Introduction 1 2 3 4 2 5 6 7 8 9 10 12 13 14 15 16 19 18 20 Materials and methods Sample collection Aliquots of 5 ml of whole blood and saliva were collected from each of five healthy volunteers (four men and one woman) of western European genetic origin under informed consent before their inclusion in the study. Native blood was collected without anticoagulation treatment to avoid disturbing effects of anticoagulation reagents on gene expression. In each sample, 75 cotton swabs were immersed. Special care was taken to shorten the time between collection and swab absorption to avoid blood coagulation. After complete absorption of the fluids, swabs were left until dry on a bench top at room temperature. When dry, the swabs were stored in dust-free nonhumid conditions (but subjected to normal daylight) for different time intervals. Swabs were visually inspected and sorted out to ensure similar liquid content between individual swabs. After 0, 1, 3, 7, 14, 21, 57, and 180 days, swabs were stored at −80°C until RNA isolation. For the time interval 0 days, samples were frozen immediately after drying. Semen and vaginal secretion samples were collected from one male and one female individual absorbed with cotton swabs and dried overnight before RNA isolation. RNA isolation RNA was isolated using the Qiagen RNeasy kit (Qiagen Benelux B.V.) according to the manufacturer’s instructions with minor modifications. These included cutting up the cotton swab into 1 × 1-mm pieces and soaking them in RLT buffer for 1 h at 4°C before the extraction. Trial experiments to lengthen this incubation time up to 24 h did not reveal any improvement in respect to RNA quantity and quality (data not shown). Microarray hybridization and gene expression data analysis 21 22 mas5calls mas 23 24 Real-time PCR 25 1 t t Results and discussion Microarray expression data p t p 26 Selection of tissue-specific markers 20 S1a S1b S1c RT-PCR confirmation of tissue-specific markers 1 Fig. 1 a b c t p B S  Expression of the candidate markers in other body fluids t 2 27 28 29 Fig. 2 a b t t t t t t Gray bars black bars Dotted bars t  30 35 36 38 2 40 18 18 19 41 43 19 Comparison with previously suggested mRNA markers 18 19 18 39 18 Conclusions In summary, whole-genome expression analysis in time-wise degraded samples from blood and saliva stains in combination with RT-PCR verification of various forensically relevant body fluids has resulted in the identification of stable tissue-specific mRNA markers from five genes for saliva (SPRR3, SPRR1A, KRT4, KRT6A, and KRT13) and nine genes for whole blood (CASP1, AMICA1, C1QR1, ALOX5AP, AQP9, C5R1, NCF2, MNDA, and ARHGAP26). For the first time, mRNA markers were ascertained considering almost the entire human transcriptome and based on experimental data of genome-wide gene expression as well as considering the degradation stability of mRNAs. We could demonstrate that the candidate genes identified here provide informative mRNA markers for blood and saliva identification for stains up to 180 days of age. We would like to propose their application in forensic case work (with the potential practical limitation of coamplification in vaginal secret) for stains of at least 6 months of age. However, we expect that the proposed mRNA markers will successfully identify older blood and saliva stains (respective experiments are currently in progress). Finally, we would like to remark that tissue identification in forensics should be performed in a reciprocal way; so that a tissue is identified because of the presence of markers specific for the relevant tissue together with the absence of markers specific for all other tissues in question. Clearly, more research should be dedicated towards finding the most suitable markers for tissue identification in forensics. Electronic supplementary material Below is the link to the electronic supplementary material.
 Table S1 RT-PCR primers for the genes amplified (DOC 79 kb). Figure S1a GNF Symatlas expression profiles of blood-specific genes (GIF 15 kb) High resolution image file (TIF 82 kb) Figure S1b GNF Symatlas expression profiles of blood-specific genes (GIF 14 kb) High resolution image file (TIF 77 kb) Figure S1c GNF Symatlas expression profiles of saliva-specific genes (GIF 12 kb) High resolution image file (TIF 74 kb)