Forensic Science International: Genetics Supplement Series 6 (2017) e55–e56

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Forensic Science International: Genetics Supplement Series

journal homepage: www.elsevier.com/locate/fsigss

Microarray expression profile of circular RNAs in human body fluids T

1 1 ⁎
Feng Song , Haibo Luo , Mingkun Xie, Hong Zhu, Yiping Hou
Institute of Forensic Medicine, West China School of Basic Science and Forensic Medicine, Sichuan University, Chengdu, China

A R T I C L E I N F O A B S T R A C T

Keywords: In forensic science, the reliable identification of human body fluids could provide important information for
Forensic science crime investigation. Circular RNAs (circRNAs), with an abundant, stable and tissue/developmental-stage-specific
CircRNAs expression, may be as potential markers for forensic body fluid identification. In this study, microarray ex-
Body fluid identification pression profiles of circRNAs were explored with total RNA extraction from venous blood, semen, saliva, vaginal
Microarray
secretion, and menstrual blood samples. As results, semen, saliva and venous blood could be easily distinguished
from each other based on the circRNAs expression profiles, but vaginal secretions and menstrual blood displayed
similar signatures. This study suggested that circRNAs extraction from forensic samples was possible and the
microarray screening of circRNAs could enrich the candidates for forensic body fluid identification.

1. Introduction data processing. Hierarchical clustering was performed using TM4

In forensic casework, analysis the presence and possible cellular
origin of crime-related biological material can be of crucial to reveal the
criminal nature of an event. Recently, studies have been interested in a
new class of non-coding RNA that is endogenously expressed as cova-
lently closed circular molecules and single-stranded, named Circular
RNAs (circRNAs) [1]. Based on sequencing studies, circRNAs are re-
vealed prevalently and abundantly expressed across life, featured stable
structure and in a high tissue-and developmental stage-specific manner.
In addition, CircRNAs lack of polyadenylated tail and hence are pre-
dicted to be less prone to degradation by exonucleases [2,3]. In this
study, we aim to explore potential circRNAs markers using Arraystar
Human circRNA Array (Human Circular RNA Array, Arraystar, USA) as
capture probes for 5396 known human circRNAs for five forensically
relevant body fluid samples: venous blood, semen, saliva, vaginal se-
cretions and menstrual blood.
Samples of body fluids were collected from ten unrelated in-
dividuals with them full informed written consent. Total RNA was
isolated from body fluid samples using TRIzol reagent (Invitrogen,
Carlsbad, CA, USA) following the instruction of the manufacturer and
was quantified and quality was verified with the NanoDrop ND-1000
(NanoDrop, Wilmington, DE, USA). The samples labeling and micro-
array hybridization were performed based on the manufacturer's pro-
tocol. The Agilent Feature Extraction software (version 11.0.1.1) was
used to analyze the acquired array images. R software package (R
version 3.1.2) was used for the quantile normalization and subsequent
software [4] to show the distinguishable circRNAs expression pattern
among samples.
2. Results
In this study, the amount of total RNA extracted from five types of
body fluids ranged from 4891.60 ng to 46630.50 ng, based on the es-
timation from the NanoDrop ND-1000. The purity and concentration of
total RNA samples from all the samples could yield sufficient quality
and quantity to prepare microarray detection. Unsupervised quantile
hierarchical clustering of normalized hybridization values revealed that
samples belonging to the same body fluid, i.e., saliva, venous blood,
semen tend to cluster together. Unfortunately, vaginal secretions and
menstrual blood displayed the similar expression pattern (Fig. 1).
3. Discussion
Determination of the origin and type of the body fluids left at a
crime scene can provide important insights into crime scene in-
vestigation by making the connection between the sample donors and
the actual criminal acts. Bahn et al. [5] identified more than 400 cir-
cRNAs in human cell free saliva samples using high-throughput RNA
sequencing and in-depth bioinformatics analysis method. Lately,
Memczak et al. detected circRNAs in human blood based RNA se-
quencing studies [6]. However, it is not yet known whether circRNAs
present in other body fluid. In consideration of unsupervised

⁎
Corresponding author at: 3 16 Renmin South Road, Chengdu 610041, China.
E-mail addresses: forensic@scu.edu.cn, profhou@yahoo.com (Y. Hou).
1
Feng Song and Haibo Luo contributed equally to this study.

http://dx.doi.org/10.1016/j.fsigss.2017.09.005
Received 10 August 2017; Accepted 10 September 2017
Available online 11 September 2017
1875-1768/ © 2017 Elsevier B.V. All rights reserved.
F. Song et al.
Fig. 1. Unsupervised hierarchical clustering of circRNAs from microarray analysis of five
forensically relevant body fluids, i.e., saliva (SA), semen (SE), menstrual blood (MB),
vaginal secretion (VS) and venous blood (VB).
hierarchical clustering result showed vaginal secretions and menstrual
blood displayed the similar expression pattern, there may be several
explanations for the similarity expression pattern between vaginal
e56
Forensic Science International: Genetics Supplement Series 6 (2017) e55–e56
secretions and menstrual blood. First, menstrual blood is comprised of a
complex mixture of different tissue types, especially the epithelial cells
from the vaginal lumen are present [7]. Second, it may be due to the
amount of sample material on the collection swab, differences in
marker expression over time, and the inter-individual differences. This
study, although limited to distinguish the vaginal secretions and men-
strual blood, indicate that circRNAs have great potential to become
suitable biomarkers for body fluid identification.
4. Conclusion
In this study, semen, saliva and venous blood could be easily dis-
tinguished from each other based on the circRNAs expression profiles,
but vaginal secretions and menstrual blood displayed similar sig-
natures. This result suggested that circRNAs extraction from forensic
samples was possible and the microarray screening of circRNAs could
enrich the candidates for forensic body fluid identification.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Acknowledgements
This study was supported by grants from National Key R & D
Program of China (No.2016YFC0800703) and from the National
Natural Science Foundation of China(No.81330073 and 81571854).
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