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RNA in forensic sciences 2012 Katalin Sipos Why is it difficult to introduce new methods? • Need for basic data – Normal controls with the same method • Sample collection – Not to forget – Availability – Special containers • Special problems – Previous diseases, time, temperature… RNA in Forensic Medicine • Tissue specific gene expression analysis • Degradation of RNA • Reactive changes of gene expressions Stability of RNA in postmortal tissues • • • • Differences between tissues (brain – liver) Suitable RNA for RT-PCR (96 h) Circumstances of death, hypoxia Could not find correlation between PMI and RNA degradation Identification of stains • Method – Isolation of RNA – cDNA synthesis – Detection • PCR + elfo • Real time PCR • Conditions – Amount and quality of RNA • Disadvantages – Pseudonegative RNA – cDNA – PCR, capillary elfo Mixture of 4 samples Each body fluid has its own expression pattern The fluorescence emission spectra of semen (—), skin oil (-··-··), blood (- - -), saliva (-·-·), and urine (-----) excited at 260 nm. This technique uses no destructive reagents and makes no contact with the sample. The emission lines are broad and the sample must be analyzed at several wavelengths to be distinguishable from other body fluids. 785 nm Raman spectroscopy: high intensity monochromatic light. Fotons of light suffer collisions and will produce scattering of light, which specific for the material Identification of body fluids • Blood and salive: most frequent • Other: ejaculate, vaginal secretum, lacrimal fluid, sweat • Coisolation of DNA: identification as well • Not tissue specific markers, but quantitative differences • Use a quality marker: low level of expression Age of a blood stain • Dry blood on scene • Isolation of RNA, cDNA synthesis • Rela time PCR – Ct value: number of the cycle where detectable signal enters threshold – Lower Ct means more cDNA and RNA • Ratio of 18 S RNA and beta-actin mRNA is going to change with time Course and circumstances of death • mRNA transcrip pattern changes because of cellular demand • At least 30 min • Possible biomarkers – Strangulation – Metamphetamine related death – Hypoxia – Mechanical asphyxiation – Do not forget degradation Forensic molecular pathology of violent death • Local molecular pathology – – – – Detection of vital reactions Estimation of wound age Discrimination of traumatic factors Pathogenesis, including tissue specific injury • Systemic molecular pathology – Pathophysiology of death process • Respiration, circulation, CNS – Estimation of survival time – Drugs, poisons, environmental hazards • No visible signs at autopsy • mRNA transcripts: level change, even within short time • „ideal model”: – Stable antemortem physiological status – Cause of death-specific mRNA level alterations – „freezing” of biological phenomena at the time of death (no supravital reactions) – Gradual paralell degradations of mRN transcripts Technical issues • Stability of RNA: tissue damage, general health, medical interventions • Find stable house keeping genes: glucose transporter, GAPDH • Age of victim: biological activity difference – large scale studies • Supravital reactions (early postmortem period): impossible to estimate • Species differences: animal experiments! Local molecular pathology • Morphological signs: bleading, blood clots, inflammatory cell invason • Early: mRNA level change – Inflammatory genes – Ischemia, hypoxia, apoptosis – Wounds: cytokine a chemokine mRNAs – Special proteins at brain injury Age and weight of brain correlation CH - suicide US Nederland US – homicide NL – homicide CH Ration of the two /100 000 pop Population-based incident rates of homicide–suicide, homicide and suicide over time in the Netherlands (1992–2006), Switzerland (1992–2004) and the United States (2004–2006). homicide–suicide, homicide and suicide incident rates per 100,000 per country Netherlands Switzerland United States Homicide rate 1.38 0.87 5.06 Suicide rate 9.71 19.35 10.76 0.09 0.22 Homicide– 0.05 suicide rate SEM in exhumation • A 43 year-old white man was watching a football game at a sugarcane farm when he had a misunderstanding with another fan, a fact that was witnessed by many people. After the game, the two men met on a public way and had a further misunderstanding. According to the report of the suspect, he pushed the victim, who fell and hit his head on a stone. He denied the use of any instrument during the fight with the victim • In this article, we report an exhumation case of a homicide victim, who had been buried three and a half years ago, in which it was necessary to investigate a fracture found on the interior surface of the exhumed skull.