Selection of Suitable Reference Genes for Normalization of Quantitative Real-Time Polymerase Chain Reaction in Human Cartilage Endplate of the Lumbar Spine

<div><p>The quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most widely used methods to study gene expression profiles, and it requires appropriate normalization for accurate and reliable results. Although several genes are commonly used as reference genes (such as <i>GAPDH, ACTB,</i> and <i>18S rRNA</i>), they are also regulated and can be expressed at varying levels. In this study, we evaluated twelve well-known reference genes to identify the most suitable housekeeping gene for normalization of qRT-PCR in human lumbar vertebral endplate with Modic changes, by using the geNorm, NormFinder, and BestKeeper algorithms. Our results showed that the rarely-used <i>SDHA</i> was the most stable single reference gene, and a combination of three, <i>SDHA</i>, <i>B2M</i>, and <i>LDHA</i>, was the most suitable gene set for normalization in all samples. In addition, the commonly-used genes, <i>GAPDH</i>, <i>ACTB</i> and <i>18S rRNA</i>, were all inappropriate as internal standards. The rankings of reference genes for the three types of Modic change differed, although <i>SDHA</i> and <i>RPL13A</i> uniformly ranked in the first and last position, respectively. Further simulated expression analysis validated that the arbitrary use of a reference gene could lead to the misinterpretation of data. Our study confirmed the necessity of exploring the expression stability of potential reference genes in each specific tissue and experimental situation before quantitative evaluation of gene expression by qRT-PCR.</p>