Abstract
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.
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Acknowledgments
This work was supported by the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (Grant No. 2011BAI03B02-2) and the Scientific and Technological Developing Scheme of Ji Lin Province (Grant No. 20140101124JC) and the National Nature Science Foundation of China (Grant No. 81072829 and 81303165).
Conflict of interest
We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work; there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript.
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Online Resource 1
RPKM (reads per kilobase of exon model per million mapped reads) value distribution of five factors related with bone development (PDF 54 kb)
Online Resource 2
RT-qPCR (quantitative real-time reverse transcription-polymerase chain reaction) assays used to evaluate the factors related with bone development (PDF 73 kb)
Online Resource 3
Variability of the candidate reference genes in the different samples (PDF 56 kb)
Online Resource 4
Expression profiles of five factors related with bone development. The most stable genes identified by geNorm (RPL40 and Gpx) and NormFinder (NADH) and the least stable gene GAPDH were used for normalization. For RPL40 and Gpx, the geometric mean was calculated and used for normalization. The relative expression levels are depicted as the standard deviation calculated from three biological replicates (PDF 120 kb)
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Liu, M., Yao, B., Zhang, H. et al. Identification of novel reference genes using sika deer antler transcriptome expression data and their validation for quantitative gene expression analysis. Genes Genom 36, 573–582 (2014). https://doi.org/10.1007/s13258-014-0193-x
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DOI: https://doi.org/10.1007/s13258-014-0193-x