Micro‐RNAs (miRNAs) are a class of small noncoding RNA (ncRNA) molecules with a length of 18–24 nucleotides which play an essential regulative role for many cellular processes. Evidence suggests that the miRNome is a more precise and meaningful representation of a cell type and condition than the mRNA transcriptome. To identify miRNAs that are suitable for forensic body‐fluid identification, a global screening by microarray analysis of c. 800 miRNAs of forensic blood and saliva samples was performed, and by bioinformatic processing, three differentially expressed candidate miRNAs for saliva and blood each were selected. The six candidates were then validated and confirmed via quantitative PCR. Herein, we present miRNA assays consisting of three differentially expressed miRNAs for the identification of blood (miR‐126, miR‐150, miR‐451) and saliva (miR‐200c, miR‐203, miR‐205), respectively. We conclude that miRNA extraction from forensic samples is possible and support a “proof of concept” that body‐fluid identification by miRNA analysis may become a potent forensic technique.

BackgroundTwo major biological stressors of freshwater zooplankton of the genus Daphnia are predation and fluctuations in food quality. Here we use kairomones released from a planktivorous fish (Leucaspius delineatus) and from an invertebrate predator (larvae of Chaoborus flavicans) to simulate predation pressure; a microcystin-producing culture of the cyanobacterium Microcystis aeruginosa and a microcystin-deficient mutant are used to investigate effects of low food quality. Real-time quantitative polymerase chain reaction (QPCR) allows quantification of the impact of biotic stressors on differential gene activity. The draft genome sequence for Daphnia pulex facilitates the use of candidate genes by precisely identifying orthologs to functionally characterized genes in other model species. This information is obtained by constructing phylogenetic trees of candidate genes with the knowledge that the Daphnia genome is composed of many expanded gene families.ResultsWe evaluated seven candidate reference genes for QPCR in Daphnia magna after exposure to kairomones. As a robust approach, a combination normalisation factor (NF) was calculated based on the geometric mean of three of these seven reference genes: glyceraldehyde-3-phosphate dehydrogenase, TATA-box binding protein and succinate dehydrogenase. Using this NF, expression of the target genes actin and alpha-tubulin were revealed to be unchanged in the presence of the tested kairomones. The presence of fish kairomone up-regulated one gene (cyclophilin) involved in the folding of proteins, whereas Chaoborus kairomone down-regulated the same gene.We evaluated the same set of candidate reference genes for QPCR in Daphnia magna after exposure to a microcystin-producing and a microcystin-free strain of the cyanobacterium Microcystis aeruginosa. The NF was calculated based on the reference genes 18S ribosomal RNA, alpha-tubulin and TATA-box binding protein. We found glyceraldehyde-3-phosphate dehydrogenase and ubiquitin conjugating enzyme to be up-regulated in the presence of microcystins in the food of D. magna. These findings demonstrate that certain enzymes of glycolysis and protein catabolism are significantly upgregulated when daphnids ingest microcystins. Each differentially regulated gene is a member of an expanded gene family in the D. pulex genome. The cyclophilin, GapDH and UBC genes show moderately large sequence divergence from their closest paralogs. Yet actin and alpha-tubulin genes targeteted by our study have nearly identical paralogs at the amino acid level.ConclusionGene expression analysis using a normalisation factor based on three reference genes showed that transcription levels of actin and alpha-tubulin were not substantially changed by predator-borne chemical cues from fishes or invertebrates, although changes in expression on the protein level were shown elsewhere. These changes in protein level could be caused by others than the investigated paralogs, showing the importance of the construction of phylogenetic trees for candidate gene approac...

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