Download RNA in forensic sciences

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.