Using long RNA fragment degradation ratio to estimate the time elapsed since bloodstain deposition

When crimes that involve injuries or deaths are investigated, it is sometimes necessary to estimate the length of time that has elapsed since bloodstains were deposited at the crime site. A method based on the degree of bloodstains' RNA degradation is one of the most commonly used indicators, but the applications of general real-time polymerase chain reaction (PCR) techniques for this purpose are often limited by the techniques' inability to quantify long (>400 bp) DNA fragments, rendering them unsuitable for relatively new bloodstains. We assessed RNA degradation ratios by using a real-time PCR method that amplifies > 500-bp RNA fragments to evaluate these ratios' potential for estimating the age of relatively newly deposited bloodstains. Bloodstains were stored at room temperature (RT) or in a refrigerator for ≤ 6 months. Once-wetted bloodstains generated by wetting with water after 1-day storage were stored at RT for ≤ 7 days. Using Kodakaraensis (KOD) DNA polymerase, we amplified 51-, 578-, 998-, and 1782-bp fragments in ACTB gene and then calculated the 578:51, 998:51, and 1782:51 fragment ratios as the RNA degradation ratios. Our approach successfully quantified long amplicons (500-1800 bp) with the use of KOD DNA polymerase. All of the fragment ratios for the dried bloodstains correlated well with the length of time since the bloodstains' deposition; in particular, 1782:51 (stored ≤1 month), 998:51 (≤2 months), and 578:51 (≤6 months) were suitable for estimating the time since the bloodstain deposition. The 578-bp, 998-bp, and 1782-bp fragments for the bloodstains stored in a refrigerator were not detected in some samples that had been stored for > 10 days. The once-wetted bloodstains showed that RNA degrades rapidly immediately after being wetted and then gradually undergoes further degradation. Our results suggest that it is possible to estimate the length of time since a bloodstain's deposition over a short term (up to 6 months) by using the three above-described RNA amplicon lengths as indicators for dried bloodstains that are stored indoors.