Exploration of Rhythmic Patterns of Gene Expression to Estimate the Time of Day a Bloodstain Was Created

Abstract

Introduction: Interest in the analysis of RNA in forensically relevant body fluid stains has increased in recent years and we have shown that a correlation exists between the state of degradation of mRNA transcripts in dried stains and their age. In addition to estimating the age of a forensic sample, instances exist in which being able to estimate the time of day a biological sample was deposited at a crime scene would advance the investigation.Methods: In this study, a panel of 10 RNA transcripts, suspected to fluctuate in abundance during a daily circadian rhythm, were quantified by qPCR in blood stains collected from unrelated donors over a 24-hour period. The abundance of a transcript in the circadian marker panel was normalized against the abundance of either S100A12 or CLOCK transcripts expressed among blood cell populations. These studies aimed to detect and explore patterns of abundance changes in transcripts during a 24-hour daily cycle. The reference pattern could then be matched against the pattern of transcript abundance produced from a crime scene bloodstain in an attempt to estimate the time of day the stain was deposited.Results: Transcript abundance did not vary significantly in blood stains prepared from donors collected repeatedly at the same time of day on different days. Likewise, the age of the donor did not seem to significantly affect the transcript profile in stains collected at one time of day. Of 10 transcripts in the panel evaluated, PER3 in females showed a statistically significant change in abundance over a 24-hour cycle if qPCR results were normalized against the CLOCK transcript and changes in transcript abundance suggested a rhythmic pattern. Although some patterns of transcript abundance suggested a rhythmic pattern, they did not achieve statistical significance using repeated measures ANOVA, likely due to the variance in gene expression among individuals over a 24-hour period.Discussion: The change in PER3 abundance in females suggests that the expression of this gene follows a circadian rhythm. The pattern of PER3 expression could allow a blood stain deposited by a female during the afternoon to be distinguished from one deposited in the early morning based upon these qPCR results. The results also underscore the importance of normalizing qPCR data against the appropriate target. Analysis of the transcriptome in blood stains from males and females through RNA sequencing may allow other gene transcripts to be identified that cycle in abundance in a way comparable to PER3 in blood from females.