Abstract
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 GAPDH, ACTB, and 18S rRNA), 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 SDHA was the most stable single reference gene, and a combination of three, SDHA, B2M, and LDHA, was the most suitable gene set for normalization in all samples. In addition, the commonly-used genes, GAPDH, ACTB and 18S rRNA, were all inappropriate as internal standards. The rankings of reference genes for the three types of Modic change differed, although SDHA and RPL13A 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.
Highlights
Signal intensity changes in the vertebral endplate and subchondral bone marrow on magnetic resonance imaging, known as Modic changes (MCs), are often observed in patients with degenerative spinal diseases [1]
Type I changes are hypointense on T1-weighted imaging (T1WI) and hyperintense on T2-weighted imaging (T2WI) and indicate edema and hypervascularity in the lesions as confirmed histologically
The expression levels of these 12 reference genes varied widely with Ct values ranging from 17.6 (18S rRNA) to 33.5 cycles (ACTB), and most of the Ct values were between 24 and 33 cycles. 18S rRNA was the most abundantly transcribed with a mean Ct value of 20.2 cycles
Summary
Signal intensity changes in the vertebral endplate and subchondral bone marrow on magnetic resonance imaging, known as Modic changes (MCs), are often observed in patients with degenerative spinal diseases [1]. Type I changes are hypointense on T1-weighted imaging (T1WI) and hyperintense on T2-weighted imaging (T2WI) and indicate edema and hypervascularity in the lesions as confirmed histologically. Type II changes are hyperintense on T1WI and isointense or hyperintense on T2WI and reflect fatty replacement of the red bone marrow. Type III changes are hypointense on both T1WI and T2WI and represent subchondral bone sclerosis. Identification of the mechanisms and factors involved in the progression of MCs is of great importance for clinical interventions to repair or retard the development of MCs. To exploit the mechanisms of MCs, it is necessary to understand the pathophysiological changes of the vertebral cartilage endplate with MCs at the molecular level
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