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The effects of different storage methods on both DNA degradation and the results of preliminary blood tests were evaluated. Bloodstains stored at room temperature, 4 °C, –20 °C, and –80 °C for 20 years; whole blood samples stored at –20 °C and –80 °C for 20 years; and fresh whole blood samples were analyzed. DNA degradation was evaluated by quantifying the ratios of 305 and 129 base pair (bp) fragments to 41 bp fragments. Preliminary testing included leuco-malachite-green staining, anti-human hemoglobin (Hb) testing by immunochromatography, and the detection of the hemoglobin-beta (HBB) mRNA. Bloodstains stored at room temperature and 4 °C were more highly degraded than fresh blood, as indicated by the ratio of 129:41 bp DNA fragments and 305:41 bp DNA fragments. All samples tested positive using leuco-malachite-green staining and anti-human Hb assays. HBB was not detected in any whole blood samples stored at –20 °C or –80 °C, although HBB was detected in all bloodstain samples. Therefore, to prevent DNA degradation during long-term storage, it is recommended that blood samples be stored at below –20 °C. In addition, stored bloodstains are suitable for preliminary tests of blood, rather than storing blood.
When seeking a resolution to a long-unsolved crime, short tandem repeat (STR) genotyping of old bloodstains is evidentially important. However, forensic data may be difficult to obtain if samples have been improperly stored. In the present study, we explored the effects of different storage conditions on DNA degradation and preliminary blood tests.
2. Materials and method
2.1 Samples
Bloodstains that adhered to cotton gauze and were stored at room temperature, 4 °C, –20 °C, and –80 °C for 20 years (n = 6, each); whole blood samples stored at –20 °C and –80 °C for 20 years (n = 6, each); and fresh whole blood samples (n = 6) were analyzed. This study was approved by the Ethics Committee at Saitama Medical University.
2.2 DNA extraction and assessing DNA degradation
DNA was extracted and purified from a 1 × 1-cm piece of gauze or 30 μL of whole blood using the EZ1 DNA Investigator Kit (QIAGEN, CA, USA), according to the manufacturer’s instructions, and was eluted in 50 μL water. DNA degradation was assessed by calculating the ratios of 129 and 305 bp DNA fragments to 41 bp DNA fragments using the KAPA Human Genomic DNA Quantification and QC Kit (Kapa Biosystems, Woburn, MA, USA), according to the manufacturer’s instructions. The 41-, 129- and 305-bp DNA fragments were quantified using the StepOne-Plus Real-Time PCR System.
2.3 Discrimination test for blood
Preliminary testing was performed using the leuco-malachite-green test, anti-human Hb testing by immunochromatography, and detection of blood-specific mRNAs. The leuco-malachite-green reagent was spotted directly onto bloodstains and incubated at room temperature. A positive result was taken as the visible detection of blue–green coloration after a 1 min incubation.
Anti-human Hb testing by immunochromatography was performed using OC-Hemocatch S (Eiken Chemical, Tokyo, Japan) with a 0.2 × 0.2-cm bloodstain, according to the manufacturer’s protocol.
Total RNA was extracted from a 1 × 1-cm piece of gauze or 30 μL of whole blood using the RNeasy Mini Kit (QIAGEN). DNase I digestion and reverse transcription were performed using the RNase-Free DNase Set (QIAGEN) and High-capacity RNA-to-cDNA™ Kit (Life Technologies, Carlsbad, CA, USA), respectively, according to the manufacturer’s instructions. To investigate the expression of blood-specific mRNA, the hemoglobin-beta (HBB; Hs00758889_s1) gene was selected; and the 18S rRNA (18S; Hs99999901_s1) gene was chosen as an endogenous control. All primers and probes were supplied in a commercially designed kit (Life Technologies). Real-time PCR was performed in 20-μL reaction mixtures containing 2× TaqMan® Fast Advanced Master Mix (Life Technologies), a 20× TaqMan® Gene Expression Assay reagent (primers and probe; Life Technologies) and 2 μL of cDNA. PCR amplification was performed using the StepOne-Plus Real-Time PCR System (Life Technologies) programmed for 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. When the threshold reached 0.2, HBB detection was defined as a sample that had a Ct value of ≤34 cycles, whereas 18S detection was defined as a sample that had a Ct value of ≤33 cycles.
3. Results and discussion
Fig. 1 shows the relationship between storage conditions and DNA degradation. Bloodstains stored at room temperature and 4 °C were significantly more degraded than fresh blood samples, as determined by the 129:41 bp and 305:41 bp fragment ratios. Consistent with the mRNA data, sample storage at –20 °C or lower is suitable for maintaining the integrity of DNA. Hara et al. reported that the Identifiler kit did not detect all loci when the 305:41 bp ratio was ≤0.0118 [
M. Hara, H. Nakanishi, S. Takahashi et al. Relationship between DNA degradation ratios and the number of loci detectable by STR kits in extremely old seminal stain samples, Legal Med. In press.
]. In this study, the 305:41 bp ratio of bloodstains stored at room temperature for 20 years was 0.0220 ± 0.0130 (mean ± standard deviation) (data not shown); thus, storage under these conditions might affect STR genotyping.
Fig. 1The DNA degradation ratio in bloodstains stored at room temperature, 4 °C, –20 °C, and –80 °C for 20 years; of whole blood samples stored at –20 °C and –80 °C for 20 years; and of fresh whole blood. Mean values ± SD for six samples each are shown. Dark gray bars represent the 129:41 bp ratio, whereas the light gray bars represent the 305:41 bp ratio. *Significant differences were calculated using the t-test (P < 0.05), compared with fresh whole blood.
Table 1 shows the number of positive reactions in preliminary tests examining various storage conditions. All bloodstains and whole blood samples tested reacted positively with leuco-malachite-green staining and anti-human Hb testing. In whole blood samples stored at –20 °C and –80 °C, 18S was detected in all samples, whereas HBB was not detected. In comparison, HBB and 18S were detected in all bloodstain samples. These data suggest that stored bloodstains are suitable for preliminary tests of blood, rather than storing blood. In conclusion, to prevent DNA degradation, blood samples should be stored at below –20 °C for long-term storage. In addition, stored bloodstains are suitable for preliminary tests of blood, rather than storing blood.
Table 1The number of positive reaction of preliminary tests in various storage conditions.
M. Hara, H. Nakanishi, S. Takahashi et al. Relationship between DNA degradation ratios and the number of loci detectable by STR kits in extremely old seminal stain samples, Legal Med. In press.
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