Identification of novel reference genes using sika deer antler transcriptome expression data and their validation for quantitative gene expression analysis

The most commonly used normalization strategy for quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) is to select a stable reference gene. However, to date, no suitable reference genes have been identified in sika deer antler tissues. Thus, the aim of this study was to identify the most stable gene or a set of genes to be used as reference genes for RT-qPCR analysis in sika deer antler tissues. We first selected candidate reference genes using sika deer antler gene expression data from an Illumina sequencing platform (Hiseq 2000); twenty-one reference genes from the antler tips of Chinese sika deer were selected to test for the normalization of expression levels during different growth stages. These genes were tested by RT-qPCR and ranked according to the stability of their expression using two different methods (implemented in geNorm and NormFinder). Based on different algorithms and analytical procedures, our results clearly indicate RPL40 and Gpx as the most stable reference genes of our pool.