 (link)} Ct values. Relative transcript abundance of each gene of interest was determined using the ΔΔCt method^{80 (link)}. Briefly, transcript expression in individual mice is presented relative to the mean expression value in Mal− mice. When possible, primer pairs were designed to span large introns such that amplification exclusively represented cDNA template. Primer sequences and amplicon sizes are listed in Supplemental Table 1.