Amelioration for an ignored pitfall in reference gene selection by considering the mean expression and standard deviation of target genes

Routine tissue-specific reference genes are often used in expression studies, but target genes are not taken into account. Using the relative RT-qPCR approach, we evaluated the expression of three target genes. At the same time, meta-analyses were conducted in various ethnic groups, genders, and thyroid cancer subtypes. When eight common reference genes were examined, it was discovered that some of them not only lacked consistent expression but also had considerable expression variance. It is worth noting that while choosing a reference gene, the mean gene expression and its standard deviation should be carefully addressed. An equation was developed based on this, and it was used to perform statistical analysis on over 25,000 genes. According to the subtype of thyroid cancer and, of course, the target genes in this investigation, appropriate reference genes were proposed. The intuitive choice of GAPDH as a common reference gene caused a major shift in the quantitative expression data of target genes, inverting the relative expression values. As a result, choosing the appropriate reference gene(s) for quantification of transcription data, and especially for relative studies of the expression of target gene(s), is critical and should be carefully considered during the study design.