mRNA profiling in ancient bloodstains

Our work involved old bloodstains, dated 50 years and 60 years back respectively, on three blood-specific markers ALAS2, CD93, and HBB, together with two housekeeping genes (ACTB; 18S-rRNA). A total of 20 aged bloodstain samples stored in the Institute of Legal Medicine of Ferrara, Italy, were evaluated. Samples were divided into two sets: 10 samples (60 years group) and 10 samples (50 years group). DNA/RNA co-extraction was performed using a half of every trace. Remaining ones were tested using HemDirect Hemoglobin test (SERATEC®) to compare results. DNA/RNA were extracted using AllPrep DNA/RNA Mini Kit (QIAGEN®) adopting a modified protocol developed in the laboratory. For the reverse transcription reaction, random primers and RETROscript (Ambion®) were used. After cDNA quantification, samples were amplified using Multiplex PCR Mastermix (Qiagen®) according to the manufacturer's instructions. Primer sequences and concentrations were adopted from Ven den Berge et al. [1] and previously tested in an ISFG Italian Working Group - GEFI collaborative exercise. DNA was STR typed using the AmpFlSTR® NGM™ amplification kit (Thermo Scientific®). Detection of all amplified fragments was performed using an ABI PRISM 310 Genetic Analyzer (Thermo Scientific®). Results showed all bloodstains positive for mRNA identification. The most stable was HBB, returning positive results in over 80% of the 60 years and over 90% of the 50 years back samples. All membranes were all weakly positive for blood, except for five 60 years back samples. Full STR profiles were obtained for all blood samples.