medoct group, Medical University Vienna, Jack Ballantyne, PhD National Center for Forensic Science, Orlando, FL USA

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1 medoct group, Medical University Vienna, Jack Ballantyne, PhD National Center for Forensic Science, Orlando, FL USA Association of Forensic DNA Analysts and Administrators San Antonio, TX June 2012

2 Perceived to be the result of DNA obtained from shed skin cells STR profiles can be obtained Tissue source not typically confirmed Lack of molecular based strategies for skin identification Some claim that identification of tissue source of origin from trace biological material is not possible Failure to identify tissue source could have undue influence on perception of the circumstances of the crime Aim: identification of skin using RNA biomarkers mrna and mirna 2

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4 Literature searches ( biased approach) Whole transcriptome sequencing (RNAseq) ( unbiased approach) Human skin total RNA samples Two donors, each run in duplicate Comparison with two vaginal secretions samples Sequence data compared to NCBI reference database Count values indicate expression levels ( DGE ) Our strategy: genes with high counts in skin but low counts in vaginal secretions 4

5 * literature 20 RNAseq *some candidates rejected after sequence review since suitable primers could not be designed higher count values in skin samples compared to vaginal samples (other epithelial cell containing fluid) + ID d from literature search; confirmed with RNAseq data once available ++ ID d from RNAseq data directly 5

6 LCE 1C, 1D, 2D Late Cornified Envelope genes Groups 1 and 2 (in contrast to Group 3) are dominant in skin (external epithelia) Vulva intermediate expression (between internal/external) CCL27 Chemokine Most abundant expression of CCL27 observed in keratinocytes of epidermal basal layers recruits CLA+ memory T cells to normal or inflamed skinbinds to receptor CCR10 IL1F7 Interleukin 1 family, member 7 (IL37) Predominant role in suppression of inflammatory responses IL1F7 synthesized by, inter alia, keratinocytes 6

7 LCE1C (RT+) 15ng (RT-) 15ng LCE1D (RT+) 25ng (RT-) 25ng LCE2D (RT+) 25ng (RT-) 25ng CCL27 (RT+) 25ng (RT-) 25ng IL1F7 (RT+) 25ng (RT-) 25ng 7

8 Only small number of positives in other fluids Signals still significantly less than skin (10-45 X less) Some samples were not quantitated Possible that some samples came in contact with external surfaces or hands during collection? 8

9 * Human skin total RNA samples * Header colors indicate CE channel 9

10 Specificity of all markers re-evaluated Optimal input based on sensitivity data No cross-reactivity observed with the other body fluids using this input * * *re-designed primers 10

11 > 50 RFUs RNA Isolation: Manual, AllPrep, RNeasy, Pin Point ~50% success rate 11

12 Glove, female Fingerprint - glass Coffee Pot Handle Forehead Candy jar lid 12

13 Multiple products obtained with initial primer set Possible amplification of other variants/isoforms? Sequencing of expected-sized product confirms LCE1C Primer set re-designed to improve specificity Still two products (56/58bp), but improved sensitivity Original primers New primers 13

14 100% detection in sample set (N=12) with the redesigned LCE1C primer set Small sample size need to test additional samples 14

15 High sensitivity of redesigned LCE1C Need to ensure that sensitivity is not too great False positives Areas presumed to be negative for skin contact were tested 1 st amp hood False positive, since re-testing of multiple areas all negative Base board Reagents stored in this area Possible that skin cells are present in this area 15

16 4-plex With alternative polymerase (Advantage HD, Clontech) 16

17 Skin 25ng RT+ 25ng RT- *Pentaplex using Qiagen Multiplex PCR kit 17

18 Visser et al (Int J Leg Med (2011), 125 (2): p253-63) CDSN, LOR, KRT9 Report strong over-expression in skin samples relative to samples from forensic body fluids Detection in thumbprints High Avg. Ct values LOR ~36, CDSN ~ 35, KRT9 ~ 36, ACTB~36 We had previously evaluated CDSN, LOR, KRT9 CDSN detection in vaginal secretions and menstrual blood Gel based assays LOR similar expression levels in vaginal secretions Using same ABI real time PCR assay and gels KRT9 only detectable with >25ng? 18

19 Evaluated > 100 potential skin mrna biomarkers 5 highly specific for skin LCE1C, LCE1D, LCE2D, CCL27 and IL1F7 Sensitivity Most detected down to 5 25pg total RNA Touch/Contact Samples Skin samples: Face, arm, forehead, knee, foot Touch/contact samples: Mouse, keyboard, phone handles, coffee pot handle, pencil, thumbprint, inside of glove LCE1C most sensitive for real touch samples Multiplex development 2 assays: 4plex and 5plex 19

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21 Touch/Contact samples Test additional samples-also correlate with STR results Optimize RNA recovery Characterize the amount and quality of RNA In progress Some preliminary data demonstrating the ability to obtain DNA and RNA profiling results from touch/contact samples Develop mirna assays for the identification of skin 21

22 Samples: Sterile cotton swab moistened with nuclease free water Surface of touched/contacted surfaces and items swabbed Whole swab used for extraction Extraction: Organic co-extraction developed by NCFS Aqueous layer split into DNA and RNA fractions Processed separately to obtain DNA and total RNA Analysis DNA Identifiler, 12.5 ml reaction, cycles RNA housekeeping gene, LCE1C singleplex 22

23 Many samples with no DNA profile, but LCE1C detected 23

24 LCE1C and B2M detected No DNA profile obtained 28 cycles 36 cycles 24

25 DNA profile and skin marker (LCE1C) ALL samples All mixture profiles (DNA) 25

26 * + LCE1C and B2M detected DNA profile of primary pen user + * Allele Drop-in + Allele Drop-out Donor profile confirmed with reference profile 26

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28 Explosion of interest in a newly discovered class of small RNAs micrornas ~20-25 nt in length May be useful in the analysis of degraded or compromised forensic samples Reports in current literature of tissue specific mirnas Possibility to discover mirnas specific to forensically relevant biological fluids Blood, semen, saliva, vaginal secretions, menstrual blood, skin 28

29 Original screening: mirbase v mirnas Didn t include skin Screening of new mirnas for novel candidates mirbase v 11.0 Additional 364 mirnas mirbase v.13 and v.14 Additional 30 mirnas mirbase v.15 and v.16 Additional 352 mirnas Total = 1,198 mirnas All body fluids: blood, semen, saliva, vaginal, menstrual, skin 29

30 mir-451 mir-16 mir-135b mir-10b mir-658 mir-205 mir-124a mir-372 mir-451 mir-412 Normalization with RNU6b 30

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32 Axes are DCt of mirna candidate and normalizer 32

33 Axes are DCt of mirna candidate and normalizer 33

34 NEW High Spec buffer 34

35 Expression may not be the same in deep vs. surface layers Test touch/contact samples to determine if expression of skin mirna candidates can be detected Samples Door handle, water faucet, water fountain button, computer mouse, pen, phone, laptop surface, drinking container, stair railing Arm as a positive control 35

36 1) door-321; 2) water faucet; 3) mouse-322; 4) water fountain; 5) door-office; 6) phone-322; 7) pen; 8) laptop; 9) drink container; 10) stair railing; 11) arm; 12) door frame 36

37 Validation of novel mirna assays New candidates, new miscript II RT kit Sensitivity, specificity, mock casework samples Comparison to mrna Environmentally compromised or degraded samples Touch/contact samples Evaluate input into RT Evaluate skin assay 2 Novel candidate screening? mirbase now v. 18 New mirnas deposited New potential candidates? 37

38 Erin Hanson NCFS Also Samantha Nelson, Laura Guzman Cordula Haas and Rinaldo Jucker Institute of Legal Medicine, Zurich, Switzerland Funding: State of Florida NIJ 2008-DN-BX-K007, 2010-DN-BX-K139 This project was supported by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. The opinions, findings, and conclusions or recommendations in this presentation are those of the author(s) and do not necessarily reflect those of the Department of Justice. 38