1 st Mae Fah Luang University International Conference 2012 1 EVALUATION OF ALPHA-GLOBIN MRNA AS A MOLECULAR MARKER FOR IDENTIFICATION OF HUMAN BLOODSTAINS Unyarat Tuckprakhon, Khemika Lomthaisong * Forensic science program, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand * e-mail: Khemlo@kku.ac.th Abstract Identification of questioned stain collected from crime scene as whether or not human blood is meaningful for DNA analysis. Anti-human serum and human specific DNA sequence had been successfully used for human bloodstain identification. However, none of these had information on the age estimation of bloodstain. Therefore, this research aims to evaluate alpha-globin mRNA as a molecular marker for both human blood identification and age estimation of bloodstain. The oligonucleotide primers of human alpha-globin mRNA were designed using Primer3. The designed primers were then blast with alpha-globin mRNA from four animal species (cow, pig, dog and chicken) using Primer-Blast. No sequence homology was found which confirmed that amplification of non-specific sequence would not be occurred. PCR reactions were then performed using synthesized human and animal cDNA as DNA template. Interestingly, a 168 bp amplified fragment was observed only in human blood sample. This result suggested that human blood identification was successfully performed by using alpha-globin mRNA as a molecular marker. However, for the age estimation of bloodstain, further investigation is needed. Keywords: Human alpha-globin mRNA, Human blood identification, Forensic science Introduction Identification and age estimation of human bloodstains is valuable for investigator in order to 1) reduce number of samples used for DNA analysis 2) exclude suspect from the investigation and/or 3) disprove statement of suspects in which they insist that their bloodstains presented before the crime has occurred. The analyses of bloodstains age were mostly based on the changes of hemoglobin to its derivatives which could be observed by the color change and the solubility of bloodstains (Anderson et al. 2005). In addition, the activities of enzymes and proteins found in bloodstains were also used to detect the age of bloodstain (Rajamannar 1977). However, none of these methods could discriminate human blood from animals and their results also depended on the quantity of samples. Therefore, if any of methods can 1) identify human bloodstains 2) estimate the age of bloodstain and 3) analyze sample in a small quantity, in a single step, it will be very useful. Although, studies had shown that DNA is a good source for species identification and the results can be obtained even small amount of sample is present (Crouse and Schumm 1995; Nakaki et al. 2007). But, DNA lacks information on age estimation due to its stability. Therefore, mRNAs have been used to estimate the age of bloodstains because it degrades with time. Previous study had shown that the age of bloodstain could be determined by the reduction of -actin mRNA number using real-time RT-PCR (Anderson et al. 2005). Hence, this study aims to use human alpha-globin mRNA as a target for identification and age