Development of a mRNA profiling multiplex for the inference of organ tissues

Abstract

Forensic characterisation of organ tissue generally occurs through histological and immunological assays of limited sensitivity. Here, we explore an alternative approach and examine a total of 41 candidate mRNA markers for their ability to differentiate between brain, lung, liver, skeletal muscle, heart, kidney and skin. Various selection rounds are applied involving 85 organ tissues (36 excised autopsy specimens and 49 frozen tissue sections, with at least ten specimens for each organ type), 20 commercially available RNAs from different human tissues and at least two specimens of blood, saliva, semen, vaginal mucosa, menstrual secretion or touch samples. Finally, 14 markers are regarded tissue-specific and included in an endpoint RT-PCR multiplex together with one general muscle, one blood and one housekeeping marker. This 17-plex is successfully used to analyse a blind test set of 20 specimens including mixtures, and samples derived from stabbing of organ tissues. With the blind test set samples, it is shown that an earlier described interpretation strategy for RNA cell typing results [1] is also effective for tissue inference. As organ-typing is embedded in a procedure of combined DNA/RNA extraction and analysis, both donor and organ type information is derived from the same sample. Some autopsy specimens presented DNA profiles characteristic for degraded DNA. Nevertheless, the organ-typing multiplex could generate full RNA profiles, which is probably due to small sizes of the amplicons. This assay provides a novel tool for analysis of samples from violent crimes.

Extended specificity studies of mRNA assays used to infer human organ tissues and body fluids

“…Housekeeping marker 18S‐rRNA was detected in most animal amplifications and is, thus, not a human specific housekeeping marker, which is consistent with the results described in . The main function of this housekeeping marker in these end‐point RT‐PCR assays is to confirm successful RNA analysis by indicating the presence of RNA, and the absence of factors inhibiting cDNA synthesis or PCR, which is especially informative when none of the other markers give a signal . Few signals were observed for blood marker HBB, lung marker SFTPD, and skeletal muscle marker TNNI2, but these signals were sporadic.…”

“…The use of messenger RNA (mRNA) profiling for the identification of body fluids and skin has been increasingly investigated in the past decades [1][2][3][4][5][6][7][8][9][10][11]. More recently, an mRNA assay was developed which allows for the inference of seven organ tissue types [12][13][14]. Previously, enzyme-linked immunosorbent assays or histological methods were used to infer the type of cell [15][16][17][18].…”

Development of Highly Sensitive and Specific mRNA Multiplex System (XCYR1) for Forensic Human Body Fluids and Tissues Identification

“…UMOD is expressed in renal tubules and secreted in urine [32], [33], [38]. Akutsu et.al firstly detected the expression of THP in urine samples by ELISA and real time PCR (RT-PCR) [28].…”

“…However, further work is required to seek new candidates and include more markers for the identification of vaginal secretion, skin, sweat, tear and urine stains. For example, CYP2B7P1 for vaginal secretion [19], [47] and LCE1C for skin [17], [19], [33] should be considered in future multiplex development. Other more sensitive markers would contribute to the reliable identification of LCN samples in forensic casework.…”

“…Uromodulin, also known as Tamm-Horsfall glycoprotein, is the most common protein excreted in the urine of healthy individuals [28], [32], [33].…”