mRNA and MicroRNA for Body Fluid Identification

BIOLOGY/DNA/RNA

mRNA and MicroRNA for Body Fluid Identification C Haas, University of Zu¨rich, Zu¨rich, Switzerland E Hanson and J Ballantyne, University of Central Florida, Orlando, FL, USA ã 2013 Elsevier Ltd. All rights reserved.

Glossary Ct value Cycle number at which the fluorescence signal generated by the amplification of a gene passes a preset threshold (point at which fluorescent signal is distinguishable from baseline). Delta Ct (dCt) method The dCt is obtained by subtracting the average body fluid gene Ct from the average housekeeping gene Ct. A positive dCt value indicates that a body fluid gene is present in higher abundance than the housekeeping gene, whereas a negative dCt value indicates that a body fluid gene is present in lower abundance than the housekeeping gene. Microarray analysis Hybridization of a nucleic acid sample to a very large set of oligonucleotide probes, which are attached to a solid support (usually a glass slide or silicon thinfilm cell), to determine the sequence or to detect variations in a gene sequence or expression or for gene mapping.

Introduction The presence of body fluids can indicate the location of potential sources of DNA that, once recovered, may be used to identify the donor of the stain. While the most common body fluids found at crime scenes are blood, saliva, and semen, other fluids such as vaginal secretions, menstrual blood, urine, and sweat can be encountered. Conventional methods for body fluid stain analysis involve the use of enzymatic or immunological tests (Table 1). Due to limited specificity (i.e., cross-reactivitywith other species or tissues), many of these tests are presumptive in nature. Furthermore, no routinely used tests are available for the identification of vaginal secretions, menstrual blood, and sweat. Identification of the tissue source of a biological stain can be crucial to the investigation and prosecution of a case. An example demonstrating the importance of identifying the body fluid could be that DNA from a sexual assault victim is found in a suspect’s vehicle and the suspect claims it was present due to casual contact since the victim had ridden in his car numerous times. However, the significance of this evidence would increase if the source of the DNA could be shown instead to originate from the victim’s vaginal secretions, a circumstance which would be more difficult to attribute to casual contact as opposed to a sexual assault. To provide operational forensic laboratories

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Minor groove binder (MGB) probes Hybridized to internal regions of qPCR products during the annealing stage, MGB probes incorporate a 50 reporter dye and a 30 nonfluorescent quencher. Following hybridization to their complementary DNA template, they are degraded by the 50 –30 exonuclease activity of the Taq polymerase during extension, releasing the fluorescence of the reporter dye. Pseudogenes Nonfunctional homologues of known genes that have lost their protein-coding ability. So-called processed pseudogenes are retrotransposed from mRNA and incorporated into the chromosome; they do not have introns or promoters. Real-time PCR (qPCR) Monitors the accumulation of PCR product (changes in fluorescent signal) formation during amplification using either a fluorescently labeled probe (specific sequence within the target region; e.g., MGB probe) or an intercalating dye specific for DNA (e.g., SYBR green).

with improved methodologies for body fluid identification, attempts to utilize molecular-based identification techniques are being developed. Messenger RNA (mRNA) and microRNA (miRNA) profiling are promising new methods for the identification of body fluids in biological stains. These are considered as confirmatory tests that conclusively identify a body fluid. mRNA profiling is based on the premise that each single tissue type is comprised of cells that have a unique transcriptome or gene expression (i.e., mRNA) profile. A number of markers have been identified for the forensically most relevant body fluids, that is, blood, saliva, semen, vaginal secretions, menstrual blood, and sweat. mRNA is notorious for its rapid postmortem and in vitro decay, due to ubiquitously present RNases. This was expected to be especially problematic as biological stains from casework are often challenged by moisture, UV light, temperature, and suboptimal environmental pH, thus potentially resulting in mRNA of insufficient quality for analysis. Quite unexpectedly, however, a high stability of mRNA in dried stains, even from old and compromised samples, has been reported. Recently, there has been an explosion of interest in a class of small noncoding RNAs, miRNAs, whose regulatory functions in various developmental and biological processes have been identified. Numerous studies have indicated that miRNAs are also expressed in a tissue-specific manner and therefore

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Biology/DNA/RNA | mRNA and MicroRNA for Body Fluid Identification

Table 1

403

Summary of conventional enzymatic and immunological methods for body fluid analysis

Classification

Protein

Method

Heme Heme Heme Hemoglobin

Luminol, fluorescein, Bluestar Benzidine, Kastle–Meyer, O-toludine, tetramethyl-benzidine/hemastix, leucomalachite green Teichman, Takayama HemeSelect, ABAcard, HemaTrace, Hexagon OBTI, RSID-blood

a-Amylase a-Amylase

Starch-iodine, Phadebas, Amylose azure, Rapignost-Amylase, SALIgAE RSID-saliva

Chemical

Acid phosphatase

Crystal tests Microscopic Immunological

Choline, spermine

Alpha-naphthyl phosphate/Brentamine Fast Blue, beta-naphthol/Fast garnet B, alpha-naphtol/Fast Red AL Florence, Barberio, Puanen’s tests Christmas tree, haematoxylin/eosin, Baecchi’s, Papanicolaou’s staining Biosign, ABAcard, Chembio, Medpro, Onco-screen, PSA-check-1, Seratec PSA Semiquant RSID-semen, Nanotrap Sg

Blood Chemiluminescent Chemical Crystal test Immunological Saliva Chemical Immunological Semen

Prostate-specific antigen Semenogelin

Urine Chemical Immunological

Urea Creatinine Tamm–Horsfall glycoprotein

Nessler’s reagent, p-dimethylaminocinnamaldehyde, urease/bromthymol blue Jaffe test, Salkowski test RSID-urine

Source: Adapted from Virkler et al. (2009).

may be used to identify body fluids. Due to their small size (18–24 bases in length), miRNAs may be more stable than mRNAs and therefore more suitable for use with degraded and environmentally compromised samples frequently encountered in forensic casework.

Messenger RNA mRNA is a single-stranded nucleic acid, consisting of four kinds of nucleotides: uracil, guanine, adenine, and cytosine. mRNA is transcribed from a DNA template and carries coding information to the ribosomes, where it is translated into a protein (Figure 1). During transcription, RNA polymerase II makes a complementary copy of a gene from DNA to pre-mRNA. Eukaryotic pre-mRNA undergoes extensive processing: 50 cap addition, splicing, editing, and polyadenylation. The 50 cap is critical for recognition by the ribosome and protection from RNases. During splicing, certain stretches of noncoding sequences (introns) are removed. Pre-mRNA can sometimes be spliced in several different ways; as a result, a single gene can encode multiple proteins (alternative splicing). In some instances, an mRNA is edited by changing its nucleotide composition. The poly(A) tail at the 30 end helps to protect mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. The mature mRNA is exported from the nucleus to the cytoplasm, where it is translated into protein by the ribosome. During translation, the sequence of nucleotides in the mRNA molecule is read in sets of three nucleotides (called codons), each of which codes for a specific amino acid. The resulting polypeptide will later fold into an active protein. mRNA is

degraded by several decay pathways. The limited lifetime of mRNA enables a cell to alter protein synthesis rapidly in response to its changing needs.

Detection Technology mRNA profiling includes three main steps: RNA extraction, reverse transcription (RT), and amplification/detection. Total RNA and DNA can be coextracted from the same stain, either manually or with an extraction kit. The RNA extract should be cleaned from residual DNA using a DNase treatment. It is beneficial to quantify the RNA and put a defined amount into the RT reaction to avoid false positive and negative signals. In the RT reaction, mRNA is reverse-transcribed into complementary DNA (cDNA) using random primers. cDNA is then amplified with fluorescence-labeled, tissue-specific primers (end-point polymerase chain reaction – PCR) and amplicons are separated and detected with capillary electrophoresis. Alternatively, cDNA may be analyzed using real-time quantitative PCR (qPCR) with tissue-specific primers and a minor groove binder (MGB) probe. qPCR assays include a housekeeping gene to normalize the expression of the tissue-specific genes. mRNA markers can be combined in multiplexes, for end-point PCR as well as qPCR, which amplify several markers for one or several body fluids in a single PCR. Coextracted DNA can be analyzed according to standard Short tandem repeat (STR) protocols (see chapters 40 and 50).

Tissue-Specific mRNA Markers A number of mRNA markers have been identified for the forensically most relevant body fluids, that is, blood, saliva, semen, vaginal secretions, menstrual blood, and sweat. Table 2 lists a

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Nucleus Transcription RNA Pol II

Antisense

C T G A C G G A T C A G C C G C A A G C GG A A T T GG C G A C A T A A G A C U G C C U A G U C GG C G U U

RNA transcript G A C T G C C T A G T C GG C G T T C G C C T T A A C C G C T G T A T T

Sense strand

3⬘ and 5⬘ end processing Splicing

Poly(A)

CAP

AAAA(n) EJC

Export Nuclear pore Localization

Cytoplasm

PABP AA A

AA

Translation

Xm1

Decay

(n) A

5⬘ to 3⬘ decay

3⬘ to 5⬘ decay exosome

Figure 1 The life cycle of mRNA in eukaryotic cells: In the nucleus, RNA polymerase II transcribes a chromosomal gene sequence to pre-mRNA. mRNA processing includes 50 cap addition, intron splicing, editing, and addition of the poly(A) tail. The mature mRNA is then transported to the cytoplasm involving the exon–junction complex (EJC). The poly(A) tail of the mRNA interacts with poly(A)-binding protein (PABP), resulting in mRNA circularization and translation initiation. The small ribosomal subunit scans the mRNA until an appropriate start codon has been reached. Once the ribosomal preinitiation complex is formed, the large ribosomal subunit joins and translation begins. mRNA is degraded by several decay pathways. Adapted from Brooks SA (2010) Functional interactions between mRNA turnover and surveillance and the ubiquitin proteasome system. Wiley Interdisciplinary Reviews: RNA 1: 240–252, copyright 2010, with permission from John Wiley & Sons Ltd.

selection of commonly used markers that have been reported as fluid-specific and, in addition, some mRNA markers that have not yet been extensively tested or shown to cross-react with other body fluids. Approaches to find tissue-specific markers include searches for tissue-specific proteins or bacteria in the literature or online expression databases (e.g., BioGPS), wholegenome expression analysis, or whole transcriptome shotgun sequencing. Primers are designed to overlap exon–exon junctions or span an intron to ensure that the obtained products are not due to the presence of contaminating DNA. The tissuespecific genes should not possess pseudogenes (nonfunctional homologues of the genes) because processed pseudogenes cannot be distinguished from cDNA by mRNA profiling.

Results and Interpretation The evaluation of mRNA markers for forensic use includes testing their sensitivity and specificity, as well as performance with casework samples. The above-mentioned blood, saliva, and semen markers show a high degree of specificity and no relevant cross-reaction with other human tissues/body fluids or with other species. It has been demonstrated that the corresponding mRNA markers can be detected in samples as small as 0.001 ml blood, 0.05 ml saliva, and 0.01 ml semen. The

analysis of sperm and seminal plasma markers allows for the differentiation between samples from aspermic (absence of sperm in semen) men and normospermic men (with normal sperm production). The vaginal markers mucin 4 (MUC4) and human beta-defensin 1 (HBD1) cross-react with buccal cells (i.e., saliva) and are therefore only of limited use in forensic casework. The matrix metalloproteinases (MMP) markers confirm the presence of menstrual blood, but a minor expression is also found in vaginal cells. This complicates the interpretation of a positive MMP result, but could be overcome by comparing the result to a housekeeping gene and/or the vaginal secretion markers HBD1 and MUC4, which are expressed quite constantly and strongly during the whole menstrual cycle. The blood markers are rather weak in menstrual blood samples, because whole blood accounts for only 30–50% of the total flow in most women. Additional mRNA markers have been suggested for the identification of vaginal secretion, menstrual blood, and sweat, but have not yet been extensively tested for forensic use. To date, no mRNA markers for the identification of urine are available, probably due to the limited number of cells and/or low mRNA expression in dried urine stains. False positive and negative signals can be caused by the fact that expression of tissue-specific transcripts is indeed abundant in the respective body fluid, but not necessarily absent in others.

Biology/DNA/RNA | mRNA and MicroRNA for Body Fluid Identification

Table 2

405

Genes that have been reported to be expressed in a tissue-specific manner or body fluid-specific bacteria

Gene

Function/localization of protein

Blood Hemoglobin a (HBA), hemoglobin b (HBB) Aminolevulinate synthase 2 (ALAS2) CD3 gamma molecule (CD3G) Ankyrin 1 (ANK1) Porpho-bilinogen deaminase (PBGD), erythroid form b-spectrin (SPTB) Glycophorin A (Glyco A)a

Alpha- and beta-subunit of hemoglobin A Erythroid-specific mitochondrial enzyme that catalyzes the first step in the heme biosynthetic pathway Part of the T-cell receptor-CD3 complex, which plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways Erythrocyte membrane protein, which provides the primary linkage between the membrane skeleton and the plasma membrane Third enzyme of the heme biosynthetic pathway Erythrocyte membrane protein Glycophorin A is a major sialoglycoprotein of the human erythrocyte membrane and carries the M or N group antigen

Saliva Histatin 3 (HTN3) Statherin (STATH) Mucin 7 (MUC7)

Part of the nonimmune host defense system in the oral cavity Salivary protein that binds hydroxyapatite and acts as an inhibitor of precipitation of calcium phosphate salts in the oral cavity Small salivary mucin, which is thought to play a role in facilitating the clearance of bacteria in the oral cavity and to aid in mastication, speech, and swallowing

Semen Protamine 1 and 2 (PRM1, PRM2) Transglutaminase 4 (TGM4) Prostate-specific antigen, kallikrein 3 (PSA) Semenogelin 1 (SEMG1)

During spermatogenesis, histones are replaced by protamines, which are essential for the highly condensed chromatin structure in the sperm nucleus The transglutaminase family catalyzes the irreversible cross-linking of peptide-bound glutamine residues either with peptide-bound lysines or with primary amines; TGM4 is specific to the prostate Serine protease, found in seminal fluid, prostatic fluid, male serum, and male and female urine Protein originating in the seminal vesicles

Vaginal secretions Mucin 4 (MUC4)b Human beta-defensin 1 (HBD1)b Lactobacillus crispatusa, Lactobacillus gasseria

Mucins protect the surfaces of the reproductive tract epithelium from pathogen penetrance and modulate sperm entry into the uterus Antimicrobial peptide of urogenital tissues Lactobacilli are specific to the vagina

Menstrual blood Matrix metalloproteinases 7, 10, 11 (MMP7, MMP10, MMP11)

Endopeptidases involved in the breakdown of extracellular matrix components

Sweat Dermcidin (DCD)a

Antibiotic peptide, secreted by sweat glands

a

Indicates markers from single studies that have not yet been extensively tested. Indicates markers that have been shown to cross-react with other body fluids.

b

Therefore, a defined amount of RNA should be used in the RT reaction for standardization. To date, no robust human-specific RNA quantification system is available. Fluorescence-based RNA quantification methods are sensitive and convenient, but might have limited casework utility because they are not humanspecific. Thus, bacterial RNA, which is found in abundance in buccal or vaginal swabs, will result in an underestimation of the amount of human RNA in these sample types. A positive control consisting of a marker expressed in all tissue types would confirm a successful analysis. However, no housekeeping gene has been described that is suitable for the detection of all tested body fluids. Saliva and semen, for example, show very little or no expression of common housekeeping genes (glyceraldehyde 3-phosphate dehydrogenase – GAPDH, 18S ribosomal RNA, transcription elongation factor 1a – TEF, ubiquitin-conjugating enzyme – UCE), presumably

because of limited cell metabolism in spermatozoids and the desquamated cells of the cheek mucosa. In addition, housekeeping genes are likely to have pseudogenes. Coextracted DNA might serve as a positive control for the presence of human cells. A concurrent negative RNA result would suggest that none of the analyzed human body fluids is present or that the mRNA is degraded and no result can be obtained. In the case of degradation, housekeeping genes would also be affected. Several negative controls can be included during mRNA analysis to identify possible contamination: extraction blank (no sample added to lysis and purification reaction); RT blank (water in place of RNA added to RT reaction); RT minus (no reverse transcriptase added to RT reaction), to identify possibly contaminating DNA (frequently a larger size than the expected RNA product) or the presence of pseudogenes (same size

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as RNA product); and amplification blank (water in place of cDNA added to PCR reaction). Since casework material is often limited, an important advantage of body fluid identification by mRNA profiling is the possibility of simultaneously isolating RNA and DNA from the same stain and the ability to multiplex numerous mRNA markers for the identification of one or several body fluids. The quantity and quality of coextracted DNA is also sufficient for casework and environmentally exposed samples, even though the yield might be lower compared to non-coextraction methods. The mRNA/DNA coextraction strategy is a reliable, confirmatory, and time-saving option for the identification of body fluids in forensic casework that requires less sample consumption and is also compatible with the current DNA analysis methodology.

MicroRNA miRNAs are a class of small noncoding RNAs, 18–24 nucleotides in length, which regulate many cellular processes at the posttranscriptional level. They bind to complementary sequences on target mRNA transcripts, usually resulting in translational repression and gene silencing (Figure 2). The human genome encodes over 1000 miRNAs. Most miRNA genes are found in intergenic regions or introns and are transcribed by RNA polymerases to primary miRNA (pri-miRNA). Pri-miRNAs are processed by the enzyme ‘Drosha,’ resulting in long hairpin-containing precursor miRNA (pre-miRNA). ‘Exportin-5’ transports pre-miRNA from the nucleus to the cytoplasm. An RNase called ‘Dicer’ cleaves the pre-miRNA and produces double-stranded mature miRNA. Argonaute protein (Ago2) and Dicer together form the ribonucleoprotein RNA-induced silencing complex (RISC). One strand of the mature miRNA is incorporated in the RISC and guides the complex to its RNA target by interacting with the three prime untranslated regions (30 UTR) of the mRNA. miRNA-mediated gene regulation depends on the complementarity between miRNA and target mRNA: If there is perfect complementarity,

Ago2 can cleave the mRNA and lead to direct mRNA degradation; if there is only partial complementarity, the silencing is achieved by preventing translation. This mechanism implies that any given miRNA can bind to a broad spectrum of different mRNAs and any given mRNA can be bound by several miRNAs, thereby expanding miRNA regulatory potential. In addition, miRNA regulation can fine-tune and optimize protein levels using a ‘rheostat’-like mechanism.

Detection Technology The analysis steps of miRNA profiling are similar to those of mRNA profiling: RNA extraction, miRNA-specific RT, and amplification/detection. Total RNA is extracted, DNase-treated, and quantified in exactly the same way as described for mRNA. The short length of mature miRNAs (22 nucleotides) prohibits the conventional design of a random-primed RT step followed by a specific qPCR assay. Two different approaches can be used for RT of miRNAs. The first involves the use of miRNA-specific stem-loop primers. Alternatively, miRNAs are simultaneously polyadenylated and all RNAs (miRNA, mRNA, and other small RNAs) are reverse-transcribed using both random and oligo-dT primers. cDNA is analyzed with qPCR using miRNA-specific primers and an MGB probe or SYBR green for detection. Currently, one or several small RNAs are used for normalization of miRNA abundances across tissues, employing the delta Ct method. miRNA markers and reference genes (small RNAs used for normalization) can also be included in a multiplex system for simultaneous amplification and detection, the only restriction being the limited number of dyes available for qPCR assays.

Tissue-Specific miRNA Markers Over 16 000 miRNAs, across 153 species, have been entered into the miRBase, a searchable database of miRNA sequences and annotations, with hairpin and mature sequences available.

Transcript degradation (requires perfect complementarity)

Target transcript

Cytoplasm Nucleus

or Translational repression (requires only partial complementarity)

Exportin 5

Pasha Drosha PrimiRNA

PremiRNA

RISC

Argonaute

Dicer Mature-22-base-miRNA

Figure 2 The life cycle of miRNA in eukaryotic cells: A primary miRNA (pri-miRNA) transcript is encoded in the cell’s DNA and transcribed in the nucleus, processed by the enzyme Drosha, and exported into the cytoplasm where it is further processed by Dicer. After strand separation, the mature miRNA represses protein production either by blocking translation or causing mRNA degradation. Reprinted from Mack GS (2007) MicroRNA gets down to business. Nature Biotechnology 25: 631–638, copyright 2007, with permission from Macmillan Publishers Ltd.

Biology/DNA/RNA | mRNA and MicroRNA for Body Fluid Identification

Table 3

407

Human miRNAs that have been reported to be expressed in a tissue-specific manner

Blood

Saliva

Semen

Vaginal secretion

Menstrual blood

Reference genes

hsa-miR-451 hsa-miR-16 hsa-miR-144 hsa-miR-185

hsa-miR-658 hsa-miR-205

hsa-miR-135b hsa-miR-10b hsa-miR-135a hsa-miR-891a

hsa-miR-124a hsa-miR-372

hsa-miR-412 hsa-miR-451

RNU6b RNU24 RNU44 RNU48

Through studies of these miRNAs, the biomedical research community has suggested that the miRNome may be a more precise representation of a cell type and condition than the transcriptome. Numerous miRNAs have been reported to be tissue-specific and could be used to identify the body fluid origin of forensic biological stains. Human miRNA markers were screened in forensically relevant body fluids (blood, semen, saliva, vaginal secretion, menstrual blood) by two different research groups using different approaches, resulting in differing body fluid-specific miRNA markers (Table 3). One approach surveyed the expression of 452 miRNAs in several body fluids through qPCR. The other approach detected the expression levels of 718 miRNAs in several body fluids on a microarray and validated potential candidates through qPCR.

Results and Interpretation The evaluation of miRNA markers for forensic use is based on a few publications. Hanson et al. propose a panel of differentially expressed miRNAs that are characteristic of an individual body fluid. None of the selected miRNAs is truly body fluidspecific in the sense that it is expressed uniquely in one tissue and not in any other. Expression levels of miRNA pairs are displayed in 2D scatter plots, and distinct clustering of each body fluid can be observed, separating the body fluid of interest from the others. Twenty-one human tissues also exhibited differing expression profiles. Zubakov et al. present two miRNA markers each for the identification of blood and semen. These miRNA markers are not prone to degradation in samples stored for 1 year. A high sensitivity of the qPCR assay has been shown in that miRNAs could be detected from as little as 0.1 pg total RNA. Notably, the sets of miRNAs proposed by Hanson et al. and Zubakov et al. are completely exclusive and nonoverlapping. While the specificity of the Hanson et al. blood and semen miRNA candidates was confirmed, the specificity for the Hanson et al. saliva, vaginal secretions, and menstrual blood miRNA candidates could not be reproduced by Zubakov et al. However, Zubakov et al. also reported discrepancies between the microarray data and subsequent specificity confirmation testing for numerous miRNA candidates. These discrepancies may, in part, be due to the differing technologies used to detect and analyze miRNA expression. Therefore, additional work will be necessary to determine the most suitable candidates and analytical methodologies. In addition to an evaluation of analytical methods and platforms, further studies are necessary to evaluate miRNA profiling with forensic samples. A proposed advantage of miRNAs is the suitability for use with challenging samples, such as environmentally exposed and heavily degraded

biological stains. However, extensive evaluations of such samples will be necessary before definitive conclusions can be made. Additionally, direct comparison studies examining the stability and sensitivity of miRNA and mRNA markers in these challenging samples will need to be performed to determine which RNA biomarker may be most suitable for use in forensic casework. It will also be necessary to examine potential expression effects caused by diseased or nonhealthy tissue or fluids because of the potential role of miRNAs in various disorders or cancers. Furthermore, the species specificity of the identified candidates will need to be demonstrated when possible. Despite the need for additional work, these initial studies indicate that miRNA profiling may play a role in the identification of forensically relevant biological fluids. As new miRNAs are identified, novel body fluid-specific candidates may be found. Some miRNA analytical methods allow for the simultaneous evaluation of mRNA and miRNA and, therefore, a combination of both RNA biomarkers may provide additional and necessary specificity for the identification of all forensically relevant biological fluids and tissues.

Other Applications of RNA in Forensic Science In forensic casework analysis, it has become necessary to obtain genetic (i.e., STR) profiles from increasingly smaller amounts of biological material left behind by persons involved in criminal offenses. The ability to obtain profiles from trace biological evidence is routinely demonstrated with so-called touch DNA evidence, generally perceived to be the result of DNA obtained from shed skin cells transferred from donor to an object or person during physical contact. Several studies clearly demonstrate the ability to obtain genetic profiles from trace biological evidence but they, out of necessity, bypass the critical identification of the source of the biological material. mRNA profiling may also be applied to the identification of skin cells and some promising candidates have already been described. Other forensic applications using RNA are also being explored, such as using RNA degradation as a parameter for the determination of the postmortem interval and the age of biological stains (time since deposition). Good correlations have been demonstrated under controlled circumstances, but in forensic settings, unfortunately, conditions are not predictable. Additionally, it has been demonstrated that it is possible to identify age-specific expression patterns allowing for a possible determination of the biological age of a bloodstain donor. The age of external and internal wounds is another important question in forensic pathology, which could be answered by measuring mRNAs coding for proteins involved in the cell reaction to injury. Some suitable markers have already been

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evaluated. Another area of interest is the cause and mechanism of death. mRNA profiling could offer insight into the pathological mechanisms leading to death by disclosing the functional and morphological changes of cells immediately preceding death. Postmortem tissue undergoes immediate changes, a fact which has to be considered when studying premortem gene expression. This is a complex field, currently at its very beginning.

See also: Biology/DNA: Basic Principles; DNA Extraction and Quantification; Short Tandem Repeats; Biology/DNA/Methods/ Analytical Techniques: Capillary Electrophoresis in Forensic Genetics; Methods: Capillary Electrophoresis: Basic Principles; Capillary Electrophoresis in Forensic Biology.

Further Reading Ballantyne J (2000) Serology overview. In: Siegel JA, Saukko PJ, and Knupfer GC (eds.) Encyclopedia of Forensic Sciences, pp. 1322–1331. London: Academic Press. Bauer M (2007) RNA in forensic science. Forensic Science International: Genetics 1: 69–74. Courts C and Madea B (2010) Micro-RNA – A potential for forensic science? Forensic Science International 203: 106–111. Fleming RI and Harbison S (2010) The use of bacteria for the identification of vaginal secretions. Forensic Science International: Genetics 4: 311–315. Haas C, Hanson E, Anjos MJ, et al. (2012) RNA/DNA co-analysis from blood stains – Results of a second collaborative EDNAP exercise. Forensic Science International: Genetics 6(1): 70–80. http://dx.doi.org/10.1016/j.fsigen.2011.02.004.

Haas C, Hanson EK, and Ballantyne J (2011) Capillary electrophoresis of a multiplex reverse transcription–polymerase chain reaction to target messenger RNA markers for body fluid identification. In: Alonso A (ed.) DNA Electrophoresis Protocols for Forensic Genetics. New York: Humana Press. Haas C, Klesser B, Maake C, Ba¨r W, and Kratzer A (2009) mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR. Forensic Science International: Genetics 3: 80–88. Hanson EK and Ballantyne J (2010) RNA profiling for the identification of the tissue origin of dried stains in forensic biology. Forensic Science Review 22: 145–158. Hanson EK, Lubenow H, and Ballantyne J (2009) Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs. Analytical Biochemistry 387: 303–314. Juusola J and Ballantyne J (2005) Multiplex mRNA profiling for the identification of body fluids. Forensic Science International 152: 1–12. Landgraf P, Rusu M, Sheridan R, et al. (2007) A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129: 1401–1414. Sakurada K, Akutsu T, Fukushima H, Watanabe K, and Yoshino M (2010) Detection of dermcidin for sweat identification by realtime RT-PCR and ELISA. Forensic Science International 194: 80–84. Vennemann M and Koppelkamm A (2010) mRNA profiling in forensic genetics I: Possibilities and limitations. Forensic Science International 203: 71–75. Virkler K and Lednev IK (2009) Analysis of body fluids for forensic purposes: From laboratory testing to non-destructive rapid confirmatory identification at a crime scene. Forensic Science International 188: 1–17. Zubakov D, Boersma AW, Choi Y, et al. (2010) MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation. International Journal of Legal Medicine 124: 217–226.

Relevant Websites www.biogps.gnf.org – BioGPS. www.mirbase.org – microRNA database.