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Development of RNA profiling tools and the implementation in forensic casework

Lindenbergh, P.A.

Publication date2014Document VersionFinal published version

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Citation for published version (APA):Lindenbergh, P. A. (2014). Development of RNA profiling tools and the implementation inforensic casework.

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Chapter 1A multiplex (m)RNA profiling system for the forensic identification of body fluids

and contact traces

Alexander LindenberghMirjam de PagterGeeta Ramdayal

Mijke VisserDmitry ZubakovManfred Kayser

Titia Sijen

Forensic Science International: Genetics (2012) 6:565-577

(m)RNA profiling system for body fluids and contact traces

17

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Abstract

In current forensic practice, information about the possible biological origin of forensic traces is mostly determined using protein-based presumptive testing. Recently, messenger RNA profiling has emerged as an alternative strategy to examine the biological origin. Here we describe the development of a single multiplex mRNA-based system for the discrimination of the most common forensic body fluids as well as skin cells. A DNA/RNA co-isolation protocol was established that results in DNA yields equivalent to our standard in-house validated DNA extraction procedure which uses silica-based columns. An endpoint RT-PCR assay was developed that simultaneously amplifies 19 (m)RNA markers. This multiplex assay analyses three housekeeping, three blood, two saliva, two semen, two menstrual secretion, two vaginal mucosa, three general mucosa and two skin markers. The assay has good sensitivity as full RNA profiles for blood, semen and saliva were obtained when using ≥0.05 µL body fluid starting material whereas full DNA profiles were obtained with ≥0.1 µL. We investigated the specificity of the markers by analysing 15 different sets of each type of body fluid and skin with each set consisting of 8 individuals. Since skin markers have not been incorporated in multiplex endpoint PCR assays previously, we analysed these markers inmore detail. Interestingly, both skin markers gave a positive result in samplings of the hands, feet, back and lips but negative in tongue samplings. Positive identification (regarding both DNA and RNA profiling) was obtained for specimens stored for many years, e.g. blood (28 years-old), semen (28 years-old), saliva (6 years-old), skin (10 years-old) and menstrual secretion (4 years-old). The described approach of combined DNA and RNA profiling of body fluids and contact traces assists in the interpretation of forensic stains by providing information about not only the donor(s) that contributed to the stain but also by indicating which cell types are present.

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Introduction

Next to DNA typing results, knowledge about the cellular origin of crime-related biological stains can be of significant importance for the reconstruction of the events at a crime scene. Conventional methods of body fluid identification, like the tetramethylbenzidine test, hexagon-obti and RSID-blood for blood stains [1,2], the PSA and semenogelin test for semen [3] and the amylase tests (Phadebas or RSID-saliva) for saliva [4] are protein or enzymebased, presumptive in nature and not always human-specific. Most of these methods rely on a colour-forming reaction which can be difficult to interpret, especially when dealing with coloured extracts or samples containing very little amounts of target material. Within forensic genetics, messenger RNAs (mRNAs) have increasingly gained popularity regarding their potential to distinguish between human body fluids and other forensically relevant tissues [5–10]. Alternative methods for cell typing include tissue-specific miRNAs, DNA methylation [11,12] and microbial markers [13]. miRNAs are small (20–24 nucleotides) regulatory RNAs which are strongly associated with members of a class of proteins called Argonautes [14], which makes them very stable and advantageous when dealing with degraded forensic samples [15,16]. Also epigenetic DNA methylation markers have been described that can differentiate between some tissue types [11,12]. Both miRNA and DNA methylation markers seem promising, but still in its infancy as for instance more markers are needed to discriminate the forensic range of body fluids. The use of microbial markers has been suggested for the identification of especially vaginal mucosa [13,17–19]. However, it is not yet established whether the same microbes also occur on skin surfaces that are in close proximity of, or in contact with the vaginal microbial flora, such as skin surfaces of the hands, groin or penis. For these reasons, we regard tissue-specific mRNA analysis as the most versatile cell-typing approach. mRNA profiling has evolved from a singleplex PCR technique to a multiplex RT-PCR platform, providing expression of data on multiple genes simultaneously. mRNA profiling is readily combined with DNA genotyping since RNA and DNA can be obtained from the exact same sample [6,9,20,21]. The different multiplexes that have been developed include markers for venous blood, saliva, semen, vaginal epithelia and menstrual secretion [9,10,13], and their selection was mainly based on the function described in literature [9,10] or the tissue-specific expression as reported in expression databases [22]. Dedicated whole-genome expression array analysis in samples from forensically relevant body fluids that were stored for various time intervals has previously shown to deliver stable mRNA markers useful for forensic tissue identification [23,24].


19

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Skin is an additional forensically important cell type. Recently three mRNA transcripts (LOR, CDSN and KRT9) were reported to show high expression in skin samples relative to other forensically relevant cell types [25]. The addition of skin markers to an RNA-based cell typing multiplex would increase the practical forensic value of the assay for two reasons: (1) a more complete view on all cell types present in an evidentiary trace is established which is important because skin cells are expected to occur in many crime scene samples and (2) an indication for the presence of contact DNA can be obtained. The most often used method to show the presence of contact traces is through dactyloscopic fingerprint analysis but also other microscopical and immunocytological techniques [26] have been described which can identify skin cells. Fingerprint visualisation methods do not apply to all types of substrates, and some can have negative effects on the nucleic acids in the skin cells [27] while others can introduce contamination [28]. To efficiently and objectively assess the biological origin of a forensic evidentiary trace, a single profiling assay was developed that assays both the forensically relevant body fluids and skin cells.

Materials and methods

Sample collection

Body fluids and tissues from eight individuals were collected with their informed consent. Ten, 5, 1 and 0.5 µL blood, semen and saliva were collected on cotton swabs (Deltalab, Barcelona, Spain). The 1/10, 1/20, 1/100 and 1/1000 body fluid dilutions were prepared in phosphate buffered saline. Blood was collected through a finger prick (Accu-chek, Softclix Pro, Roche Diagnostics GmbH, Germany). Vaginal mucosa and menstrual secretions (day two of the menstrual cycle) were collected on cotton swabs. Skin samples were collected from the palm of the hand, the middle of the back and on the sole of the foot by means of a damp cotton swab or omniswab (Whatman Inc, United Kingdom). Additional skin samples from fingers were obtained by fiercely rubbing a piece of cotton for 15 s and by tape lift according to de Bruin et al. [29]. Mixtures of skin and saliva were collected by swabbing a plastic cup after a drink simulation and by swabbing the palm of the hand after transfer of 10 µL saliva. Mixtures of blood and skin were obtained by swabbing finger prick areas. All samples were dried overnight at room temperature and processed immediately or stored at -80 ºC.

Chapter 1

20

Cha

pter

1

after a drink simulation and by swabbing the palm of the hand after transfer of 10 µL saliva. Mixtures of blood and skin were obtained by swabbing finger prick areas. All samples were dried overnight at room temperature and processed immediately or stored at -80 °C.

Aged stains

Blood and semen were spotted on cotton cloth and stored at room temperature. After 28 years, areas of ~0.5 cm2 in size were cut out for analysis (courtesy A.D. Kloosterman). Used items like jewellery, musical instruments and pacifiers that were left untouched at room temperature for 6–20 years, were swabbed according to standard procedures using a water-hydrated swab. Other samples include stamps on postal cards stored for 3–12 years, buccal swabs stored for 10 years and menstrual secretion stains stored for 4 years, all kept dry and dark at a constant humidity and at room temperature.

DNA/RNA co-isolation

In order to deviate as little a possible from the current certified in-house procedures for DNA isolations, a DNA/RNA co-isolation protocol was developed incorporating the widely used QIAampDNA mini Kit (QIAGEN Benelux B.V., Venlo, The Netherlands) and the mirVana miRNA Isolation Kit (Applied Biosystems™, Ambion®, Austin, TX,USA). Cells were lysed in 300–600 µL Lysis Binding Buffer (mirVana miRNA Isolation Kit, Ambion1 (MRIKA)) plus 20–40 µL proteinase K (20 mg/mL, QIAampDNA mini Kit, Qiagen (QDKQ)) and incubated for 2 h at 56 ºC. Following addition of 1/10 volume homogenate additive (MRIKA) and 10 min incubation on ice, the lysate was transferred to a shredder column (QIAGEN Benelux B.V., Venlo, The Netherlands) to separate the carrier material from the lysate. DNA was separated from the RNA by transfer of the lysate to a QIAamp DNA column and 1 min centrifugation at 8000 rpm. The RNA containing flow-through was processed first because of the fragile nature of RNA while the DNA columns were stored at 4 ºC until further processing. Next, 300–600 µL phenol/chloroform (pH 4.5, MRIKA) was added to the RNA fraction, mixed for 1 min and centrifuged for 5 min at 10,000 rpm. The aqueous phase was carefully collected and absolute ethanol was added to a final concentration of 55.5 %. The lysate/ethanol mixture was applied to an RNA filter cartridge (MRIKA), centrifuged ~15 s at 10,000 rpm and washed once with 700 µL wash buffer 1 (MRIKA) and twice with 500 µL wash


21

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buffer 2/3 (MRIKA). The RNA was eluted in 60 µL of nuclease-free water (Ambion®) pre-heated to 95 ºC. The DNA columns were washed once with 500 µL wash buffer 1 and 500 µL wash buffer 2 (QDKQ). The DNA was eluted in 100 µL of a 25 % AE solution (QDKQ), pre-heated to 70 ºC. Standard DNA extractions were performed using the QIAamp DNA mini Kit (QIAGEN Benelux B.V., Venlo, The Netherlands) according to the manufacturer’s protocol.

DNase treatment

The RNA extracts were treated with 4 U DNase (TURBO DNA free™, Ambion®) for 30 min. DNase was inactivated using Inactivation Buffer (TURBO DNA free™, Ambion®) according to the manufacturer’s protocol.

RNA integrity

RNA integrity was assessed by an Agilent 2100 Bioanalyzer using the RNA 6000 pico or the 6000 nano kit according to the manufacturer’s protocol. For both kits, 1 µL of RNA was used.

cDNA synthesis

cDNA was synthesized using the RETROscript® Kit (Ambion®) in a total volume of 20 µL. The reaction contained a maximum volume of 10 µL RNA extract, random decamers (5 mM), RT-buffer (1x), dNTPs (0.5 mM of each), RNase Inhibitor (10 U) and MMLV-RT (100 U). For each sample a negative control was included to which no MMLV-RT was added. The Quantifiler® human genomic DNA (gDNA) quantification was used as an indicator to determine input in the cDNA synthesis reaction. While the amount of genomic DNA in a diploid cell is fixed (namely 6 pg), the RNA quantity per cell type varies. Consequently the RNA input for the cDNA synthesis reaction in this study (which uses samples of known origin) was empirically determined to be the volume of the RNA extract equivalent to 0.2 ng of human gDNA in the DNA extract for blood or 4 ng for saliva, semen, menstrual secretion and vaginal mucosa. For all the skin samplings (including mixtures) and the tongue samplings a maximum amount of 10 µL was used. According to the manufacturer, the RNA capacity in the cDNA reaction is a least 2 µg. Since this will seldom be reached for forensic stains, over-loading of the cDNA synthesis reaction is not expected.

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Multiplex PCR

Primers for HBB, GAPDH, ACTB, 18S-rRNA, HTN3 and STATH were adopted from the literature [10,25,30,31]. For all other markers, primer sets were developed using Perlprimer software [32], and set to span at least one intron or to reside at an exon–exon junction. Forward primers were 5’-labeled with 6-FAM™, VIC®, NED™ or PET® and purified by HPLC (Applied Biosystems™, Warrington, United Kingdom). The primer sequences, concentrations and product sizes are indicated in Table 1. The primers appear human-specific (with exclusion of primates) when performing in silico BLAST analyses. In the multiplex PCR, up to 7.5 µL cDNA was amplified in the presence of 12.5 µL 2x QIAGEN Multiplex reaction mix and 5 µL 5x primer mix, resulting in a total volume of 25 µL. A GeneAmp1 9700 PCR System (ABTM) was used with the following cycling conditions: 95 ºC for 15 min, 33 cycles of 94 ºC 20 s, 60 ºC 30 s, 72 ºC 40 s and a final soak at 60 ºC for 45 min.

PCR purification

Prior to capillary electrophoresis, excessive salts and dye remnants were removed from the RT-PCR mixtures, by Performa® DTR V3 96-Well Short Plates or 1.5 mL columns (Edge BioSystems, Gaithersburg, USA) according to the manufacturer’s protocol with minor adjustments as described by Westen et al. [33].

DNA quantification

Human-specific genomic DNA concentrations were determined using the Quantifiler® human DNA Quantification Kit (AB™) using an ABI 7900HT real-time PCR system (AB™) according to the manufacturer’s protocol. Two microlitres of gDNA was analysed in a 25 µL reaction.

Nucleic acid precipitation

Nucleic acids were precipitated using ethanol according to our in-house protocol. Water was added to a desired volume of DNA or RNA extract making a total of 100 µL. Next, 2 µL GlycoBlue (Ambion®), 10 µL 3 M NaAc and 300 µL of 96 % ethanol was added, mixed and incubated at -20 ºC for a minimum of 1 h. After 25 min centrifugation at 16,000 x g, the pellet was washed with 500 µL of 70 % ethanol and subsequently dissolved in 10 µL nuclease free water.


23

Chapter 1

Tabl

e 1.

Pri

mer

sequ

ence

s use

d fo

r RNA

mar

ker a

mpl

ifica

tion

in th

e 19

-ple

x.Table

1PrimersequencesusedforRNAmarkeramplificationin

the19-plex.

Gene

[Primer]mM

Location

Tissue

Sequence(5

0!

30 )

Size(bp)

Dye

Reference

HBB

0.035

11p15.5

Blood

fw:GCACGTGGATCCTGAGAAC

rv:ATG

GGCCAG

CACACAGAC

61c

6FAM

TM

[10]

CD93

0.25

20p11.21

Blood

fw:ACCAGTACAGTCCGACACb

rv:TTG

CTAAGATTCCAG

TCCAG

151

NEDTM

a

AMICA1

0.15

11q23.3

Blood

fw:TCTCCTGCTCCAAGATGTG

rv:GACCATGAG

CTCTTTGGGb

136

PETTM

a

KRT4

0.2

12q12-q13

Mucosa

fw:AAAGTCCGG

ACG

GAAGAG

rv:TAAGAACTG

CACCTTGTCGb

81

6FAM

TM

a

SPRR2A

0.1

1q21-q22

Mucosa

fw:AGAACCTGATCCTGAGACTb

rv:ATG

GCTCTG

GGCACTTT

106

VIC1

a

KRT13

0.4

17q12-q21.2

Mucosa

fw:CCTACTACAAGACCATTG

AAG

AG

rv:CTCATTCTCATACTTGAG

CCTb

131

NEDTM

a

HTN3

0.2

4q13

Saliva

fw:GCAAAG

AGACATCATGGG

TAb

rv:GCCAGTCAAACCTCCATAATC

134

VIC1

[10]

STATH

0.3

4q11-q13

Saliva

fw:TTTGCCTTCATCTTG

GCTCT

rv:CCCATAACCGAATCTTCCAAb

93

6FAM

TM

[10]

SEMG1

0.8

20q12-q13.2

Semen

fw:GGAAGATGACAG

TGATCG

T

rv:CAACTG

ACACCTTGATATTGGb

121

6FAM

TM

a

PRM1

0.3

16p13.2

Semen

fw:AGACAAAGAAGTCGCAGAC

rv:TACATCGCG

GTCTGTACC

91c

NEDTM

a

HBD1

0.8

8p23.1

Vaginalmucosa

fw:GAAATCCTG

GGTGTTGCC

rv:AAAGTTACCACCTGAGGCCb

101

6FAM

TM

a

MUC4

0.8

3q29

Vaginalmucosa

fw:CTG

CTACAATCAAGG

CCA

rv:AAG

GGAAGTTCTAGG

TTG

ACb

141

6FAM

TM

a

MMP7

0.8

11q21-q22

Menstrualsecr.

fw:GAACAG

GCTCAG

GACTATCTCb

rv:TAACATTCCAGTTATAGG

TAG

GCC

126

VIC1

a

MMP11

0.4

22q11.23

Menstrualsecr.

fw:CAACCG

ACAGAAGAG

GTTCG

rv:GAACCG

AAG

GATCCTGTAGGb

76

NEDTM

a

CDSN

0.6

6p21.3

Skin

fw:CTG

GCTGGTCTCCTCCTG

rv:GGG

TCCTTACAAGGG

TCTGA

71c

VIC1

a

LOR

0.6

1q21

Skin

fw:CTTTGG

GCTCTCCTTCCTb

rv:AGAGGTCTTCACGCAGTC

89

PETTM

a

GAPDH

0.2

12p13

Housekeeping

fw:GTCCACTGG

CGTGTTCACCA

rv:GTG

GCAGTG

ATG

GCATGG

AC

261c

6FAM

TM

[30]

18S-rRNA

0.025

22p12

Housekeeping

fw:CTCAACACG

GGAAACCTCAC

rv:CGCTCCACCAACTAAGAACG

110

PETTM

[10]

ACTB

0.2/0.6

7p22

Housekeeping

fw:TGACCCAGATCATGTTTG

AGb

rv:CGTACAGGG

ATAGCACAG

75

PETTM

[25]

PrimersequencesusedforRNAmarkeramplificationin

the19-plex.

aDesignedforthis

studyusingPerlPrimer.

bPrimeratexon–exonjunction.

cPrimerpair

resultingin

differentsizeampliconsforsplicedandunsplicedtemplates.

A.Lindenberghetal./ForensicScienceInternational:Genetics6(2012)565–577

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Chapter 1

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STR-profiling

DNA profiles were generated using the AmpFℓSTR® NGM™ PCR Amplification Kit (AB™) according to the manufacturer’s recommendations. The gDNA input was normalised to 500 pg. For samples that contained a gDNA concentration


25

Chapter 1

isolate RNA, the miRNA Isolation Kit was used due to its ability to isolate both high (>200 nt) and low molecular weight RNA fragments. This feature of the kit is important as the RNA recovered from forensic samples may be compromised. Agarose gel analysis and 2100 Bioanalyzer results of various samples showed that indeed both high and low molecular weight RNAs were extracted. For most samples, when using the Bioanalyzer, no robust RNA quantifications were obtained (results not shown).

Marker selection and multiplex development

First, a 17-plex was developed containing markers selected from the literature. From studies using time-wise degraded blood and saliva samples, stable mRNA markers were selected; CD93 and AMICA1 for blood and KRT4, KRT13 and SPRR2A for saliva [23,24]. The other markers were selected based on their relevance as reported in other forensic mRNA studies. Thereby, the blood markers in the 17-plex consist of HBB, AMICA1 and CD93. Both CD93 and AMICA1 are expressed in the leukocyte and function, respectively, as a mediator during phagocytosis [34] and inducer for adhesion to endothelial cells [35]. HBB functions in red blood cells (erythrocytes which are around 700 times more abundant than leukocytes) as one of the globins that make up hemoglobin. Erythrocytes do not contain a nucleus nor mRNA but developing erythrocytes (reticulocytes) which make up 1 % of the red blood cells, do carry mRNA. The saliva markers in this 17-plex are KRT4, KRT13 and SPRR2A. KRT4 and KRT13 are both members of the keratin family and are mainly expressed in the tongue and in the suprabasal layers of non-cornified stratified epithelia [36]. SPRR2A is a member of the gene family encoding the human epidermal differentiation complex, which is responsible for the maturation of the human epidermis with high expression in the tongue [37]. For semen analysis the markers PRM1 and SEMG1 were selected. PRM1 encodes protamine which substitutes histones in spermatozoa [38], and SEMG1 encodes a semenogelin, an abundant protein in seminal fluid involved in the gelatinous entrapment of ejaculated spermatozoa. While PRM1 mRNA is only expressed in fertile men, SEMG1 mRNA is present in semen of both fertile and vasectomised men [39]. For vaginal mucosa MUC4 encoding a major constituent of mucus and HBD1 encoding a vaginal antimicrobial peptide of the beta defensin family were selected [6]. Although previous studies have discussed the possible cross reactivity of these markers with other mucus-like membranes, such as mouth epithelia, resulting in non-specific signals for saliva [40], tissue inference may be possible if the saliva markers do not show

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1

cross reactivity with the vaginal mucosa markers. For menstrual secretions the markers MMP7 and MMP11 were used. Matrix metalloproteinases (MMPs) are key effectors of cell differentiation, cyclic growth and cell death of the endometrium and are regulated by ovarian steroids and cytokines [41]. It has been shown that both markers are excellent for the determination of menstrual secretions [10,42]. In addition, the blood and vaginal markers are expected to be present in menstrual secretion. LOR and CDSN are the markers used for skin within this mRNA multiplex. LOR encodes loricrin which is a component of the cornified cell envelope found in terminally differentiated epidermal cells [43]. CDSN encodes corneodesmosin [44] that is involved in desquamation, the process which induces shedding of the outer membrane layer of skin [45]. These markers were selected due to their specificity and sensitivity in skin samplings [25]. As positive controls, ACTB, GAPDH and 18S-rRNA were chosen. All three markers have been previously described and used as endogenous controls. The corresponding amplicons have different size ranges thereby providing an indication for RNA integrity in the RNA profile. Initial testing of the 17-plex revealed regular cross-reactivity for the saliva markers KRT4, KRT13 and SPRR2A with skin samplings and mucous membranes found in vaginal samples and menstrual secretion. Therefore, we decided to add two additional markers for saliva: STATH (statherin), encoding a calcium regulator in saliva [46] and HTN3 (histatin 3), encoding a histidine rich antibacterial peptide [47]. These mRNAs are predominantly expressed in human parotid and sublingual–submandibular glands and are distinct from the set of saliva markers from which KRT4, KRT13 and SPRR2A were selected [23], which are predominantly expressed in the tongue (BIOGPS analysis [22]). This resulted in a final 19-plex. It was decided to retain KRT4, KRT13 and SPRR2A in the 19-plex as mucosa markers and investigate their performance in degraded samples, as these 3 markers have been shown to be useful in degraded samples [23]. The multiplex was optimised by testing different primer concentrations, reaction buffers and Mg2+ concentrations. We found the Qiagen multiplex PCR Kit to achieve the most reproducible results. In order to obtain specificity for mRNA derived cDNA molecules, one primer spanned an exon–exon junction for most amplicons (Table1). A DNase treatment still proved important, particularly for the markers serving as positive controls, since both 18S-rRNA and genome-integrated copies of spliced ACTB and GAPDH variants could result in DNA-derived signals (results not shown) [48].


27

Chapter 1

Sensitivity testing of blood, semen and saliva markers

To assess the sensitivity of the 19-plex for blood, salvia and semen (vaginal mucosa, menstrual secretion and skin not tested), a dilution series was analysed in which 10–0.001 µL body fluid was applied to a cotton swab. During RT-PCR, 1, 3.5 or 7.5 µL cDNA was amplified for each sample. Results are shown in Table 2. HBB was found to be the most sensitive blood marker, still detectable in a 0.001 µL blood stain. The minimal volume of blood for detection of CD93 was 0.5 µL, and for AMICA1 0.01 µL. For semen, both PRM1 and SEMG1 are detected up to a volume of 0.05 µL. Since PRM1 levels depend on the amount of spermatozoa produced and the marker is only expressed in fertile men, it is not relevant to compare the sensitivity of the two semen markers. In the dilution series for saliva, signals were obtained for the three mucosa and two saliva markers. Saliva markers STATH and HTN3 were found to be equally sensitive. As expected, the sensitivity of the markers increased with rising amounts of input in the PCR. However, high inputs are accompanied by a rise in baseline noise and the formation of non-specific amplification products. Therefore, 3.5 µL or 7.5 µL cDNA inputs are only advised when low signals are obtained with 1 µL input.

Specificity testing of the endpoint multiplex assay

Marker specificity of the above described 19-plex was assessed using 10 µL stains of blood, saliva and semen, cotton swab samplings of vaginal mucosa and menstrual secretion, various skin samplings and tongue scrapings. For each body fluid or tissue type, specimens from eight individuals were used. For semen the eight donors consisted of six fertile and two sterile donors. The electropherograms obtained for all the investigated tissue types are shown in Figure 1. The most robust positive control marker was 18S-rRNA as signals were observed in all samples. For ACTB, peaks were found in all except the skin samples, where the signals were low. Stronger ACTB signals have been observed previously [25] when analysing skin samplings by Taqman assays. The 19-plex is optimised to analyse six different cell types. Consequently, a concentration of 0.2 µM for the ACTB primers was selected which is suboptimal to detect ACTB in skin samplings. When the multiplex contained 0.6 µM of each ACTB primer, positive signals were observed for skin (data not shown), but for other body fluids over-amplification, signal saturation and by-product formation was observed. The low ACTB signal in skin does not compromise the performance of the multiplex since 18S-rRNA serves as a good positive control in skin.

Chapter 1

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1

Table 2. Sensitivity of the blood, saliva and semen markers used in the 19-plex. Presented here are the minimum amounts of body fluid needed on a swab to obtain an RNA profile with the indicated number of markers. 1, 3.5 or 7.5 µL (maximum volume) cDNA was used as input in the endpoint multiplex PCR.

a For all body fluids a series of 10, 5, 1, 0.5, 0.1, 0.05, 0.01 and 0.001 µL were tested in duplicateb Fertile donor

The GAPDH marker was found in all blood, semen, vaginal and menstrual samples. Lower expression of this marker was found in saliva samples and sporadic expression was observed in skin samplings (Table 3). The relatively large size of the GAPDH amplicon and the relatively low transcript abundance compared to ACTB and 18S-rRNA [30] may underlie these findings. As shown in Table 3, the blood markers HBB, CD93 and AMICA1 were detected consistently in all blood samples analysed. In two instances, expression of blood markers was detected in saliva samples (Table 3). This was probably due to the presence of minor amounts of blood of the donor in the saliva samples (Supplementary Table 1). The blood markers were also detected in the menstrual secretion samples (Table 3). Menstrual secretion is comprised of a complex mixture of different tissue types; next to blood (30–50 %) and degraded endometrial tissue, epithelial cells from the vaginal lumen are present [10]. The three blood markers HBB, CD93 and AMICA1 showed relatively low signals in these samplings. The PCR process may have been affected by the complex composition of this body fluid. Alternatively, mRNA degradation in blood cells possibly caused by themicrobial vaginal flora or acidic environment may explain these results. Both the menstrual secretion markers MMP7 and MMP11 showed high specificity for menstrual blood as they were not detected in any other tissue (Table 3). These findings are different compared to a previous study where high levels of MMP11 were found in vaginal mucosa [10]. Multiple studies have discussed suitability of MMP7 and MMP11 in forensic casework [10,42], but in our dataset (Table 3), MMP7 and MMP11 appear equally useful in the determination of menstrual secretion although MMP7 seems to be more sensitive as higher signals were obtained when compared to MMP11 (Supplementary Table 1).

Body Fluida Number of markers

All markers positive 1 µL input

All markers positive 3.5 µL input

All markers positive 7.5 µL input

More than 2 markers positive 7.5 µL input

Only 1 marker positive 7.5 µL input

Blood 3 0.5 µL 0.1 µL 0.05 µL 0.01 µL 0.001 µL Saliva 2 0.5 µL 0.5 µL 0.05 µL 0.05 µL 0.05 µL Semenb 2 0.1 µL 0.05 µL 0.05 µL 0.05 µL 0.001 µL


29

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Fig. 1. Typical overlay electropherograms of single source blood, saliva, semen, skin, menstrual secretion and vaginal mucosa as produced by the 19-plex. The blank corresponds to an RT-PCR with no RNA input (no RNA RT-PCR). Peaks representing marker signals are filled. Not-filled peaks represent bleed-through signals or by-products (e.g. red peak at 72 and 105 nt) that are both due to over-amplification. All signals are cDNA-derived, as no signals were observed in the minus RT controls (results not shown). Indicated by an asterisk in the blank are dye blobs which are visible in all subsequent electropherograms.

Chapter 1

30

Cha

pter

1

Tabl

e 3.

Res

ults

show

ing

spec

ifici

ty o

f the

mar

kers

use

d in

the

19-p

lex

in d

iffer

ent t

issu

e ty

pes.

For a

ll m

arke

rs 1

5 di

ffere

nt

sam

ple

sets

con

sist

ing

of e

ight

indi

vidu

als w

ere

anal

ysed

. For

sem

en sa

mpl

es th

e se

t com

pris

ed si

x fe

rtile

and

two

ster

ile

dono

rs. F

or m

ixed

spe

cim

ens,

cell

mat

eria

l of t

he s

ame

dono

r w

as u

sed.

The

per

cent

age

of in

divi

dual

s in

whi

ch m

arke

r ex

pres

sion

was

obs

erve

d is

indi

cate

d pe

r sa

mpl

e se

t as

a g

reys

cale

in w

hich

eac

h sh

ade

of g

rey

repr

esen

ts a

spe

cific

pe

rcen

tage

. Cor

resp

ondi

ng a

vera

ge a

ppro

xim

ate

peak

hei

ght d

ata

can

be fo

und

in S

uppl

emen

tary

Tab

le 1

.

a Fo

r al

l the

ski

n sa

mpl

ings

, drin

k sim

ulat

ion

and

tong

ue s

ampl

ings

, 0.

2 µM

AC

TB w

as

used

inst

ead

of 0

.6 µ

M.

b Fo

r va

gina

l m

ucos

a sa

mpl

es t

he H

BD1

prim

er s

et w

as a

lso te

sted

in s

ingl

eple

x.

c n.

d.: n

ot d

eter

min

ed

0%12

,5%

25%

37,5

%50

%62

,5%

75%

87,5

%10

0%

Hou

seke

epin

gB

lood

Muc

osa

Saliv

aSe

men

Men

stru

al

Secr

etio

nSk

inVa

gina

l Muc

osa

Sam

ple

sets

n18

S-rR

NA

AC

TBG

APD

HH

BB

AM

ICA

1C

D93

KR

T4SP

RR

2AK

RT1

3ST

ATH

HTN

3PR

M1

SEM

G1

MM

P7M

MP1

1C

DSN

LOR

MU

C4

HB

D1

HB

D1

1ple

x

1B

lood

8n.d

2aSe

men

-fert

ile6

n.d

2bSe

men

-ste

rile

2n.d

3Sa

liva

8n.d

4M

enst

rual

Secr

etio

n8

n.d

5Va

gina

l Muc

osa

8

6B

lood

-ski

n8

n.d

7Sk

in-c

otto

n8

n.d

8Sk

in-c

otto

n-st

ub8

n.d

9Sk

in-w

ashe

d8

n.d

10Sk

in-u

nwas

hed

8n.d

11Sk

in-fo

ot8

n.d

12Sk

in-b

ack

8n.d

13Sk

in +

10µ

l Sal

iva

8n.d

14D

rink

Sim

ulat

ion

8n.d

15To

nque

Sam

plin

gs8

n.d


31

Chapter 1

STATH and HTN3 appear highly specific for saliva (Table 3) which is in concordance with the literature [5,6,9,10]. In contrast to the findings of Haas et al. [10], the HTN1 isoform that has 95 % sequence identity with HTN3 was never detected in our amplifications which is probably due to the higher annealing temperature used during the RT-PCR resulting in more stringent primer binding conditions. The mucosa markers KRT4, KRT13 and SPRR2A [23,24] were detected in saliva, vaginal mucosa, menstrual secretion and skin samplings (Table 3) all of which, with the exception of skin, represent mucous membranes. Tongue scrapings were used to assess the expression of the mucosa markers in tongue cells which can be found on licked items. All three mucosa markers were found to be highly expressed in these samplings which confirms BIOGPS expression data [22]. Some STATH and HTN3 expression was also detected, possibly due to remnants of saliva on the samples as both markers are not reported to be expressed on the tongue [22]. In semen samples, PRM1 signals were restricted to samples from fertile men whereas SEMG1 was also detected in samples from vasectomised men (Table 3). Both markers exhibited a high level of specificity with generally lower expression levels for SEMG1 than for PRM1. Since the expression of PRM1 is directly related to the number of spermatozoa present in semen, this may be because all donors in our sample set have a relatively high number of spermatozoa. Vaginal samplings represent difficult specimens for cell-typing due to the high degree of biochemical similarity between vaginal and oral mucosa [23]. Despite the fact that both vaginal mucosa markers used in our assay were previously reported to give signals for saliva [49,50], in our dataset MUC4 expression was only observed in vaginal samples (Table 3). HBD1 expression was seldom found using the 19-plex in the vaginal samplings. However, as can be seen in Table 3, singleplex experiments using the same primer concentration did show HBD1 expression pointing to poor multiplexing capability of the used HBD1 primer-set. An alternative way to assess vaginal origin of forensic samples relies on microbial markers. These can readily be incorporated in mRNA multiplexes [13] if a limited number of species suffice to identify the vaginal origin irrespective of the donor (for instance also in case of bacterial vaginosis). To assess the performance of the skin markers, we collected a large sample set comprising a range of sampling sites and various forensically relevant sampling methods. By nature, skin is in contact with the external environment and thus skin samplings may contain body fluids. Therefore, both the palms of the hands (washed and unwashed) and usually covered skin areas (sole of the foot, middle of the back) were sampled. Strong LOR and CDSN signals were obtained for nearly all tested

Chapter 1

32

Cha

pter

1

individuals with occasional low expression of the mucosa markers KRT4, KRT13 and SPRR2A (Fig. 2). Sampling methods (swab, tape lift or cotton cloth) did not affect the RNA profiling results. The skin markers were not expressed in blood, saliva and semen, but occasionally vaginal mucosa and menstrual secretion samples revealed LOR and/or CDSN expression, as can be seen in Table 3. Skin cells can be also present when skin or touched surfaces are sampled. Indeed when blood was swabbed from a finger after finger prick, both blood and skin markers are detected with relatively high blood marker signals compared to skin marker signals (Fig. 3). In addition, when deposited saliva was collected from the palm of the hand, markers for both tissue types were detected. In drinking simulation samples, saliva signals were predominant but skin markers were detected when using a high cDNA input (results not shown). RNA profiles were often unbalanced due to unequal expression of the different RNAs occurring in a tissue. For example, the 18S-rRNA peak was often found to be relatively high compared to the other markers, but lowering the 18S-rRNA primer concentrations resulted in marker drop-out. The inter-marker balance may be improved by changing assay parameters such as primer concentrations or changes in Tm although this will not fully eliminate imbalance. Large multiplexes have an increased chance for the formation of primer dimers and dye blobs, with the latter caused by disassociated primer dyes. Despite the use of a dye removal method, not all dye blobs were removed as apparent from the relatively wide peaks that occur at more or less fixed positions in the electropherogram. The dye blobs can easily be recognised and do not affect the analysis and interpretation of an RNA profile. Next to RNA profiling, all samples were subjected to NGM DNA profiling. Full single donor DNA profiles concordant with the reference profiles of the donors were obtained for all except some skin samples (these few exceptions gave partial profiles). Animal specimens (including various species and body fluids) were not analysed with our 19-plex. Instead, a database search was performed to assess primer specificity. Although positive signals for our marker set cannot be excluded for animal samples, it is very unlikely that these will generate full RNA profiles (i.e. signals for housekeeping genes and the multiple tissue-specific markers). Furthermore, an RNA profile is always interpreted in combination with a DNA profile from the same sample, and studies have indicated that for non-primates, no allele calls are obtained when using NGM SElect [51] (which includes all NGM markers [52]).


33

Chapter 1

Fig. 2. Typical overlay electropherograms of 19-plex amplifications of samplings from different areas of skin. Only relevant bins are shown. Apart from the amplification products for the skin markers LOR and CDSN, amplification products for the mucosa markers KRT4, SPRR2A and KRT13 are visible. As the GAPDH marker was not amplified in these samplings, the x-axis was adopted to fit the amplification range. Also, housekeeping marker ACTB was not amplified in these skin specimens. Indicated by an asterisk in the blank are dye blobs which are visible in all subsequent electropherograms. Blank refers to no RNA RT-PCR.

Chapter 1

34

Cha

pter

1

Fig. 3. Typical overlay electropherograms of 19-plex amplifications of mixed skin–blood and skin–saliva samplings. Only relevant bins are shown. Indicated by an asterisk in the blank are dye blobs which are visible in all subsequent electropherograms. Blank refers to no RNA RT-PCR.


35

Chapter 1

Old samples

Forensic samples can be exposed to various factors that can reduce RNA integrity such as high humidity, light (UV) and longtime storage. We assayed the cell-typing ability of the 19-plex for a range of old samples. DNA-typing was also carried out. Results are summarised in Table 4. RNA and DNA profiles were generated for blood and semen samples that had been in storage for 28 years. Saliva present on 6-year-old pacifiers and 10-year-old buccal swabs were positively typed successfully generating both RNA and DNA profiles. Interestingly, the mucosa markers KRT4, KRT13 and SPRR2A performed better in these old stains than the saliva markers STATH and HTN3 (Supplementary Table 2), which is probably due to the fact that the mucosa markers were selected using aged samples [23,24]. The skin marker signals detected in the pacifier samplings most likely represent skin material of the lips. No DNA and RNA profiles were obtained from stamps of postal cards which may be due to the interference of glue or paper remnants in the isolation procedure. Menstrual secretion stains stored for 4 years were positively typed for RNA and DNA. Both the menstrual secretion and the vaginal mucosa markers were expressed. Positive skin-typing results and STR data were obtained from jewellery and musical instruments that had been left untouched for up to 12 years.

RNA in DNA samples

Cell typing through RNA profiling is a relatively new forensic application. RNA typing information may also be of value when old cases are revisited. When evidentiary items are still available, specimens can be taken and subjected to the DNA/RNA co-isolation protocol. However, often the only remaining specimens are the stored DNA extracts. It is conceivable that RNA was isolated together with the DNA although the level of co-extraction may depend on the extraction procedure used. It is unclear whether the RNA remains stable in these extracts. To assess these issues, 10 mock casework DNA extracts (>1-year-old) of known cell type (three blood QIAamp, three blood Chelex, two saliva QIAamp, two semen QIAamp) with a (human) DNA concentration ranging between 0.05 and 4.84 ng/µL were analysed with the 19-plex. Prior to cDNA synthesis the samples were treated with DNase. For only one extract (DNA concentration 2.24 ng/µL) a full RNA profile was obtained that confirmed cellular origin. To assess whether this low cell-typing success was due to RNA ending up in the flow-through fractions of the QIAamp column, 24 fresh flow-through fractions were analysed.

Chapter 1

36

Cha

pter

1

Tabl

e 4.

Ana

lysi

s of

mRN

A ex

trac

ts fr

om o

ld s

tain

s ob

tain

ed b

y en

dpoi

nt R

T-PC

R us

ing

the

19-p

lex.

Age

of t

he s

tain

and

re

sults

of N

GM D

NA p

rofil

ing

are

show

n, in

dica

ted

as th

e pe

rcen

tage

of t

he d

etec

ted

loci

. Eac

h bl

acke

ned

cell

in th

e ta

ble

repr

esen

ts a

pos

itive

cel

l-typ

ing

resu

lt fo

r the

cor

resp

ondi

ng m

RNA

mar

ker.

Corr

espo

ndin

g ap

prox

imat

e pe

ak h

eigh

t dat

a ca

n be

foun

d in

Sup

plem

enta

ry T

able

2.

OLD

STA

INS

Hou

seke

epin

g Bl

ood

Muc

osa

Saliv

a Se

men

M

enst

rual

secr

etio

n Sk

in

Vagin

al m

ucos

a

Sam

ple

Age

D

NA

18

s-rR

NA

A

CTB

G

APD

H

HBB

A

MIC

A1

CD

93

KRT4

SP

RR2A

KR

T13

STA

TH

HTN

3 PR

M1

SEM

G1

MM

P7

MM

P11

CD

SN

LOR

MU

C4

HBD

1

BLO

OD

Stain

1

28 y

rs

100%

Stain

2

28 y

rs

100%

Stain

3

28 y

rs

100%

Stain

4

28 y

rs

100%

Stain

5

28 y

rs

100%

SEM

EN

St

ain 1

28

yrs

10

0%

St

ain 2

28

yrs

10

0%

St

ain 3

28

yrs

10

0%

St

ain 4

28

yrs

10

0%

St

ain 5

28

yrs

10

0%

SA

LIVA

Pacif

ier 1

6

yrs

<50

%

Pacif

ier 2

6

yrs

>50

%,

mix

Pacif

ier 3

6

yrs

>50

%

Pacif

ier 4

6

yrs

100%

, m

ix

Pacif

ier 5

6

yrs

<50

%

Pa

cifie

r 6

6 yr

s 0%

Pacif

ier 7

6

yrs

100%

Bucc

al 1

10 y

rs

>50

%

Bu

ccal

2 10

yrs

10

0%

Bu

ccal

3 10

yrs

10

0%

Bu

ccal

4 10

yrs

10

0%

Bu

ccal

5 10

yrs

>

50%

MEN

STRU

AL

SEC

RETI

ON

Swab

day

2a

8 m

ths

100%

n.dc

n.

d

Sw

ab d

ay 7

8

mth

s 10

0%

n.

d n.

d

st

ain

4 yr

s 10

0%

n.

d

n.d

n.d

n.d

n.d

SKIN

b

Chi

n pi

ece

violin

12

yrs

>

50%

Ank

le

brac

elet

8

yrs

>50

%

Wat

ch

6 yr

s >

50%

Earri

ngs

8 yr

s 10

0%

a C

alcul

ated

from

the

star

t of t

he m

enst

rual

cycle

b Sa

mpl

es a

nalys

ed a

fter e

than

ol p

recip

itatio

n (fu

ll D

NA

and

RN

A e

xtra

cts u

sed)

c n.

d.: n

ot d

eter

min

ed


37

Chapter 1

The fractions corresponding to three blood and five skin samples and eight non-sperm and eight sperm fractions were ethanol precipitated and subjected to RT-PCR analysis. Positive results were obtained for two blood samples and two sperm fractions. The flow-through fractions were found to contain little RNA suggesting co-extraction of RNA with the DNA fraction, which remained unseen in the stored DNA extracts due to instability of the RNA. Indeed, RNA profiles were obtained when 24 QIAamp DNA extracts, that were stored at -80 ºC immediately after extraction, were assessed. We also investigated whether we could perform RNA profiling on DNA extracts from stored DNA extracts obtained by Chelex isolation. Again, hardly any RNA signals were obtained. We infer that RNA profiling is seldom compatible with old stored DNA extracts due to instability of the co-extracted RNA for the methods we described above.

Profiling approach and interpretation

To accomplish optimal RT-PCR results, we propose the following strategy: during cDNA synthesis a fixed amount of RNA input (10 µL) is used unless one is dealing with a large forensic stain. Then, RNA concentrations lie within the range applicable to Bioanalyzer and nanodrop measurements, and RNA amounts are determined by one of these methods. A maximum of 2 µg RNA is taken. With that, the main aim of the RNA measurement used for large stains is to prevent overloading of the cDNA reaction. Subsequently, multiple PCRs with a range of different cDNA inputs (e.g. 0.02 µL, 0.1 µL, 0.5 µL, 1 µL, 3.5 µL and 7.5 µL) are performed as illustrated in Figure 4 for a saliva on skin sample. The RNA profiles that have a too low or too high cDNA input and are without signal or over-amplified, are discarded, while the informative profiles served as confirmatory analyses. For each tissue type in the multiplex we included a minimum of two markers. Not only does this provide a confirmation of the findings by independent markers (as expression of the various mRNAs is not regulated by other markers in the multiplex), it also provides a safeguard for theoretical (silent) point mutations at the primer binding sites. Furthermore, as RNA profiling is based on PCR, peaks may drop out when limited template is available. The use of more than one marker per body fluid is then functional. From the results of the single source stains, an overview was derived to guide interpretation of results obtained with the 19-plex (Textbox 1). Interpretation of the RNA profiles, and thus conclusions about the cell type is based on both the presence and absence of markers.

Chapter 1

38

Cha

pter

1

Fig. 4. Overlay electropherograms of 19-plex amplifications of a saliva–skin mixture using different amounts of cDNA input. In this example the most informative profile is generated using 1 µL of cDNA input. Using 0.02, 0.1 and 0.5 µL partial RNA profiles were generated whereas 3.5 and 7.5 µL result in over-amplification. Over-amplified profiles should not be used for interpretation. LOR was not amplified in this sample. Only relevant bins are shown.


39

Chapter 1

Box 1. Overview of 19-plex results per cell type to guide RNA profile interpretation. Blood: 3 blood markers: HBB, CD93, AMICA1; HBB most sensitive

3 housekeeping markers: 18S-rRNA, ACTB, GAPDH Saliva: 2 saliva markers: STATH, HTN3 3 mucosa markers: KRT4, KRT13, SPRR2A

2 housekeeping markers: 18S-rRNA, ACTB, GAPDH; very low GAPDH Semen: 2 semen markers: PRM1, SEMG1; PRM1 fertile men only

3 housekeeping markers: 18S-rRNA, ACTB, GAPDH Skin: 2 skin markers: CDSN, LOR

1 housekeeping marker:18S-rRNA; low expression ACTB and GAPDH possible expression of 3 mucosa markers: KRT4, KRT13, SPRR2A

Menstrual secretion: 2 menstrual secretion markers: MMP7, MMP11

3 mucosa markers: KRT4, KRT13, SPRR2A 2 vaginal mucosa markers: HBD1, MUC4; HBD1 may drop out: perform singleplex 3 blood markers: HBB, CD93, AMICA1; all relatively low signals 3 housekeeping markers: 18S-rRNA, ACTB, GAPDH

Vaginal mucosa: 2 vaginal mucosa markers: HBD1, MUC4; HBD1 may drop out: perform singleplex

3 mucosa markers: KRT4, KRT13, SPRR2A 3 housekeeping markers: 18S-rRNA, ACTB, GAPDH

Concluding remarks

In this study, we describe a procedure to assess an evidentiary trace for two aspects: (1) who is the donor and (2) what cell type is present. From one sample, DNA and RNA are separately extracted using an extraction method that isolates both high and low molecular weight RNA molecules. While DNA is used for conventional STR profiling, RNA is used for determining the cellular origin. For cell typing, we designed a 19-plex RNA assay and showed that it profiles forensically relevant body fluids and skin with high sensitivity and specificity. The developed single multiplex assay targets six different cellular origins provides an unbiased cell type assessment, which is important in forensic casework. A challenging next step will be to explain the results and interpretation of RNA-based forensic tissue identification to criminal justiceprofessionals.

Chapter 1

40

Cha

pter

1

Acknowledgements

The authors are grateful to all volunteers who donated samples for this work and thank Ate Kloosterman for providing stored samples. We thank Rolla Voorhamme for critically reading the manuscript. Role of funding: This study was supported by a grant from the Netherlands Genomics Initiative (NGI)/Netherlands Organization for Scientific Research (NWO) within the framework of the Forensic Genomics Consortium Netherlands (FGCN).

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43

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38. Wykes SM, Krawetz SA. The structural organization of sperm chromatin. J Biol Chem 2003; 278:

29471–29477.

39. Lilja H, Abrahamsson PA, Lundwall A. Semenogelin, the predominant protein in human semen.

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Chapter 1

44

Cha

pter

1

Supp

lem

enta

ry t

able

1. R

esul

ts s

how

ing

spec

ifici

ty o

f the

mar

kers

use

d in

the

19-

plex

in d

iffer

ent

tissu

e ty

pes.

For

all

mar

kers

15

diffe

rent

sam

ple

sets

con

sist

ing

of e

ight

indi

vidu

als

wer

e an

alys

ed. F

or s

emen

sam

ples

the

set c

ompr

ised

six

fe

rtile

and

two

ster

ile d

onor

s. Fo

r mix

ed sp

ecim

ens,

cell

mat

eria

l of t

he sa

me

dono

r was

use

d. T

he p

erce

ntag

e of

indi

vidu

als

in w

hich

mar

ker e

xpre

ssio

n w

as o

bser

ved

is in

dica

ted

per s

ampl

e se

t as a

gre

ysca

le in

whi

ch e

ach

shad

e of

gre

y re

pres

ents

a

spec

ific p

erce

ntag

e. In

dica

ted

in e

ach

cell

is th

e av

erag

e ap

prox

imat

e pe

ak h

eigh

t.

Hous

ekee

ping

Bloo

dM

ucos

aSa

liva

Sem

enM

enst

rual

secr

etio

nSk

inVa

gina

l muc

osa

Sam

ple

sets

n18

S-R

NA

ACTB

aG

APD

HH

BB

AMIC

A1C

D93

KR

T4SP

RR

2AK

RT1

3ST

ATH

HTN

3PR

M1

SEM

G1

MM

P7M

MP1

1C

DSN

LOR

MU

C4

HB

D1

HB

D1b

1ple

x

1Bl

ood

8>6

000

>600

0>6

000

>400

0>4

000

>400

0~1

000

n.dc

2aSe

men

-fert

ile6

>600

0>6

000

~300

0600

0>6

000

>600

0n.

d

3Sa

liva

8>6

000

>600

0~1

000

6

000

>400

0400

0>6

000

>400

0~3

000

~100

0~3

000

~300

0n.

d

5Va

gina

l Muc

osa

8>4

000

>600

0~3

000

>400

0>6

000

>600

06

000

~100

0


45

Chapter 1

Supp

lem

enta

ry ta

ble

2. A

naly

sis

of m

RNA

extr

acts

from

old

sta

ins

obta

ined

by

endp

oint

RT-

PCR

usin

g th

e 19

-ple

x. A

ge

of th

e st

ain

and

resu

lts o

f NGM

DNA

pro

filin

g ar

e sh

own,

indi

cate

d as

the

perc

enta

ge o

f the

det

ecte

d lo

ci. E

ach

blac

kene

d ce

ll in

the

tabl

e re

pres

ents

a p

ositi

ve c

ell-t

ypin

g re

sult

for

the

corr

espo

ndin

g m

RNA

mar

ker.

Indi

cate

d in

eac

h ce

ll is

the

appr

oxim

ate

peak

hei

ght.

OLD

STA

INS

Hou

seke

epin

g Bl

ood

Muc

osa

Saliv

a Se

men

M

enst

rual

Secr

etio

n Sk

in

Vagin

al M

ucos

a

Sam

ple

Age

D

NA

18

s-rR

NA

A

CTB

G

APD

H

HBB

A

MIC

A1

CD

93

KRT4

SP

RR2A

KR

T13

STA

TH

HTN

3 PR

M1

SEM

G1

MM

P7

MM

P11

CD

SN

LOR

MU

C4

HBD

1

BLO

OD

Stai

n 1

28 y

rs

100%

>

6000

>

6000

>

4000

>

6000

~

3000

~

3000

Stai

n 2

28 y

rs

100%

>

6000

>

6000

>

6000

>

6000

>

6000

>

6000

Stai

n 3

28 y

rs

100%

>

6000

>

4000

>

6000

>

6000

>

6000

>

6000

Stai

n 4

28 y

rs

100%

>

6000

>

6000

~

1000

>

6000

~

2000

~

2000

Stai

n 5

28 y

rs

100%

>

6000

>

6000

>

4000

>

6000

~

1000

>

6000

SEM

EN

St

ain

1 28

yrs

10

0%

>60

00

>40

00

St

ain

2 28

yrs

10

0%

>60

00

>60

00

>60

00

Stai

n 3

28 y

rs

100%

>

6000

>

6000

>

6000

<50

0

>

4000

>

6000

St

ain

4 28

yrs

10

0%

>60

00

~30

00

~20

00

>

6000

St

ain

5 28

yrs

10

0%

>60

00

~20

00

>

4000

~

1000

SA

LIVA

~

Pa

cifie

r 1

6 yr

s <

50%

>

6000

<20

0 <

500

~

2000

~

1000

Paci

fier 2

6

yrs

>50

%,

mix

>

6000

<

200

~30

00

~20

00

~10

00

~10

00

>

4000

~

1000

Paci

fier 3

6

yrs

>50

%

>60

00

~10

00

<50

0 <

500

~

2000

Paci

fier 4

6

yrs

100%

, m

ix

>60

00

<50

0

>

4000

~

1000

<

200

~

1000

>

6000

<

500

Paci

fier 5

6

yrs

<50

%

>60

00

<

500

<

200

~10

00

Pa

cifie

r 6

6 yr

s 0%

>

6000

Pa

cifie

r 7

6 yr

s 10

0%

>60

00

<50

0

~

2000

~

2000

~

1000

<

500

~10

00

Bucc

al 1

10

yrs

>

50%

~

2000

<

200

>40

00

>40

00

>60

00

~10

00

~10

00

Bucc

al 2

10

yrs

10

0%

~10

00

~

3000

~

3000

>

4000

~

2000

~

1000

Bu

ccal

3

10 y

rs

100%

~

1000

<

200

~30

00

~30

00

~30

00

<20

0 <

500

<

200

Bucc

al 4

10

yrs

10

0%

~10

00

<50

0

~

3000

~

2000

~

3000

<

500

~10

00

Bucc

al 5

10

yrs

>

50%

~

1000

~20

00

~20

00

~30

00

<

500

MEN

STRU

AL

SEC

RETI

ON

Swab

day

2a

8 m

ths

100%

>

6000

>

6000

~

1000

~

1000

~

3000

>

6000

>

6000

n.

dc

n.d

~

1000

~

1000

~

3000

Swab

day

7

8 m

ths

100%

>

4000

48

63

~10

00

>60

00

>60

00

n.d

n.

d

<50

0

stain

4

yrs

100%

~

3000

n.

d ~

3000

<

500

~10

00

~10

00

~30

00

>60

00

>60

00

n.d

n.

d

~30

00

~10

00

n.d

n.

d

~30

00

>60

00

SKIN

b

Chi

n pi

ece

viol

in

12 y

rs

>50

%

>60

00

~20

00

~

2000

>60

00

>60

00

Ank

le

brac

elet

8

yrs

>50

%

>60

00

<50

0

<50

0

Wat

ch

6 yr

s >

50%

>

6000

~

2000

>

4000

Ea

rrin

gs

8 yr

s 10

0%

~30

00

<

500

~10

00: 5

00-1

500

rfu

~20

00: 1

500-

2500

rfu

~

3000

: 250

0-40

00 r

fu

>40

00: 4

000-

6000

rfu

>

6000

: >60

00 r

fu

a C

alcu

late

d fro

m th

e st

art o

f the

men

stru

al c

ycle

b

Sam

ples

ana

lyse

d af

ter

etha

nol p

reci

pita

tion

(full

DN

A a

nd R

NA

ext

ract

s us

ed)

c n.

d.: n

ot d

eter

min

ed

~

1000

: 500

-150

0 rfu

~

2000

: 150

0-25

00 r

fu

~30

00: 2

500-

4000

rfu

>

4000

: 400

0-60

00 r

fu

>60

00: >

6000

rfu

a C

alcu

late

d fro

m th

e st

art o

f the

men

stru

al c

ycle

b

Sam

ples

ana

lyse

d af

ter

etha

nol p

reci

pita

tion

(full

DN

A a

nd R

NA

ext

ract

s us

ed)

c n.

d.: n

ot d

eter

min

ed

Chapter 1

46

Cha

pter

1

Supplementary table 3. RNA analysis of freshly extracted DNA samples from a body fluid dilution series. DNA extracts of blood, saliva and semen isolated with the QIAamp method are compared. Indicated in grey-scale are the number of tissue-specific markers which have been found in the respective DNA sample.

Volume body fluid on swab

(µL)

Blood

Saliva

Semen

10 5 1

0.5 0.1 0.05 0.01 0.001

Two or more tissue-specific markers detected One tissue-specific marker detected No tissue-specific markers detected

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Transcript of UvA-DARE (Digital Academic Repository) Development of RNA … · tetramethylbenzidine test,...