A method to estimate the age of bloodstains using quantitative PCR

  • Jun Fu
    School of Forensic Sciences, Center for Health Sciences, Oklahoma State University, Tulsa, OK, United States
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  • Robert W. Allen
    Corresponding author at: School of Forensic Sciences, Oklahoma State University, 1111 West 17th Street, Tulsa, OK 74107, United States.
    School of Forensic Sciences, Center for Health Sciences, Oklahoma State University, Tulsa, OK, United States
    Search for articles by this author
Published:December 20, 2018DOI:


      • Novel qPCR assay was developed and able to estimate the age of a bloodstain.
      • Method exploits the degradation of mRNA in dried stains.
      • mRNA degradation proceeds preferentially from the 5′ end of the message.
      • The approach presented greatly reduces stochastic effects that arise during qPCR.


      The value of RNA analysis in the forensic laboratory as one means of identifying the nature of biological evidence of forensic relevance has been well established. The degradation of RNA in dried body fluid stains has also been an area of forensic interest because of the potential to estimate the age of a stain recovered from a crime scene. Here we describe a qPCR assay that demonstrates it is possible to estimate the age of bloodstains with reasonable accuracy. The 5′–3′ qPCR assay exploits the observation the 5′ end of an mRNA transcript degrades in dried stains faster than the 3′ end. This differential degradation pattern can be followed with a qPCR assay that quantifies ∼90 bp amplicons produced from the 5′ and 3′ ends of 4 transcripts chosen from the transcriptome of blood because of their degradation kinetics, determined initially using RNA sequencing. Statistical analysis of degradation kinetics suggests, depending upon the age of the sample, the age of blood stains can be accurately estimated to within 2–4 weeks for stains less than 6 months of age and 4–6 weeks for stains 6 months to 1 year old.


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