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Background <p>Bloodstains are among the most frequently encountered forms of biological evidence, and their analysis plays a central role in forensic identification and reconstruction. However, the stability of nucleic acids in bloodstains decreases over time and is influenced by environmental conditions. Understanding degradation patterns of blood-specific DNA and RNA markers may support time-since-deposition (TSD) estimations and improve evidentiary preservation.</p> Methods and results <p>This study quantified time-dependent degradation of DNA and RNA extracted from bloodstains stored under three conditions: −20&#xa0;°C (freezing), 25&#xa0;°C (room temperature), and natural exposure (variable light/temperature). Bloodstains from six healthy donors were sampled at seven time points over 30 days. Degradation of the blood-specific genes HBA and ALAS2 was quantified using real-time PCR, with Day 0 normalized to 1.00. The storage conditions significantly influenced degradation rates, with the highest stability at − 20&#xa0;°C, intermediate decay at 25&#xa0;°C, and most rapid loss under natural exposure. By Day 30, reductions were greatest under natural exposure (DNA: 86–97%; RNA: 97–99%) and lowest at − 20&#xa0;°C (DNA: 33–47%; RNA: 46–47%). RNA degraded faster than DNA across all non-frozen conditions. A gene-specific effect was observed for DNA, with HBA exhibiting greater stability than ALAS2; no gene effect was detected for RNA. Log-linear modeling of ln(stability) produced negative slopes for all series, with storage-condition-dependent half-lives.</p> Conclusions <p>These findings provide quantitative decay parameters for forensic bloodstains and support the complementary use of DNA and RNA for TSD estimation. RNA serves as a sensitive marker of recent deposition, whereas DNA offers improved stability for identification. The results highlight the importance of prompt collection and cold, dark storage to preserve evidentiary value.</p>

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Molecular analysis of time-dependent DNA and RNA degradation in bloodstains: differential stability of HBA and ALAS2 gene targets and its forensic implications

  • 期刊论文

  • 发表日期：

    2025年12月18日

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  • Rahat Bukhari,
  • Rahat Abdul Rehman,
  • Midhat Salman,
  • Allah Rakha,
  • Anam Munawar,
  • Shahid Nazir,
  • Muhammad Farhan Khan,
  • Zunaira Batool

摘要

Background

Bloodstains are among the most frequently encountered forms of biological evidence, and their analysis plays a central role in forensic identification and reconstruction. However, the stability of nucleic acids in bloodstains decreases over time and is influenced by environmental conditions. Understanding degradation patterns of blood-specific DNA and RNA markers may support time-since-deposition (TSD) estimations and improve evidentiary preservation.

Methods and results

This study quantified time-dependent degradation of DNA and RNA extracted from bloodstains stored under three conditions: −20 °C (freezing), 25 °C (room temperature), and natural exposure (variable light/temperature). Bloodstains from six healthy donors were sampled at seven time points over 30 days. Degradation of the blood-specific genes HBA and ALAS2 was quantified using real-time PCR, with Day 0 normalized to 1.00. The storage conditions significantly influenced degradation rates, with the highest stability at − 20 °C, intermediate decay at 25 °C, and most rapid loss under natural exposure. By Day 30, reductions were greatest under natural exposure (DNA: 86–97%; RNA: 97–99%) and lowest at − 20 °C (DNA: 33–47%; RNA: 46–47%). RNA degraded faster than DNA across all non-frozen conditions. A gene-specific effect was observed for DNA, with HBA exhibiting greater stability than ALAS2; no gene effect was detected for RNA. Log-linear modeling of ln(stability) produced negative slopes for all series, with storage-condition-dependent half-lives.

Conclusions

These findings provide quantitative decay parameters for forensic bloodstains and support the complementary use of DNA and RNA for TSD estimation. RNA serves as a sensitive marker of recent deposition, whereas DNA offers improved stability for identification. The results highlight the importance of prompt collection and cold, dark storage to preserve evidentiary value.