Reference gene study for forensic body fluid identification

Abstract

Reference genes are used in forensic body fluid identification studies to normalise data generated during gene expression experiments. The use of reference genes improves the reliability of qRT-PCR. In this study, 10 most common reference genes UCE, TEF, GAPDH, 18S rRNA, ACTB, B2M, B-Actin, OAZ1, RPS 29 and S15 widely used in forensic body fluid identification studies were selected from relevant literature and qPCR efficiency and sensitivity of all the reference genes was tested using SYBR Green detection. Stability was also assayed using samples stored at room temperature for 6 months using Taqman assay probes. All the markers except TEF displayed high sensitivity and were detected down to 25 pg of RNA input. Stability study demonstrates that B2M, ACTB, RPS29, and UCE are ideal markers for normalization in forensic body fluid identification studies. The study confirms that reference genes should be selected only upon adequate validation of their suitability.2015 Elsevier Ireland Ltd. All rights reserved.

Evaluation of genetic markers for forensic identification of human body fluids

“…The genes selected include GAPDH, 18S rRNA, β-Actin, OAZ1, S15, ACTB, B2M, RPS29, TEF and UCE. The results showed that ACTB, B2M, RPS29, TEF and UCE passed the reaction efficiency test using SYBR Green and were further detectable in all the body fluids up to 25 pg using Taqman probes (Afolabi et al, 2015). However, as shown in the present study, TEF did not demonstrate high sensitivity as other markers, especially on semen, blood and menstrual blood.…”

RNA‐based approaches for body fluid identification in forensic science

“…Additionally, in an EDNAP study, B2M was the only reference gene detected in challenging (trace) samples (Haas et al, 2015). B2M was the most stable in a small study by Afolabi, Roeder, Iyengar, and Hadi (2015) where a panel of reference genes was assessed for their efficiency and sensitivity across blood, menstrual fluid, vaginal material, saliva, and semen. However, B2M has been shown to be up‐ or downregulated in individuals with tumors, and when the immune system is compromised (Larsson et al, 1994; Momburg & Koch, 1989).…”

Tissue Typing by Means of PCR and Capillary Electrophoresis: New Aspects, Reality and Issues

“…Специальных требований для базовой линии не существует, хотя условия ПЦР стараются подгонять таким образом, чтобы высота минимального сигнала была не ниже 150RFU, хотя некоторые опуска-ют и до 50RFU [25]. Затруднения в точности установки базовой линии, в частности, обусловлены некоторой лабильностью экспрессии генов «домашнего хозяйства» (ГДХ), амплификация которых служит своеобразным внутренним контролем [26]. Было показано, что компо-зиция их экспрессии зависит от типа тканей, поэтому в разных модификациях панели «CellTyper» и аналогов может присутствовать разный состав в виде двух-трёх типов ГДХ, которые специфично проявляются в том или ином типе тканей [8,23].…”