Absolute qpcr sybr low rox mix

We extracted total RNA from HT22 cells or mouse brain (hippocampal region; see [18 (link)] for method) using the TRIzol reagent (Invitrogen) following the manufacturer’s instructions. We treated total RNA with DNase 1 (20U; Roche, Basel, Switzerland) to remove contaminating genomic DNA and conducted reverse transcription with 1 μg RNA using the High Capacity Reverse Transcription kit with ribonuclease inhibitor (Applied Biosystems, Life Technologies Corp, Foster City, CA). For RTqPCR we used Taqman assays for Gapdh and Klf9 [7 (link)] and SYBR green assays for all other genes. All oligonucleotide primer sequences are given in Additional file 18: Table S9. We conducted RTqPCR using an ABI 7500 fast real-time PCR machine with Absolute qPCR low ROX mix (for Taqman assays) or Absolute qPCR SYBR low ROX mix (ABgene, Epsom, UK). We designed SYBR green assays using Integrated DNA Technology’s RealTime qPCR Assay tool; where possible we designed assays to span exon-exon boundaries. We used a relative quantitation method using serial dilutions of a cDNA pool to generate standard curves. We normalized all genes to the reference gene Gapdh whose mRNA was unaffected by treatments (data not shown.)