DNA co-analysis from blood stains—Results of a second collaborative EDNAP exercise

Forensic Science International: Genetics 6 (2012) 70–80

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RNA/DNA co-analysis from blood stains—Results of a second collaborative EDNAP exercise C. Haas a,*, E. Hanson b, M.J. Anjos m, W. Ba¨r a, R. Banemann i, A. Berti g, E. Borges p, C. Bouakaze f, A. Carracedo q, M. Carvalho m, V. Castella t, A. Choma e, G. De Cock s, M. Do¨tsch i, P. Hoff-Olsen j, ˜ as q, D. Moore r, M.-L. Morerod t, P. Johansen h, F. Kohlmeier o, P.A. Lindenbergh c, B. Ludes f, O. Maron h k s k d N. Morling , H. Niedersta¨tter , F. Noel , W. Parson , G. Patel , C. Popielarz p, E. Salata g, P.M. Schneider o, T. Sijen c, B. Sviezˇena e, M. Turanska´ l, L. Zatkalı´kova´ l, J. Ballantyne b a

Institute of Legal Medicine, University of Zu¨rich, Switzerland National Center for Forensic Science, University of Central Florida, Orlando, USA c Netherlands Forensic Institute, The Hague, The Netherlands d Forensic Science Service, Birmingham, UK e Institute of Forensic Science, Department of Criminalistic Biology and Genetic Analysis, Bratislava, Slovakia f Institute of Legal Medicine, University of Strasbourg, France g Reparto Investigazioni Scientifiche di Roma, Rome, Italy h Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health Sciences, University of Copenhagen, Denmark i Bundeskriminalamt, Wiesbaden, Germany j Institute of Forensic Medicine, University of Oslo, Norway k Institute of Legal Medicine, Innsbruck Medical University, Austria l Institute of Forensic Sciences, Ministry of the Interior, Department of Biology and DNA analysis, Slovenska´ Lupca, Slovakia m Forensic Genetic Service, Centre Branch, National Institute of Legal Medicine, Portugal o Institute of Legal Medicine, University Hospital, University of Cologne, Germany p Institut National de Police Scientifique, Laboratoire de Police Scientifique de Lyon, France q Forensic Genetics Unit, Institute of Legal Medicine, University of Santiago de Compostela, Spain r LGC Forensics, Middlesex, UK s National Institute for Criminalistics and Criminology, Brussels, Belgium t Unite´ de ge´ne´tique forensique, Centre universitaire romand de me´decine le´gale, Lausanne et Gene`ve, Switzerland b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 22 September 2010 Received in revised form 7 February 2011 Accepted 21 February 2011

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5–0.001 ml blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology. ß 2011 Elsevier Ireland Ltd. All rights reserved.

Keywords: Forensic science Body fluid identification Blood RNA/DNA co-extraction EDNAP exercise mRNA profiling

1. Introduction * Corresponding author at: Institute of Legal Medicine, Forensic Genetics, University of Zu¨rich, Winterthurerstrasse 190, 8057 Zu¨rich, Switzerland. Tel.: +41 44 635 56 56; fax: +41 44 635 68 58. E-mail address: [email protected] (C. Haas). 1872-4973/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigen.2011.02.004

The analysis of cell-specific mRNA expression has been proposed as a promising method for the identification of body fluids [1–17], even for the analysis of old and environmentally

C. Haas et al. / Forensic Science International: Genetics 6 (2012) 70–80

compromised samples [18–20]. Previously, a first collaborative exercise was performed by the European DNA Profiling Group (EDNAP) in order to evaluate the robustness and reproducibility of mRNA profiling for blood identification using the 3 blood-specific markers HBB, SPTB and PBGD [21]. Fifteen of sixteen laboratories, most of which had no prior experience with RNA, were able to successfully isolate and analyze RNA from the provided samples. While sensitivity varied between laboratories, the method proved to be reproducible and sensitive using different analysis strategies. The initial exercise was limited to three blood specific-markers using singleplex reactions. Additional putative blood-specific markers have been reported in the forensic literature, some of which may provide increased sensitivity compared to the markers included in the first EDNAP study. Therefore, in a further study, additional mRNA blood markers (alpha- and beta-subunits of hemoglobin A (HBA, HBB), aminolevulinate synthase 2 (ALAS2), CD3 gamma molecule (CD3G), ankyrin 1 (ANK1), the erythroid form of porphobilinogen deaminase (PBGD), b-spectrin (SPTB) and Aquaporin 9 (AQP9)) were critically evaluated and compared in terms of sensitivity, specificity and performance with casework samples [22]. Despite differences in the observed sensitivity and specificity of the blood markers examined in this study, a number of the candidate mRNA markers appeared to be suitable for forensic purposes. This resulted in the development of two multiplex systems, a ‘high sensitivity’ duplex (HBB, HBA) and a ‘moderate sensitivity’ pentaplex (ALAS2, CD3G, ANK1, PBGD and SPTB) [22]. A second collaborative exercise was organized by the Institute of Legal Medicine, University of Zu¨rich, Switzerland, on behalf of the European DNA Profiling Group (EDNAP), in order to implement a RNA/DNA co-extraction strategy and to test additional forensically suitable blood markers. The inclusion of the RNA/DNA coextraction strategy in this second exercise allowed for an evaluation of the simultaneous analysis of DNA profiles from blood stains without consumption of additional sample. Blood is a frequently encountered body fluid in forensic analyses and most laboratories had previous experience with the RNA extraction of blood from the first EDNAP mRNA exercise. The previously developed blood duplex and pentaplex systems [22] were utilized in this study to provide participants with the ability to evaluate a set of mRNA biomarkers that possess either ‘moderate’ (pentaplex) or ‘high’ (duplex) sensitivity’ while concomitantly evaluating their use with bona fide forensic casework samples. 2. Materials and methods 2.1. Samples and materials provided The organizing laboratory (Institute of Legal Medicine, University of Zu¨rich, Switzerland) sent 6 blood stains (1–6) and two blood dilution series (5, 1, 0.1, 0.01, 0.001 ml) on cotton swabs (A) and on FTA paper (B) to the participating laboratories. Fresh blood and EDTA blood samples were collected from 7 healthy volunteers. Two non-EDTA treated blood samples were immediately deposited onto a white cotton T-shirt (washed once with detergent at 95 8C) and dried overnight. Subsequently, 5 mm (stain 1) and 2 mm (stain 4) circles were punched. Stain 2 was 10 ml EDTA blood on a bandage, stain 3 was 10 ml EDTA blood on a swab, stain 5 was 10 ml non-treated blood on a swab stored for 3 years at room temperature in the dark (in box, stored in lab), stain 6 was EDTA blood on white cotton cloth stored for 11 years at room temperature in the dark (in envelope, stored in office). For the dilution series, EDTA blood was diluted in 0.9% NaCl to a final volume of 5 ml per sample and placed on either swabs or Whatman FTA cards (Roth AG, Arlesheim, Switzerland). The laboratories were

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requested, but not required, to examine a few additional samples: bona fide or mock casework material that could include human and/or non-human blood stains, or other forensically relevant body fluids (semen, saliva, vaginal secretions or menstrual blood). HPLC-purified primers were purchased from Microsynth (St. Gallen, Switzerland). The organizing laboratory prepared primer mixes for the two multiplexes and aliquots were provided to the participating laboratories for use in amplification reactions. The primers and samples were sent by regular post since pre-testing indicated stability of the primer mixes for at least 2 weeks at room temperature. 2.2. RNA(/DNA co-)extraction and reverse transcription The participating laboratories were asked to use the entire swabs or stains for extraction (RNA only or RNA/DNA coextraction). The organizing laboratory provided an example protocol for extraction and reverse transcription. The laboratories could, however, use methods of their own choice. The example protocol included the following: total RNA and DNA is extracted with the AllPrep RNA/DNA Mini Kit (Qiagen, Hombrechtikon, Switzerland) according to the manufacturer’s instructions with the following adaptations: the piece of stain is placed in 345 ml RLTbuffer +5 ml Carrier-RNA in a DNA LoBind tube (VaudauxEppendorf, Scho¨nenbuch, Switzerland) and incubated up to 3 h at 56 8C. Spin baskets may be used to recover the supernatant and remove the fabric substrate. The RNase-free DNase Set (Qiagen) is used for on-column DNA digestion. RNA is eluted in 30 ml H2O and DNA in 80 ml EB buffer. For the reverse transcription reaction, the Superscript III reverse transcriptase system (Invitrogen, Basel, Switzerland) is used according to the manufacturer’s instructions: 16 ml RNA, 2 ml random primers (50 ng/ml), 2 ml 10 mM dNTPs are mixed and incubated 5 min at 65 8C, then placed on ice for 1 min. 18 ml cDNA Synthesis Mix (4 ml 10 RT buffer, 8 ml MgCl2, 4 ml 0.1 M DTT, 2 ml RNase Out (40 U/ml)) is added to the RNA/primer solution, mixed gently and briefly centrifuged. 19 ml are pipetted into a new tube and 1 ml Superscript III is added (RT+). 1 ml H2O is added to the first tube (RT minus, without reverse transcriptase), which is a control for genomic DNA contamination. The samples are incubated 10 min at room temperature, then 50 min at 50 8C. The reaction is terminated at 85 8C for 5 min. cDNA is obtained in a final volume of 20 ml. 2.3. Endpoint PCR The previously published primer sets were used for the duplex (HBB – 61 bp, HBA – 112 bp) and the pentaplex (CD3G – 154 bp, ANK1 – 165 bp, ALAS2 – 133 bp, PBGD – 177 bp, SPTB – 247 bp) [22]. The forward primers were 50 -labelled with 5-FAM. The following amplification conditions were recommended: (duplex) – the 25 ml reaction mix contains 3 ml cDNA, 2.5 ml primer mix (see below), 1 mM dNTPs (Applied Biosystems – AB), 1 Gold Buffer (AB), 3 mM MgCl2 (AB), and 1.5 U AmpliTaq Gold DNA Polymerase (AB); (pentaplex) – the 25 ml reaction mix contains 5 ml cDNA, 2.5 ml primer mix (see below), 1 mM dNTPs (AB), 1 Gold Buffer (AB), 3 mM MgCl2 (AB), and 1.5 U AmpliTaq Gold DNA Polymerase (AB). Sterile water is used in place of cDNA for non-template controls. The primer mixes were prepared using the following concentrations: (duplex) – HBB 0.1 mM, HBA 0.1 mM, (pentaplex) – ALAS2 0.15 mM, CD3G 0.25 mM, ANK1 0.2 mM, PBGD 0.8 mM, SPTB 0.25 mM. The initial denaturation is at 95 8C for 11 min, followed by 25 cycles (duplex) or 35 cycles (pentaplex) of 94 8C 20 s, 57 8C (+0.2 8C per cycle) 30 s, 72 8C 40 s and the final elongation at 72 8C for 60 min. Post PCR purification was suggested as an option, to eliminate dye blobs (e.g. MinElute PCR purification kit, Qiagen) [22,23].

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2.4. Capillary electrophoresis (CE) The participating laboratories were able to use standard, multicolor fluorescent genetic analyzers and standard electrophoresis conditions for the detection of the blood specific amplicons. Any dye set that included 5-FAM, with associated internal lane standard, could be used. Raw data were analyzed with the Genescan/Genotyper or Genemapper Software (AB). The threshold for a positive result was arbitrarily set to 100 RFUs, which is generally higher than that for STR typing, because of a noisy baseline mainly due to disassociated dye (‘dye blobs’). 2.5. DNA-amplification and -detection If DNA was co-extracted, the laboratories were able to use a standard STR typing kit, PCR and CE conditions of their choice. A peak detection threshold of 50 RFUs was used.

3. Results The participating laboratories were asked to complete a questionnaire describing the methods they used (Table 1). The primers and samples took up to 14 days for delivery by regular post. The 18 laboratories used 6 different kits for the RNA only or RNA/DNA co-extraction, 5 different reverse transcription kits, 9 slightly modified PCR protocols for RNA analysis, 6 different STR typing kits for DNA analysis, 4 different thermocyclers, 3 different genetic analyzers and 3 different polymers (Table 1). 3.1. RNA results The mRNA profiling results are summarized in Table 2. A representative electropherogram is shown in Fig. 1a, b. All participating laboratories were able to perform the method successfully as demonstrated by detection of the RNA blood markers. All 7 markers were detected by more than half of the laboratories for stains 1–3. A few of the markers in the moderate sensitivity pentaplex were not detected for stains 4–6, likely due to the small size (2 mm circle) or age of the stain (3–11 years old). The high sensitivity blood markers HBA and HBB were detected in all stains by more than half of the laboratories. HBA and HBB were detected in as little as 0.1 ml blood, the other markers (pentaplex) in as little as 1 ml blood by more than half of the laboratories (dilution series A – swabs). This sensitivity-level was not obtained for dilution series B (FTA paper) where HBA and HBB were detected only down to 1 ml blood and the other markers (pentaplex) down to 5 ml blood. Despite the overall sensitivity results obtained by a majority of laboratories, a few laboratories were able to detect single markers, namely HBA, HBB and CD3G, in the 0.001 ml samples. Amplicon size estimates exhibited inter-laboratory variation as expected (2 bp) due to differences in the instruments and polymers used. Additional factors could be temperature, sizing standards and/or algorithms. No peaks were detected in the RT minus (no reverse transcriptase added) or PCR negative controls by a majority of the laboratories that included such controls. Only one laboratory (laboratory 16) did not include RT minus controls. Laboratories 4 and 13 had some problems with the RT minus and PCR negative controls. Laboratory 13 detected around 50 smaller peaks in HBB RT- than HBB RT+. This finding was not reproducible with HBB singleplex testing, indicating a possible issue with the multiplex PCR reaction for this laboratory that had used slightly modified reaction conditions. Laboratory 4 observed amplified products in negative controls (extraction negative control, RT minus and PCR negative control) for CD3G, PBGD and ALAS2, possibly due to

contamination (potential sources of contamination were identified and could be modified to prevent future occurrences). Due to an alternative random hexamer concentration amount suggested in the instructions, 4 laboratories used reduced amounts of random hexamers for the RT reaction (2.5 ng instead of 50 ng per sample). Three of these laboratories (laboratories 4, 5 and 6) obtained satisfactory results using this lower primer concentration (detection of single blood markers down to 0.1, 0.01 and 0.1 ml blood for dilution series A and down to 5, 0.1 and 0.1 ml blood for dilution series B). Laboratory 3 that had obtained poor results using the lower primer concentration (detection of single blood markers down to 5 ml blood for dilution series A and B) repeated the RT reaction with increased random hexamers and obtained somewhat improved results (detection of single blood markers down to 1 ml blood for dilution series A and B). Post-PCR purification resulted in increased peak heights (Fig. 2) and reduced baseline signal noise. The removal of interfering anionic species (such as dye blobs and primers, etc.) will result in a greater quantity of analyte (in this case amplified target DNA from a cDNA) being electro-kinetically injected onto the capillary. Post PCR purification did not allow for detection of additional markers that were not observed prior to post-PCR purification. Improvement in signal intensity after post-PCR purification was largely observed for low template samples (stains 4–6 and dilution series B (5, 1, 0.1 ml blood on FTA paper)). Fig. 2 shows representative pentaplex results before and after post PCR purification, analyzed by 2 different laboratories. 3.2. DNA results Thirteen laboratories performed a RNA/DNA co-extraction. The DNA profiling results are shown in Table 3 and a representative electropherogram is shown in Fig. 1c. No specifications for DNA analysis were provided and therefore various STR typing kits and cycle numbers were utilized amongst the participating laboratories. While direct sensitivity and success rate comparisons could not be made, these results demonstrate that DNA of sufficient quantity and quality for STR analysis can be simultaneously extracted with RNA from small amounts of dried blood. Full STR profiles were obtained for stains 1–6 from most laboratories, stains 2, 3 and 5 almost without allelic/locus drop outs (Table 3). The obtained genotypes were confirmed by comparison to reference profiles. Full profiles (except for one drop-out in D3S1358) were obtained from dilution series A (on swabs) from 1 ml blood stains. Partial profiles were obtained with as little as 0.1 ml blood. Single loci genotypes were even observed by some laboratories in 0.001 ml blood stains. Results for dilution series B (on FTA paper) were slightly worse, showing full profiles for 5 ml blood, partial profiles for 1 ml blood and single loci down to 0.01 ml blood, indicating that the FTA paper is a challenging medium from which to obtain DNA profiles from low quantities of dried blood stains. Laboratory 15 obtained improved sensitivity with DNA precipitation (75 ml DNA were precipitated, pellet taken up in 10 ml H2O and completely used in one PCR) and increased injection voltage/ time (normal: 3 kV/15 s, increased: 9 kV/15 s) [24]. 3.3. RNA and DNA results of the optional stain samples The laboratories were invited to analyze additional samples including bona fide and mock casework samples, non-human blood samples and non-blood body fluid samples (Table 4 and Figs. 3 and 4). Twenty-seven blood stains were analyzed by 9 different laboratories. Four of these laboratories reported lower sensitivities for the mandatory samples compared to a majority of laboratories and therefore optimal results were probably not obtained from the additional samples tested by these laboratories. HBA and HBB were

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Table 1 Summary of the kits, chemicals, quantities and instrumentation used by the participating laboratories. Method Zu¨rich

Methods used by the participating laboratories

Lab no.

1. RNA-extraction RNA extraction kit used

Allprep RNA/DNA Mini Kit (Qiagen)

* 5,11 17 1 13 15

Volume lysis buffer Carrier RNA used

350 ml Yes

Incubation in lysis buffer at 56 8C Spin baskets used

Stains 5 and 6: 3 h Yes

DNase digestion

Yes, RNase-Free DNase Set (Qiagen)

Elution in

30 ml H2O

12 Allprep RNA/DNA Mini Kit (Qiagen) 2 RNeasy micro Kit (Qiagen) 1 RNeasy mini Kit (Qiagen) 1 PureLink RNA mini Kit (Invitrogen) 1 RNAqueous Kit (Ambion) 1 mirVana miRNA isolation Kit (Ambion) 100–500 ml 11 yes 7 no some stains up to 5 h 10 no 1 QiaShredder (Qiagen) 7 yes 13 RNase-Free DNase Set (Qiagen) 1 TURBO DNA-free Kit (Ambion) 4 no 15 30 ml H2O 2 24 ml H2O 1 100 ml ES buffer

2. DNA-extraction Elution in

80 ml EB buffer

PCR kit used

SGM Plus (AB)

* 15,16,18 3,8,12,16 6,9,15,19 10,14 2 4 18

Volume DNA in PCR Total PCR volume

5 ml 25 ml

50–200 ml EB buffer (Qiagen) 3 concentrated to 10–25 ml 4 SGM Plus (AB) 4 Identifiler (AB) 2 SEfiler plus (AB) 1 NGM (AB) 1 PowerPlex16 (Promega) 1 PowerPlex16 HS (Promega) 0.5–10 ml or 0.5–3 ng 9 25 ml 4 10–12.5 ml

3. Reverse transcription Volume RNA in RT RT kit used

11 ml for RT+, 11 ml for RT Superscript III (Invitrogen)

RT primers

Random hexamers (Invitrogen)

Amount

50 ng/sample

RNase inhibitor used

RNaseOUT (Invitrogen)

Volume cDNA (RT+ and RT) 4. PCR PCR buffer

20 ml RT+, 20 ml RT

cDNA

2plex: 3 ml, 5plex: 5 ml

Total PCR volume

25 ml

Thermal cycling protocol

As provided

Thermocycler

PCR system 9700 (AB)

Post PCR purification

Yes, MinElute (Qiagen)

Elution volume after post PCR purification 5. Capillary electrophoresis Genetic Analyzer

30 ml

Polymer

POP4

PCR Gold buffer (AB)

Genetic Analyzer 3130xl (AB)

*Denotes all labs except those listed below in the same section.

0.3–11 ml 12 Superscript III (Invitrogen) 2 First Strand cDNA Synthesis Kit (Novagen) 2 RetroScript (Ambion) 1 High capacity cDNA RT Kit (AB) 1 PrimeScript (Takara) 12 random hexamers (Invitrogen) 6 others (included in kits) 15 50 ng or 5 mM/sample 3 2.5 ng/sample 15 yes 3 no 20 ml RT+, 20 ml RT

* 1,9,13–16,19 * 16 1,4,6,8,15,17,19 * 19 1,8,13,15 * 5,13 15

* 4,10,16,18

* 17,18 13,15 5 16

* 4,5,6 * 10,17,18

13 PCR Gold buffer (AB) 3 PCR buffer I (AB) 2 PCR buffer II (AB) 13 as suggested 5 variations (duplex: 1–8 ml, pentaplex: 1–12 ml) 17 25 ml 1 10 ml 16 as provided 1 as exercise 1 [21] 13 PCR system 9700 (AB) 3 Eppendorf Mastercycler 1 PCR system 2700, 2720 (AB) 1 DuoCycler (VWR) 7 MinElute (Qiagen) 11 no 10–30 ml

* 6,10,12 14,19 * 2,10,13,15,17 * 13 * 13 * 12,17,18 4 2 1,4,5,6,9,12,17 *

9 Genetic Analyzer 3130xl (AB) 5 Genetic Analyzer 3130 (AB) 4 Genetic Analyzer 3100 (AB) 13 POP4 2 POP6 3 POP7

* 2,10,17,18,19 8,9,12,16 * 2,8 4,15,19

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Table 2 mRNA profiling results (duplex and pentaplex) from 18 laboratories for the six stains and the 2 blood dilution series. Dark grey squares represent stains/markers that have been detected by all 18 laboratories, light grey squares are stains/markers that have been detected by more than half of the laboratories, and white squares represent stains/ markers that have been detected by less than half of the laboratories. Sample

HBB

HBA

ALAS2

CD3G

ANK1

PBGD

SPTB

Stains 1 2 3 4 5 6

17/18 17/18 17/18 18/18 16/18 13/18

17/18 17/18 15/18 17/18 14/18 13/18

18/18 15/18 17/18 14/18 12/18 4/18

17/18 17/18 15/18 13/18 10/18 5/18

14/18 13/18 13/18 8/18 7/18 1/18

14/18 10/18 11/18 4/18 6/18 2/18

15/18 12/18 14/18 10/18 6/18 0/18

17/18 15/18 10/18 3/18 0/18

17/18 15/18 13/18 9/18 1/18

15/18 14/18 8/18 5/18 0/18

15/18 15/18 6/18 3/18 1/18

12/18 12/18 1/18 2/18 0/18

11/18 7/18 0/18 1/18 0/18

11/18 12/18 3/18 2/18 0/18

Dilution series A 5 1 0.1 0.01 0.001 Dilution series B 5 1 0.1 0.01 0.001

16/18 12/18 6/18 2/18 1/18

16/18 14/18 8/18 2/18 1/18

13/18 9/18 3/18 0/18 0/18

detectable in almost all stains (Table 4). Variable results were obtained for the pentaplex markers using blood stains of various sizes (1–50 ml), storage periods (up to 12 years) and storage conditions (Table 4). Two laboratories analyzed menstrual blood stains (four samples) and reported the detection of 1–5 blood markers (Table 4). This is expected as peripheral blood is present in menstrual blood samples. Fifteen non-blood samples (9 saliva, 5 semen, 1 vaginal swab) were analyzed by 7 laboratories. Only two cross reactive peaks (CD3G and SPTB) in one semen sample were observed (see Discussion). In the 3 non-human blood samples (monkey, dog, cow), analyzed by 2 laboratories, none of the blood markers were detected. Full autosomal STR profiles were obtained for most samples where a RNA/DNA co-extraction was performed, with a partial profile obtained only from a 12 year old blood stain from plastic flooring. One peak for D19S433 appeared in the blood sample from the spider monkey. 4. Discussion The purpose of this exercise was to evaluate a RNA/DNA coextraction strategy with challenging blood samples and seven reportedly blood-specific mRNA markers using a highly sensitive duplex (HBB, HBA) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, PBGD and SPTB). Co-extracted DNA was analyzed with various commercial STR typing kits. All 18 participating laboratories successfully set up the method, using different kits and chemicals for RNA only or RNA/DNA coextraction, reverse transcription and PCR and using their own laboratory equipment. All of the strategies used by the participating laboratories appeared to work successfully. The high sensitivity markers, HBA and HBB, were detected by most of the laboratories, but some laboratories had problems with the pentaplex markers. RNA and DNA results were better from dilution series A (on swabs) than dilution series B (on FTA paper). Therefore FTA paper seemed not ideal for storage of small amounts of RNA and DNA, but additional work would be needed to evaluate this phenomenon. The differing sensitivities between the laboratories can be explained by the different kits and chemicals, but also the skill sets within the laboratories, since those laboratories with more

11/18 2/18 0/18 0/18 0/18

10/18 3/18 1/18 1/18 0/18

8/18 1/18 1/18 0/18 0/18

8/18 0/18 0/18 0/18 0/18

experience in RNA analysis in general produced better results than the others. The reduced random hexamer amount for the RT reaction (2.5 ng instead of 50 ng per sample), used by 4 laboratories, did not compromise the results significantly. Based on these findings, it is possible that a range of primer concentration can be utilized, however, the random hexamer should not be a limiting reagent. The multiplex PCR systems need some further optimization prior to routine use in casework analysis because of multiplex design issues (e.g. split peaks, noisy baseline). There may also have been an effect of shipment, since transport of primers took up to 2 weeks, albeit during a cold winter period. Some extraneous peaks with the pentaplex might be ascribed to this non-ideal storage of primers, but these were outside of the marker ranges and therefore did not interfere with peak interpretation. The use of post-PCR purification improved the quality of the multiplex results (reduced appearance of split peaks, dye blobs, etc.) and increased peak heights from low level samples. Therefore, it is possible that postPCR purification could be included as part of the standard protocol in order to resolve quality issues without requiring significant labor-intensive multiplex optimization experiments. One laboratory reported cross-reactivity of CD3G and SPTB in one semen sample. In a previous study occasional, but not reproducible, false positive reactions were reported for some of the 7 blood markers in other body fluids, and additionally some crossreactivity of the RNA multiplexes with human tissues and animal blood was observed [22]. The use of RNA quantification prior to the reverse transcription reaction would allow for a standardization of the input of RNA into the reaction, which could thereby eliminate some of the adverse affects of adding too much input RNA (extraneous peaks, saturation, cross-reactivity) or too little input RNA (products below the analytical detection limits). However, for this exercise RNA quantification was not included in the suggested protocol as currently a human-specific RNA quantification system is not available. The inclusion of negative controls is standard practice in operational forensic laboratories. In RNA analysis, several negative controls can be included during analysis: (1) RT blank (water in place of sample with reverse transcriptase added); (2) RT minus

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Fig. 1. Electropherograms of stain 1 (fresh blood on T-shirt, 5 mm circle punch) analyzed by the organizing laboratory, showing the blood markers HBB and HBA amplified in a duplex PCR reaction, with the corresponding RT minus control (a), ALAS2, CD3G, ANK1, SPTB and PBGD amplified in a pentaplex PCR reaction, with the corresponding RT minus control (b) and the STR profile (SGM Plus) of the co-extracted DNA (c).

(no reverse transcriptase added) – to identify possibly contaminating DNA (frequently a larger size than the expected RNA product) or the presence of pseudogenes (same size as RNA product); (3) amplification blank. The RT and amplification blanks are similar to negative controls used in DNA analysis to identify possible contamination. It could be suggested that the use of RT minus controls in RNA analysis may not always be necessary if appropriate primer design measures were applied (primers

overlap exon–exon-junctions or span an intron), if the absence of pseudogenes was demonstrated, and if the presence of possible DNA amplification products were identified. However, mRNA is currently not routinely used and therefore it would seem prudent during this stage of development to include the use of RT minus controls thereby ensuring the integrity and quality of the obtained expression results. Therefore, the use of RT minus controls was recommended in the current collaborative exercise. While most

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Fig. 2. Representative pentaplex results from stains 4–6 and dilution series B (5, 1, 0.1 ml blood on FTA paper) before and after post PCR purification, analyzed by 2 different laboratories. Laboratory 5 purified the remaining 24 ml PCR product and eluted in 10 ml H2O (A). Laboratory 12 purified the remaining 24 ml PCR product and eluted in 25 ml EB buffer (B).

Table 3 DNA profiling results from 13 laboratories for the six stains and the 2 blood dilution series, amplified with 6 different STR typing kits. Only the 10 SGM Plus loci were taken into account. Dark grey squares represent stains/loci that have been detected by all 13 laboratories, light grey squares are stains/loci that have been detected by more than half of the laboratories, and white squares represent stains/loci that have been detected by less than half of the laboratories. DNA quantification results (total ng DNA in extract) were provided by two laboratories (laboratory 8* and laboratory 198) using standard realtime PCR methods (Alu-based method [25] and Quantifiler human DNA quantification kit, AB), an indication for retrieved DNA amounts. Sample

D3

vWA

D16

D2

AML

D8

D21

D18

D19

TH01

FGA

ng DNA*

ng DNA8

Stains 1 2 3 4 5 6

10/13 12/13 13/13 11/13 13/13 12/13

10/13 13/13 13/13 12/13 13/13 12/13

11/13 13/13 12/13 10/13 13/13 11/13

9/11 11/11 11/11 9/11 10/11 9/11

11/13 12/13 12/13 11/13 13/13 12/13

10/13 13/13 13/13 12/13 13/13 12/13

11/13 12/13 13/13 11/13 13/13 12/13

10/13 11/13 12/13 11/13 13/13 11/13

9/11 10/11 11/11 10/11 11/11 10/11

10/13 12/13 13/13 11/13 13/13 11/13

10/13 12/13 13/13 12/13 13/13 11/13

50.2 42.5 67.6 30.5 127.6 10.2

11.4 14.1 14.4 2.8 62.2 2.2

Dilution series A 5 13/13 1 12/13 0.1 8/13 0.01 1/13 0.001 0/13

13/13 13/13 8/13 1/13 0/13

13/13 13/13 8/13 1/13 0/13

11/11 11/11 6/11 0/11 0/11

13/13 13/13 10/13 1/13 1/13

13/13 13/13 9/13 2/13 0/13

13/13 13/13 9/13 1/13 0/13

13/13 13/13 9/13 1/13 0/13

11/11 11/11 8/11 0/11 0/11

13/13 13/13 7/13 1/13 1/13

13/13 13/13 8/13 1/13 0/13

43.2 12.4 2.4 0.2 0.0

15.3 4.0 0.4 0.0 0.0

Dilution series B 5 13/13 1 12/13 0.1 3/13 0.01 0/13 0.001 0/13

13/13 12/13 5/13 0/13 0/13

13/13 12/13 5/13 0/13 0/13

11/11 10/11 2/11 0/11 0/11

13/13 12/13 5/13 2/13 0/13

13/13 12/13 2/13 0/13 0/13

13/13 12/13 6/13 0/13 0/13

12/13 12/13 3/13 0/13 0/13

11/11 10/11 4/11 1/11 0/11

13/13 12/13 3/13 0/13 0/13

12/13 12/13 2/13 0/13 0/13

33.2 6.4 0.5 0.1 0.0

30.3 3.4 0.0 0.0 0.0

C. Haas et al. / Forensic Science International: Genetics 6 (2012) 70–80

77

Table 4 RNA and DNA profiling results of the optional stains that were analyzed by the participating laboratories (casework or mock samples, non-human/non-blood samples). Grey squares represent RNA peaks >100 RFU and full DNA profiles (FP). Stains marked with light grey font were analyzed by laboratories who obtained poor results for the mandatory samples and therefore results should be interpreted with caution. Lab

Blood samples

HBB (rfu)

HBA (rfu)

ALAS2 (rfu)

CD3G (rfu)

ANK1 (rfu)

PBGD (rfu)

SPTB (rfu)

DNA

4203 – – – –

238 – – – –

– – – – –

– – – – –

– – – – –

– – – – –

– – – – –

FP FP FP FP FP

– 230 8630 7059

– 41 8642 5835

– 121 8609 2486

– 944 8644 1208

– 595 1079 241

– – 2736 140

– – 5475 65

FP FP FP FP

7087

8076

579

479

–

–

65

FP

668 1231 72 223 6741

1269 1614 1421 3210 7231

1029 2218 248 1691 –

4316 1134 3613 6095 724

– 60 662 64 –

8891 6759 – – –

825 588 530 – –

FP FP FP FP FP

6350 2876 324

5642 2046 1738

59 – –

– – –

– – –

– – –

– – –

FP FP FP

224

3364

–

–

–

–

–

Not done

512

7621

–

–

–

–

–

Not done

321

5628

–

–

–

–

–

Not done

–

4354

–

–

–

–

–

Not done

4148 –

2043 –

– –

– –

– –

– –

– –

FP Partial profile

19 19 19

5 ml blood on swab 5 ml blood on swab, frozen, 4.5 years old Degraded blood stain, 1 cm2, 4 years old Degraded blood stain, 1 cm2, 4 years old Frozen/defreezed human blood, on swab after >1 year Casework, human blood on plastic Casework, human blood on wool 10 ml blood on swab Blood on fabric, stored dry at ambient temp., 2 years old Blood on fabric, stored dry at ambient temp., 3 years old 10 ml blood 1 on a sterile compress, 2 days old 10 ml blood 2 on a sterile compress, 2 days old Small amount of heavy blood stain 1 on swab Small amount of heavy blood stain 2 on swab 20 ml blood on sterile pads, 3person mix (GEDNAP 38 C) 20 ml blood on sterile pad (GEDNAP 39 A) 10 ml blood on blue paper (GEDNAP 39 C) 20 ml blood on sterile pad, 3person mix (GEDNAP 39 D) 1 ml blood 1 on swab, liquid blood sample stored at 4 8C, 4–5 months old 1 ml blood 2 on swab, liquid blood sample stored at 4 8C, 4–5 months old 1 ml blood 3 on swab, liquid blood sample stored at 4 8C, 4–5 months old 1 ml blood 4 on swab, liquid blood sample stored at 4 8C, 4–5 months old Casework, blood stain on swab, 1 year old Casework, blood stain from piece of plastic flooring, 12 years old 50 ml blood on swab, 22 8C, 50% humidity, 1 week old 50 ml blood on swab, 30 8C, 90% humidity, 1 week old 50 ml blood on swab, on back seat of car, 1 week old

1290 8483 8741

5919 9061 9179

396 9141 8516

643 7986 8114

– 986 845

– 1884 699

– 650 882

FP FP FP

Lab

Menstrual blood samples

HBB (rfu)

HBA (rfu)

ALAS2 (rfu)

CD3G (rfu)

ANK1 (rfu)

PBGD (rfu)

SPTB (rfu)

DNA

3 3 15

Menstrual blood stain, degraded, 1 cm2 Menstrual blood swab, frozen, 4 years old Menstrual blood 1 swab, 10 ng RNA into RT, 0.5 ng cDNA into PCR Menstrual blood 2 swab, 10 ng RNA into RT, 0.5 ng cDNA into PCR

3944 3895 8737

– 147 7726

– 449 –

– 378 –

– – –

– 47 –

– – –

FP FP Not provided

8382

8791

5221

–

1305

182

–

Not provided

Lab

Non-human/non-blood samples

HBB (rfu)

HBA (rfu)

ALAS2 (rfu)

CD3G (rfu)

ANK1 (rfu)

PBGD (rfu)

SPTB (rfu)

DNA

6 8 12 12 14 15

Whole buccal swab 10 ml saliva directly into lysis buffer Whole buccal swab 1 Whole buccal swab 2 Buccal swab 100 ml saliva 1 on swab, 10 ng RNA into RT, 0.5 ng cDNA into PCR 100 ml saliva 2 on swab, 10 ng RNA into RT, 0.5 ng cDNA into PCR 10 ml saliva on postage stamp (GEDNAP 39 B) 1/4 buccal swab, room temp., 1 year old 10 ml sperm directly into lysis buffer 100 ml semen 1 on swab, 10 ng RNA into RT, 0.5 ng cDNA into PCR 100 ml semen 2 on swab, 10 ng RNA into RT, 0.5 ng cDNA into PCR 10 ml sperm on toilet paper (GEDNAP 38 D) Semen on shirt, in sealed bag, room temp., 5.5 years old 1/4 vaginal swab, room temp., 1 year old 5 ml blood on swab, spider monkey 5 ml blood on swab, dog Bovine blood

– – – – – –

– – – – – –

– – – – – –

– – – – – –

– – – – – –

– – – – – –

– – – – – –

FP FP FP FP Not done Not provided

–

–

–

–

–

–

–

Not provided

– – – –

– – – –

– – – –

– – 107 –

– – – –

– – – –

– – 731 –

FP FP FP Not provided

–

–

–

–

–

–

–

Not provided

– –

– –

– –

– –

– –

– –

– –

FP FP

– – – –

– – – –

– – – –

– – – –

– – – –

– – – –

– – – –

FP D19 No result No result

3 3 3 3 6 6 6 8 8 8 9 9 12 12 16 16 16 16 17 17 17 17 18 18

15

15 16 19 8 15 15 16 19 19 3 3 12

78

C. Haas et al. / Forensic Science International: Genetics 6 (2012) 70–80

Fig. 3. Electropherograms of a mock casework sample (50 ml blood on swab, on back seat of car, 1week old), analyzed by laboratory 19, showing the duplex PCR result (a), the pentaplex PCR result (b) and the STR profile (Identifiler) of the co-extracted DNA (c).

laboratories included such controls, it was within each participating laboratory’s discretion to utilize protocols deemed by them to be suitable for use. The possibility of co-extracting RNA and DNA from the same stain sample is an important advantage, since the amount of sample is often limited in forensic casework. The quantity and quality of DNA from co-extracted samples seemed to be sufficient also for casework and environmentally exposed samples, even though the results are slightly poorer than conventional DNA Chelex extraction [22]. From almost all stains, good quality DNA profiles and the positive identification of blood could be achieved; even stains exposed to un-controlled humidity and old stains could be identified as blood. The quality of the DNA data might be improved with DNA precipitation and

increased injection voltage [24]. Alternatively, post-PCR purification would increase signal strength [23]. While this was not directly evaluated by the participating laboratories, the results from post-PCR purification with the RNA multiplexes demonstrate that improved signal intensity can be achieved without modification to existing capillary electrophoretic protocols. Either approach includes simple modifications that can be performed in order to improve overall profile quality and signal intensity and therefore should be considered by operational crime laboratories. In summary, the results of this study support a RNA/DNA coextraction strategy allowing for positive identification of the tissue/fluid source of origin (blood) by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR

C. Haas et al. / Forensic Science International: Genetics 6 (2012) 70–80

79

Fig. 4. Electropherograms of a mock casework sample (blood on fabric, stored dry at ambient temperature, 2 years old), analyzed by laboratory 8, showing the duplex PCR result (a), the pentaplex PCR result (b) and the STR profile (SGM Plus) of the co-extracted DNA (c).

profiling. The co-extraction results indicate the feasibility of incorporating mRNA profiling strategies into current DNA analysis pipelines. The seven evaluated blood markers proved to be robust, reproducible and two of them (HBA and HBB) in particular were found to be very sensitive. Co-extracted DNA from the same stain provided good-quality STR profiles. For future body fluid identification systems, multiplexes for the simultaneous identification of

several body fluids and possibly tissues would be preferable. A subsequent EDNAP mRNA exercise will include an evaluation of mRNA markers for the identification of saliva and semen. The collective knowledge gained from this series of collaborative exercises is likely to facilitate the formulation of recommended practices and procedures for mRNA profiling for body fluid identification.

80

C. Haas et al. / Forensic Science International: Genetics 6 (2012) 70–80

Acknowledgements [14]

We thank A. Holmer, G.S. Ramdayal and V. Birraux for help with laboratory work.

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