Download Messenger RNA profiling: a prototype method to supplant

Messenger RNA profiling:
a prototype method to
supplant
conventional methods for
body fluid identification.
Bram Bekaert
Why identification of
body fluids?
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sexual assault involving vaginal
intercourse whereby the female victim at
the time of the assault is in menses
DNA from a victim is found on an
implement that is believed to have been
used in a sexual assault
sexual abuse of a young child by a
person living in the same residence as
the victim in which the suspect’s DNA is
found on the child’s clothing or bed linen
Existing identification
methods
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Histology
Serology
Biochemical tests (Ab-Ag and Enzsubstrate)
– immunoelectrophoresis
– iso-electric focusing
– ELISA
Disadvantages
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labour-intensive
used in series
costly in terms of time and sample
no confirmatory test for saliva and
vaginal fluids
urine id. can be problematic
organ tissues require histologists
Why use mRNA to identify
body fluids?
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terminally differentiated cells do not
express every gene located in their
nucleus
certain genes are turned off whereas
others are turned on
results in gene expression pattern
unique to each cell type
and of course: gene => mRNA
Advantages
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No multiplex for degraded proteins available
greater specificity than conventional
methods
simultaneous and semi-automatic
decreased sample consumption
compatibility with DNA extraction
methodologies
mRNA can be profiled parallel to DNA
RNA
Each cell has 2 ng RNA
1-3% RNA is mRNA
0.001 – 0.1% any particular transcript present
in mRNA pool
Method
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Gene expression of complementary DNA’s of
different tissues was monitored on microarrays
Blood, semen, saliva and vaginal samples
were collected
RNA and DNA extraction
DNase I digestion
cDNA synthesis
RNA amplified in RT-PCR and run on
agarose gel
RNA quantitation
RT-PCR
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reverse-transcriptase PCR
RNA => cDNA
Two different primers were used:
– oligo-dT primers
– random decamer primers
Genes studied
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3 housekeeping genes: S15, GAPDH,
β-actin
5 suspected tissue specific genes:
– Statherin
– Histatin 3
– PRB-1
– PRB-2
– PRB-3
Results
1. Visualisation of RNA
2. Detection of the
housekeeping mRNA’s
in all 3 bio. stains
RNA quantity and
stability
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Quantity:
– evaluated using varying quantities of input RNA
– 160 pg was enough to detect S15
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Stability:
– mRNA from β-actin was detectable up to 9
months when random decamers were used, 4
weeks for oligo-dT primers (poly A-tail)
Tissue specific mRNA
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candidate saliva specific genes obtained from
Cancer Genome Anatomy Project.
Statherin, histatin, PRB1, PRB2 and PRB3 were
amplified from saliva stain cDNA
Pseudogenes
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Pseudogenes= genes
resembling mRNA structure
but located in DNA
Control: amplify DNA, look for
bands at same regions as
mRNA
Location of ‘real genes’ =>
higher on gel as they are not
processed
Present status
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multiplex real-time PCR for id. of saliva,
blood, semen, vaginal secretions, menstrual
blood.
two body fluid-specific genes + 1 household
gene
no need for post-PCR processing or
electrophoresis
5’ nuclease assay to detect specific
amplimers
Future aims
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to develop assays to detect tissue-specific
genes to automate the id. of body fluids:
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blood
semen
saliva
vaginal secretions
skin
urine
muscle
adipose
brain
fecal matter
5-10 tissue specific genes per tissue type
Automation on ‘body fluid identification chip’
References
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Ballantyne, J. et al. (2003) Messenger RNA
profiling: a prototype method to supplant
conventional methods for body fluid
identification. For. Sci. Int. 135, 85-96
http://mafs.net/dalelaux/Meeting%20Presen
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