Simultaneous Analysis of Micro‐RNA and DNA for Determining the Body Fluid Origin of DNA Profiles,

Abstract

Micro-RNAs (miRNAs) can be specifically expressed in forensically relevant body fluids such as blood or saliva. The aim of the study was to develop a simultaneous extraction and analysis protocol that allows for the acquisition of a DNA profile and the identity of the body fluid using a single process. DNA and micro-RNA were extracted from blood and saliva before undergoing a cDNA synthesis step by using stem-loop reverse transcription PCR. The resulting extracts containing DNA and cDNA synthesized from body fluid-specific miRNA markers then underwent standard STR analysis using a modified ABI AmpFℓSTR(®) NGM SElect™ kit. In all samples, a full DNA profile was obtained along with additional peaks corresponding to the miRNA marker targeted. In all cases, blood samples profiled exhibited a peak indicating the presence of the blood-specific miRNA marker and the saliva sample profiled exhibited a peak indicating the presence of the saliva-specific miRNA marker.

Considering the effect of stem-loop reverse transcription and real-time PCR analysis of blood and saliva specific microRNA markers upon mixed body fluid stains

“…Such work includes exploring potential case work issues and development of interpretational guidelines in partner with a forensic science provider and end user organisations such as the police. Finally, a future study will incorporate DNA profiling using the single stream strategy proposed by van der Meer et al [18]. …”

“…For each marker, the mature miRNA sequence was identified using an online microRNA database (www.mirbase.org) [25][26][27][28], along with their corresponding stem-loop sequences to ensure that the mature sequences were sufficiently different, particularly with respect to the 6bp sequence on the 3' end. These markers were screened using expression data (from an in-house study) [18], and the markers which were deemed to be preferentially expressed in the relevant body fluid were selected for this study. RNU-44, a small nuclear RNA (snRNA), was used as the endogenous control [10].…”

“…If a single stream strategy, such as that suggested by van der Meer et al [18], is applied to this test and incorporates a full miRNA marker panel, then the language could be strengthened to 'In my opinion, the major DNA profile was obtained from the blood.' However, such a marker panel would need to include skin cell specific markers, and said markers need to be sufficient to differentiate between salivary and vaginal epithelia.…”

“…MicroRNA is, in theory, the preferred marker to target as it is more stable due to its small size (~22-26 bp) and relatively high copy numbers within the cells [11][12][13][14][15][16][17]. There are some concerns relating to its species specificity, but this may be resolved by using a single channel simultaneous extraction technique [18] and the associated DNA profile, which is human specific.…”

Evaluation of Stem-Loop Reverse Transcription and Poly-A Tail Extension in MicroRNA Analysis of Body Fluids

“…MicroRNA (miRNA) analysis has been demonstrated to be a viable tool for body fluid identification in forensic case work [1][2][3][4], which provides a much more reliable, robust and sensitive assay than is currently available. A full range of body fluid markers has been identified [1] and their effectiveness is evaluated in the context of body fluid mixtures [4] and alternative end-point analysis, such as capillary electrophoresis (CE) [5][6][7].…”