Reference genes to normalize body fluid identification data obtained using RT-qPCR

The use of messenger RNA for body fluid identification has been well documented. While reference genes are commonly used for normalizing gene expression, the use of reference genes in forensic science is not straightforward. Degradation of samples, mixed body fluids, and small volumes contribute to the complexity of normalizing gene expression. To demonstrate normalization of gene expression for forensic body fluid identification, a preliminary study involving two reference genes (beta-2-microglobulin, B2M and glucose-6-phosphate dehydrogenase, G6PD) was undertaken. Novel reverse-transcription quantitative PCR (RT-qPCR) assays for circulatory blood, buccal swabs, menstrual fluid, spermatozoa, seminal fluid, and vaginal material were designed. An appropriate PCR efficiency for the B2M and G6PD reference gene assays across the body fluid panel was confirmed prior to assessing their expression levels. B2M was found to be more highly expressed across the different body fluids than G6PD. The normalization of target assay expression using both genes was investigated. However, high variation and poor sensitivity, particularly in buccal and semen samples, were observed for both, which would limit sensitivity and mixture detection in forensic casework. If C_T values are the intended output rather than comparative quantification, we propose that reference gene normalization is not required and could be detrimental to interpretation.