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

Abstract

In the present work, we have evaluated eight reportedly blood-specific mRNA markers (HBB, HBA, ALAS2, CD3G, ANK1, PBGD, SPTB and AQP9) in an attempt to determine the most suitable ones for use in forensic applications based on their sensitivities, specificities and performance with casework samples.While varying levels of expression were observed, all markers were relatively sensitive requiring as little as 1 ng of RNA input into the reverse transcription (RT) reaction. In singleplex reactions, seven of the eight analyzed blood markers (all except AQP9) demonstrated a high degree of specificity for blood. In multiplex reactions, non-reproducible cross-reactivity was observed for several of the mRNA markers, which was reduced and, in most cases, eliminated when less input total RNA was used. Additionally, some cross-reactivity was observed with tissue and animal samples. Despite differences in the observed sensitivity and specificity of the blood markers examined in this study, a number of the candidates appear to be suitable for inclusion in appropriately validated multiplex mRNA-based body fluid identification systems.

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

“…The sensitivity of housekeeping gene detection was in the same range as the body-fluid-specific markers [26,39,40] and STR analysis (Table S7), though slightly reduced in saliva and semen compared to the saliva and semen specific markers [40]. Two of the housekeeping genes tested in this study, B2M and UBC, proved to be quite sensitive and were detected in most body fluid stains (Table S8).…”

“…Participating laboratories were asked to quantify RNA using one of the following methods: Quant-iT TM RiboGreen 1 RNA Assay kit (Invitrogen) using a fluorescence microplate reader (high and low range protocol option) [11], Quant-iT TM RNA assay kit (Invitrogen) using the Qubit fluorometer [26] or Bioanalyzer (Agilent) [20]. Laboratories were able to quantify DNA using a quantification system of their own choice.…”

“…Post PCR purification was optional, but recommended, to eliminate dye blobs and to concentrate/dilute the samples by variation of the elution volume (e.g. MinElute PCR purification kit, QIAGEN) [26,41].…”

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

“…Forensic RNA analysis is increasingly being used for more definitive body fluid identification (BFID) with a focus on messenger RNA (mRNA) [1][2][3][4][5] and more increasingly on microRNA (miRNA) [6][7][8][9][10][11]. 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].…”

An Investigation of Various Tissue Specific mRNA Markers in Body Fluid Identification

“…Bundan başka, menstrüel kan ve vajinal sekresyonun identifikasyonu için geliştirilmiş enzimatik veya immünolojik doku identifikasyon testi bulunmamaktadır. [10][11][12][13] Son dönemlerde, hücre spesifik mRNA belirteçlerinin analizi vücut sıvılarının identifikasyonu için umut verici yeni bir yöntem olarak ileri sürül-mektedir. Kan, tükürük, semen, vajinal sekresyon ve menstrüel kanda adli analizler için kullanılabi-lirliği test edilen çok sayıda genetik belirteç tanım-lanmıştır.…”