The Use of Forensic Messenger RNA (mRNA) Analysis to Determine Stain Age

After attending this presentation, attendees will understand the
principles of forensic mRNA analysis and learn how it may be used as a
method to determine the age of biological stains found at crime scenes.
This presentation will impact the forensic science community by
proposing a novel method for determining the age of biological stains. The
proposed method could prove crucial for forensic casework and could run
parallel to the predominant DNA analysis method. Where DNA analysis is
used to identify a suspect, mRNA analysis could be used to determine the age
of biological stains, that in turn could place the suspect at the scene of a crime
at a specific time.
RNA has been presumed to be very unstable, as one of its features is that
it degrades rapidly as it is broken down by ribonucleases. In previous
literature however; it has been shown that RNA may be extracted from
biological stains up to 16-years-old. For stain age determination, it has been
suggested that using a decay rate ratio, derived from two endogenous controls
differentially expressed within the body fluid stain, should eliminate the
effect of any external decomposition factors. This should then be expressed
as a linear change in mRNA expression over time.
The majority of previous research concentrates on determining the age
of bloodstains. In this study, blood and saliva stains were used. The goal of
the study was to try to determine the age of these biological stains by isolating
mRNA and quantifying the expression of housekeeping genes 18s, GAPDH,
and ACTB also known as β-Actin. This study aims to demonstrate proof of
principle and explore any limitations to a decay rate ratio, to see if there is any
correlation between the age of a stain and the amount of genetic material
present. Blood andsaliva samples havebeencollected ona regular basis overthe
past two years. Bloodstains were prepared using the finger prick method and
depositing blood on to a sterile filter paper. Saliva stains were prepared by
swabbing the inside of the cheek with a buccal swab. All samples were taken
from healthy individuals. The samples were stored at room temperature and
protected from sunlight. Both blood and saliva samples were extracted using
Qiagen RNeasy Mini Kit with the appropriate modifications for each body
fluid. The extracted samples then underwent RNA quantitation, DNase
digestion, and reverse transcription using M-MLV reverse transcriptase and
random hexamers. The resulting complimentary DNA (cDNA) was then
quantified using absolute quantitation on a 7500 Fast Real-Time PCR
System, using pre-designed Taqman Gene Expression Assays for human
housekeeping genes 18s, GAPDH, and ACTB (β-Actin). Only two
housekeeping genes were run together at one time, this was to compare the
differences between all three, for example the decay rate ratio between 18s
and GAPDH as well as 18s with ACTB and then GAPDH with ACTB. The
two values from quantifying the two housekeeping genes were then
expressed as a ratio. The proof of principle would be demonstrated by a
linear downward expression of the two genes over time.
The results from initial experiments show that mRNA can be extracted
from stains up to two years old. Further experiments are being carried out to
obtain enough data to determine the proof of principle.
Future work will involve optimising the protocols and identifying the
shortest stain age per body fluid as well as exploring the use of more
housekeeping genes. With bloodstains being the current focus in most of the
literature, experiments will be carried out to determine the expression levels
of human housekeeping genes in semen to see if it is possible to identify the
time of deposition. Identifying the time a semen stain was deposited in a
sexual offence case could prove to be vital when trying to convict or
exonerate an individual.