Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identification. Bram Bekaert

Why identification of body fluids? sexual assault involving vaginal intercourse whereby the female victim at the time of the assault is in menses 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 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 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 Histology Histology Serology Serology Biochemical tests (Ab-Ag and Enz- substrate) Biochemical tests (Ab-Ag and Enz- substrate) –immunoelectrophoresis –iso-electric focusing –ELISA

Disadvantages labour-intensive labour-intensive used in series used in series costly in terms of time and sample costly in terms of time and sample no confirmatory test for saliva and vaginal fluids no confirmatory test for saliva and vaginal fluids urine id. can be problematic urine id. can be problematic organ tissues require histologists organ tissues require histologists

Why use mRNA to identify body fluids? terminally differentiated cells do not express every gene located in their nucleus terminally differentiated cells do not express every gene located in their nucleus certain genes are turned off whereas others are turned on certain genes are turned off whereas others are turned on results in gene expression pattern unique to each cell type results in gene expression pattern unique to each cell type and of course: gene => mRNA and of course: gene => mRNA

Advantages No multiplex for degraded proteins available No multiplex for degraded proteins available greater specificity than conventional methods greater specificity than conventional methods simultaneous and semi-automatic simultaneous and semi-automatic decreased sample consumption decreased sample consumption compatibility with DNA extraction compatibility with DNA extraction methodologies methodologies mRNA can be profiled parallel to DNA mRNA can be profiled parallel to DNA

RNA Each cell has 2 ng RNA 1-3% RNA is mRNA – 0.1% any particular transcript present in mRNA pool

Method Gene expression of complementary DNA’s of different tissues was monitored on micro- arrays Gene expression of complementary DNA’s of different tissues was monitored on micro- arrays Blood, semen, saliva and vaginal samples were collected Blood, semen, saliva and vaginal samples were collected RNA and DNA extraction RNA and DNA extraction DNase I digestion DNase I digestion cDNA synthesis cDNA synthesis RNA amplified in RT-PCR and run on agarose gel RNA amplified in RT-PCR and run on agarose gel RNA quantitation RNA quantitation

RT-PCR reverse-transcriptase PCR reverse-transcriptase PCR RNA => cDNA RNA => cDNA Two different primers were used: Two different primers were used: –oligo-dT primers –random decamer primers

Genes studied 3 housekeeping genes: S15, GAPDH, β-actin 3 housekeeping genes: S15, GAPDH, β-actin 5 suspected tissue specific genes: 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 Quantity: Quantity: –evaluated using varying quantities of input RNA –160 pg was enough to detect S15 Stability: 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 candidate saliva specific genes obtained from Cancer Genome Anatomy Project. candidate saliva specific genes obtained from Cancer Genome Anatomy Project. Statherin, histatin, PRB1, PRB2 and PRB3 were amplified from saliva stain cDNA Statherin, histatin, PRB1, PRB2 and PRB3 were amplified from saliva stain cDNA

Pseudogenes Pseudogenes= genes resembling mRNA structure but located in DNA Pseudogenes= genes resembling mRNA structure but located in DNA Control: amplify DNA, look for bands at same regions as mRNA Control: amplify DNA, look for bands at same regions as mRNA Location of ‘real genes’ => higher on gel as they are not processed Location of ‘real genes’ => higher on gel as they are not processed

Present status multiplex real-time PCR for id. of saliva, blood, semen, vaginal secretions, menstrual blood. multiplex real-time PCR for id. of saliva, blood, semen, vaginal secretions, menstrual blood. two body fluid-specific genes + 1 household gene two body fluid-specific genes + 1 household gene no need for post-PCR processing or electrophoresis no need for post-PCR processing or electrophoresis 5’ nuclease assay to detect specific amplimers 5’ nuclease assay to detect specific amplimers

Future aims to develop assays to detect tissue-specific genes to automate the id. of body fluids: to develop assays to detect tissue-specific genes to automate the id. of body fluids: blood blood semen semen saliva saliva vaginal secretions vaginal secretions skin skin urine urine muscle muscle adipose adipose brain brain fecal matter fecal matter 5-10 tissue specific genes per tissue type 5-10 tissue specific genes per tissue type Automation on ‘body fluid identification chip’ Automation on ‘body fluid identification chip’

References Ballantyne, J. et al. (2003) Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identification. For. Sci. Int. 135, Ballantyne, J. et al. (2003) Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identification. For. Sci. Int. 135, tations/FB/Jane%20Juusola/Joint%20Meetin g%202004%20Abstract%20Body%20Fluid %20ID%20Real%20time%20PCR.pdf tations/FB/Jane%20Juusola/Joint%20Meetin g%202004%20Abstract%20Body%20Fluid %20ID%20Real%20time%20PCR.pdf