mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR

Abstract

mRNA profiling is a promising new method for the identification of body fluids from biological stains. Major advantages of mRNA profiling are the possibility of detecting several body fluids in one multiplex reaction and of simultaneously isolating DNA without loss of material. A reverse transcription endpoint polymerase chain reaction (PCR) method and a realtime PCR assay were established for the identification of blood, saliva, semen, vaginal secretions and menstrual blood, and were compared to conventional enzymatic and immunologic tests. The results for specificity, sensitivity and suitability to biological stains were satisfying and mRNA stability was demonstrated for up to 2-year-old stains. Two novel multiplex assays were created with the endpoint PCR primers: multiplex 1 amplifies two markers for each of the above mentioned body fluids and is suited for screening; multiplex 2 was designed for the detection of blood, vaginal secretions and menstrual blood. The results demonstrate that both endpoint PCR and realtime PCR are suitable for the identification of body fluids in forensic stains and represent an effective alternative to conventional enzymatic and immunologic tests.

Introduction

Identification of the biological source of a stain is an important issue in forensic casework, that may also help in predicting the success of a DNA analysis and in interpreting DNA results. In forensic practice biological stains are routinely preanalyzed with enzymatic or immunologic tests. However, there are some problems associated with these tests. Most of them are not specific, due, for example, to cross-reactions with other species or tissues. Furthermore, no such tests are available for the identification of vaginal secretions and menstrual blood. In particular, the differentiation between menstrual blood and blood due to trauma can be crucial in forensic casework, for example, in sexual assault cases.

Recently, the analysis of cell-specific mRNA expression has been proposed as a promising new technique for the identification of body fluids in biological stains. A number of markers have been identified for the forensically most relevant body fluids: blood, saliva, semen, vaginal secretions and menstrual blood [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11].

However, RNA is notorious for its rapid post-mortem and in vitro decay, because of the ubiquitously present RNases. Quite unexpectedly, however, several reports have pointed out a high stability of RNA under controlled conditions; for example, RNA could be isolated from 15-year-old dried blood stains [12]. Even from stains that were exposed to a range of environmental conditions for up to 547 days, RNA of sufficient quality and quantity could be isolated [13]. 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 piece of stain [14], [15].

In the present study a reverse transcription endpoint PCR and a realtime PCR method were developed and compared to conventional enzymatic and immunologic tests for the identification of body fluids. Endpoint PCR allows the detection of a specific transcript, when present in sufficient quantity. In contrast, realtime PCR allows the detection of relative gene expression levels in different samples and in comparison to an endogenous control. The following genes, which have been reported to be expressed in a tissue-specific manner, were analyzed: porphobilinogen deaminase (PBGD) [16], β-spectrin (SPTB) [17] and hemoglobin beta (HBB) [18] for blood, statherin (STATH) [19] and histatin 3 (HTN3) [19], [20] for saliva, protamine 1 and 2 (PRM1 and PRM2) [3], [21] for sperm, human beta-defensin 1 (HBD1) [22] and mucin 4 (MUC4) [23] for vaginal secretions and matrix metalloproteinases 7 and 11 (MMP7 and MMP11) [2] for menstrual blood. The housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S rRNA were used as endogenous controls. The endpoint PCR primers were combined and tested in two multiplexes, allowing the simultaneous amplification of several markers in one PCR reaction. Specificity, sensitivity and suitability to biological stains were analyzed and mRNA stability was recorded over time. The menstrual marker expression was investigated for a whole menstrual cycle (days 1–28).