The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids

Selection of highly specific and sensitive mRNA biomarkers for the identification of blood

“…However, currently no housekeeping gene is 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 (GAPDH, 18S rRNA, TEF, UCE), presumably because of limited cell metabolism in spermatozoids and the desquamated cells of the cheek mucosa [14,17]. Additionally, the high abundance of housekeeping genes relative to mRNA may result in the titration of critical PCR components thereby affecting the overall sensitivity of the multiplex system.…”

“…A number of mRNA 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][12][13][14][15][16][17]. RNA is notorious for its rapid post-mortem and in vitro decay, but several reports have pointed out an unexpectedly high stability of RNA in forensic stains [18][19][20].…”

“…Blood was chosen for its easy availability, stability and the number of reported markers [6][7][8][9][10][13][14][16][17][19][20]. The following blood markers were included in the study: HBA and HBB are the alpha-and betasubunits of hemoglobin A [21,22]; aminolevulinate synthase 2 (ALAS2) is an erythroid-specific mitochondrial enzyme, that catalyzes the first step in the heme biosynthetic pathway [23]; CD3 gamma molecule (CD3G) is part of the T-cell receptor-CD3 complex, which plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways [24]; ankyrin 1 (ANK1) is an erythrocyte membrane protein, which provides the primary linkage between the membrane skeleton and the plasma membrane [25]; the erythroid form of porphobilinogen deaminase (PBGD) is the third enzyme of the heme biosynthetic pathway [26]; β-spectrin (SPTB) is an erythrocyte membrane protein [27]; Aquaporin (AQP9) is a water channel protein expressed in peripheral leukocytes [28].…”

RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: Results of a fourth and fifth collaborative EDNAP exercise

“…The expression of these housekeeping genes was additionally tested in blood, saliva and semen dilution series. Several housekeeping gene markers have already been tested on body fluids [19,24,32,37], but up to now, no marker has been described that is universally suitable for all body fluids. Saliva and semen normally show reduced expression of housekeeping genes, probably because spermatozoids contain little cytoplasm and few ribosomes and the desquamated cells of the buccal mucosa have almost no cell metabolism [19].…”

Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic ‘Touch DNA’ Evidence

“…Without such a determination, the ambiguity of the tissue source of origin can be exploited as alternative circumstances of the crime (e.g., how the body fluid may have been deposited) could be suggested. The bioparticle collection and isolation protocols described here could be used to collect bioparticles or other cells (e.g., buccal, vaginal) for use in a tissue source identification (mRNA profiling [21][22][23][24][25][26][27][28][29][30] ) strategy that involves micro-volume reverse transcription (RT) and amplification reactions. With further optimization it may also be possible to develop a DNA/RNA co-isolation strategy to permit cell type identification (RNA) and STR profiling from the same sample, including bioparticles from touch DNA evidence.…”