Jack Ballantyne

Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs.

The serology-based methods routinely used in forensic casework for the identification of biological fluids are costly in terms of time and sample and have varying degrees of sensitivity and specificity. Recently, the use of a molecular genetics-based approach using messenger RNA (mRNA) profiling has been proposed to supplant conventional methods for body fluid identification. However, the size of the amplification products used in these mRNA assays (approximately 200-300 nt) might not be ideal for use with degraded or compromised samples frequently encountered in forensic casework. Recently, there has been an explosion of interest in a novel class of small noncoding RNAs, microRNAs (miRNAs, approximately 20-25 bases in length), with numerous published studies reporting that some miRNAs are expressed in a tissue-specific manner. In this article, we provide the first comprehensive evaluation of miRNA expression in dried, forensically relevant biological fluids--blood, semen, saliva, vaginal secretions, and menstrual blood--in an attempt to identify putative body fluid-specific miRNAs. Most of the 452 human miRNAs tested (approximately 67% of the known miRNAome) were either expressed in multiple body fluids or not expressed at all. Nevertheless, we have identified a panel of nine miRNAs--miR451, miR16, miR135b, miR10b, miR658, miR205, miR124a, miR372, and miR412--that are differentially expressed to such a degree as to permit the identification of the body fluid origin of forensic biological stains using as little as 50 pg of total RNA. The miRNA-based body fluid identification assays were highly specific because the miRNA expression profile for each body fluid was different from that obtained from 21 human tissues. The results of this study provide an initial indication that miRNA profiling may provide a promising alternative approach to body fluid identification for forensic casework.

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

Conventional methods of body fluid identification use a variety of labor-intensive, technologically diverse techniques that are performed in a series, not parallel, manner and are costly in terms of time and sample. Theoretically, the identification of a body fluid may be made by determining a sufficient number of mRNAs that are expressed exclusively in cells that collectively comprise that body fluid. Advantages of an mRNA-based approach, compared to conventional biochemical methods of analysis, include greater specificity, simultaneous and semi-automatic analysis through a common assay format, improved timeliness, decreased sample consumption and compatibility with DNA extraction methodologies. In this report, we demonstrate that RNA is stable in biological stains and can be recovered in sufficient quantity and quality for analysis. Messenger RNA from the housekeeping genes S15, beta-actin and GAPDH was detected in blood, semen and saliva stains using a sensitive reverse transcriptase-polymerase chain reaction assay (RT-PCR). Additionally, we have identified a number of candidate tissue-specific genes, statherin, histatin 3, PRB1, PRB2 and PRB3 that may be useful for the positive identification of saliva. Messenger RNAs from these genes were detectable in saliva stains but not in blood or semen stains. Collectively these findings constitute the basis of a prototype RNA based assay system that may eventually supplant conventional methods for body fluid identification.

mRNA Profiling for Body Fluid Identification by Multiplex Quantitative RT‐PCR*

Abstract:  An alternative approach to conventional protein‐based body fluid identification is gene expression profiling analysis. In the present work, we report the development of sensitive and robust multiplex quantitative reverse transcriptase‐PCR assays for the identification of blood, saliva, semen, and menstrual blood. Each body fluid assay comprises a triplex system that detects transcripts from two body fluid‐specific genes and a housekeeping gene GAPDH. The body fluid‐specific genes include erythroid δ‐aminolevulinate synthase (ALAS2) and β‐spectrin (SPTB) for blood, statherin (STATH) and histatin 3 (HTN3) for saliva, protamine 1 (PRM1) and protamine 2 (PRM2) for semen, and matrix metalloproteinase 7 (MMP7) and matrix metalloproteinase 10 (MMP10) for menstrual blood. Normalization of both body fluid‐specific genes to the housekeeping gene by means of appropriate cycle threshold metrics ensures the high specificity of each assay for its cognate body fluid.

Recovery and Stability of RNA in Vaginal Swabs and Blood, Semen, and Saliva Stains

Abstract:  RNA expression patterns, including the presence and relative abundance of particular mRNA species, provide cell and tissue specific information that could be used for body fluid identification. In this report, we address perceived concerns on the stability, and hence recoverability, of RNA in forensic samples. Stains were prepared from blood, saliva, semen, and vaginal secretions and exposed to a range of environmental conditions from 1 to 547 days. The persistence and stability of RNA within each type of body fluid stain were determined by quantitation of total RNA, and reverse transcriptase‐polymerase chain reaction (RT‐PCR) using eight different mRNA transcripts from selected housekeeping and tissue‐specific genes. The results demonstrate that RNA can be recovered from biological stains in sufficient quantity and quality for mRNA analysis. On average, several hundred nanograms of total RNA was recovered from 50‐μL‐sized blood and saliva stains, 1 μg from a 50‐μL semen stain and nearly 70 μg from a whole vaginal swab. Messenger RNA is detectable in some samples stored at room temperature for at least 547 days. The environmental samples that were protected from direct rain impact exhibited housekeeping and tissue specific mRNA recoverability up to 7 days (saliva and semen), 30 days (blood), or 180 days (vaginal swab). Additionally, rain had a detrimental effect on the recoverability of blood (3 days), saliva (1 day), semen (7 days), and vaginal secretions (3 days) specific transcripts, with one of the mRNA species (the semen marker PRM2) not being detectable after 1 day.

Simplified Low‐Copy‐Number DNA Analysis by Post‐PCR Purification

Abstract:  Frequently, evidentiary items contain an insufficient quantity of DNA to obtain complete or even partial DNA profiles using standard forensic gentotyping techniques. Such low‐copy‐number (LCN) samples are usually subjected to increased amplification cylces to obtain genetic data. In this study, a 28‐cycle polymerase chain reaction (PCR) was used to evaluate various methods of post‐PCR purification for their effects on the sensitivity of fluorophore‐based allelic detection subsequent to capillary electrophoretic separation. The amplified product was purified using filtration, silica gel membrane, and enzyme mediated hydrolysis purification techniques and evaluated for their effect on fluorescent allelic signal intensity. A purification method was selected and its effect on fluorescent allelic signal intensity was compared with that of the unpurified PCR product. A method of post‐PCR purification is described which increases the sensitivity of standard 28‐cycle PCR such that profiles from LCN DNA templates (<100 pg DNA) can be obtained. Full DNA profiles were consistently obtained with as little as 20 pg template DNA without increased cycle number. In mock case type samples with dermal ridge fingerprints, genetic profiles were obtained by amplification with 28 cycles followed by post‐PCR purification whereas no profiles were obtained without purification of the PCR product. Allele dropout, increased stutter, and sporadic contamination typical of LCN analysis were observed; however, no contamination was observed in negative amplification controls. Post‐PCR purification of the PCR product can increase the sensitivity of capillary electrophoresis to such an extent that DNA profiles can be obtained from <100 pg of DNA using 28‐cycle amplification.

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

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 μl 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.

mRNA profiling for the identification of blood—Results of a collaborative EDNAP exercise

A collaborative exercise on mRNA profiling for the identification of blood was organized by the European DNA Profiling Group (EDNAP). Seven blood samples and one blood dilution series were analyzed by the participating laboratories for the reportedly blood-specific markers HBB, SPTB and PBGD, using different kits, chemistries and instrumentation. The results demonstrate that HBB is expressed abundantly in blood, SPTB moderately and PBGD significantly less. All but one of the 16 participating laboratories were able to successfully isolate and detect RNA from the dried bloodstains even though a majority of the laboratories had no prior experience with RNA. Despite some expected variation in sensitivity between laboratories, the method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise support the potential use of mRNA profiling as an alternative to conventional serological tests.

Laser Microdissection Separation of Pure Spermatozoa from Epithelial Cells for Short Tandem Repeat Analysis

ABSTRACT: Short tandem repeat (STR) analysis is a valuable tool in identifying the source of biological stains, particularly from the investigation of sexual assault crimes. Difficulties in analysis arise primarily in the interpretation of mixed genotypes when cell separation of the sexual assailants sperm from the victims cells is incomplete. The forensic community continues to seek improvements in cell separation methods from mixtures for DNA typing. The feasibility of applying laser microdissection (LMD) technology to precisely separate sexual assault cell mixtures by visual inspection coupled with laser dissection was assessed through three experiments. First, various histological stains were evaluated for use with LMD and DNA analysis. Second, different DNA isolation methods were evaluated on LMD‐collected cells. Finally, STR analysis was performed on LMD‐separated sperm cells from mixtures of semen and female buccal epithelial cells. The results indicated (a) hematoxylin/eosin staining performed best in its ability to differentiate sperm and epithelial cells while exhibiting the least negative effect on further downstream analysis; (b) both QIAamp® and Lyse‐N‐Go™ methods were useful for recovery of DNA from LMD‐collected sperm cells; and (c) LMD separation provided clear STR profiles of the male donor with the absence of any additional alleles from the female donor. This report describes an efficient, low‐manipulation LMD method for the efficient separation of spermatozoa from two‐donor sperm/epithelial cell mixtures.

Novel Y-STR typing strategies reveal the genetic profile of the semen donor in extended interval post-coital cervicovaginal samples

For a variety of reasons, some victims of sexual assault provide vaginal samples more than 24-36 h after the incident. In these cases, the ability to obtain an autosomal STR profile of the semen donor from the living victim diminishes rapidly as the post-coital interval is extended. We have used a number of carefully selected Y-STR loci in a variety of multiplex or monoplex formats to extend the post-coital interval from which a genetic profile of the semen donor can be obtained. The proposed Y-STR typing strategies enable the routine detection of the male donor Y-STR haplotype in cervicovaginal samples recovered up to 4 days post-coitus. We attribute our success to a number of factors that significantly improve the sensitivity and specificity of the analysis. Firstly, we utilize a subset of Y-STR loci that have been carefully selected for their superior performance under stressed conditions in both multiplex and monoplex formats. Specifically these loci function with low copy number templates in the presence of a vast excess of potentially confounding female DNA. Secondly, sperm and non-sperm DNA is co-extracted without a differential extraction process to prevent the unnecessary loss of the small number of structurally fragile sperm remaining in the cervicovaginal tract several days after intercourse. Thirdly, low copy number detection is facilitated by increasing the cycle number to 34-35 cycles and by the ability to input up to 450 ng of co-extracted sperm/non-sperm DNA into the PCR reaction without the appearance of confounding female artifacts. Lastly, the proper collection of post-coital cervicovaginal samples, instead of the lower or mid-vaginal tract samples often taken, is required for optimal recovery of sperm for analysis. In this report we demonstrate that our previously described 19 Y-STR loci systems (MPI and MPII) permit a reliable high resolution haplotype determination of the semen donor in cervicovaginal samples taken up to 48 h after intercourse. However, as the post-coital interval is extended further, dramatic loss of signal is observed and haplotype determination of the male donor is no longer possible with MPI and MPII. Nonetheless, subsets of these 19 loci (MPA and MPB) have been developed specifically to detect the male haplotype in samples recovered 4 days after intercourse. Thus, it is possible to derive an 11-19 locus Y-STR profile of the semen donor in cervicovaginal samples recovered 2-4 days after intercourse.

RNA/DNA co-analysis from human saliva and semen stains--results of a third collaborative EDNAP exercise.

A third collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Twenty saliva and semen stains, four dilution series (10-0.01 μl saliva, 5-0.01 μl semen) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 20 participating laboratories using an RNA extraction or RNA/DNA co-extraction method. Two novel mRNA multiplexes were used: a saliva triplex (HTN3, STATH and MUC7) and a semen pentaplex (PRM1, PRM2, PSA, SEMG1 and TGM4). The laboratories used different chemistries and instrumentation and a majority (16/20) were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA from individual stains not only permitted a confirmation of the presence of saliva/semen (i.e. tissue/fluid source of origin), but allowed an STR profile of the stain donor to be obtained as well. The method proved to be reproducible and sensitive, with as little as 0.05 μl saliva or semen, using different analysis strategies. Additionally, we demonstrated the ability to positively identify the presence of saliva and semen, as well as obtain high quality DNA profiles, from old and compromised casework samples. The results of this collaborative exercise involving an RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of saliva and semen in forensic casework that is compatible with current DNA analysis methodologies.