qRT-PCR Validation of Transcriptome Analysis

To confirm the results of transcriptome analysis, selected genes were subjected to qRT-PCR (for list of genes and primers see Supplementary Table S4). RNA samples were reverse transcribed into cDNA using High Capacity RNA-to-DNA Kit (Applied Biosystems, Foster City, CA, USA). cDNA was analyzed by real-time quantitative PCR (qPCR) using an ABI 7500 Fast Real Time PCR system (Applied Biosystems, Foster City, CA, USA) and a pair of gene-specific primers for each selected gene. qPCR was performed in triplicates using EvaGreen Mix (Bio&SELL, Feucht, Germany) and 20 ng of cDNA per reaction. The transcript levels were normalized to the geometric mean of three housekeeping genes (Hmbs, Ywhaz, Actb) [35 (link),36 (link)] from the same sample used as an internal reference, and the fold change of mutant relative to the WT mice was calculated as 2^−ΔΔCT [37 ]. Statistical analysis was performed with GraphPad Prism 5.0 software; unpaired Student’s t-test was used to estimate significance.