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Forensic Science International: Genetics

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Short communication

A model for data analysis of microRNA expression in forensic body fluid

identification
Zheng Wang a, Haibo Luo a, Xiongfei Pan b, Miao Liao a, Yiping Hou a,*
a
Department of Forensic Genetics, West China School of Basic Science and Forensic Medicine, Sichuan University (West China University of Medical Sciences), Chengdu 610041,
Sichuan, China
b
Department of Epidemiology, West China School of Public Health, Sichuan University, Chengdu 610041, Sichuan, China

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

Article history: MicroRNAs (miRNAs, 18–25 bases in length) are small, non-coding RNAs that regulate gene expression at
Received 30 July 2010 the post-transcriptional level. MiRNA expression patterns, including presence and relative abundance of
Received in revised form 9 August 2011 particular miRNA species, provide cell- and tissue-specific information that can be used for body fluid
Accepted 18 August 2011
identification. Recently, two published studies reported that a number of body fluid-specific miRNAs had
been identified. However, the results were inconsistent when different technology platforms and
Keywords: statistical methods were applied. To further study the role of miRNAs in identification of body fluids, this
miRNA
study sets out to develop an accurate and reliable model for data analysis of miRNA expression. To that
Body fluid identification
TaqMan
end, the relative expression levels of three miRNAs were studied using the mirVanaTM miRNA Isolation
RT-qPCR Kit, high-specificity stem-loop reverse transcription (RT) and high-sensitivity hydrolysis probes
Forensic medicine (TaqMan) quantitative real-time polymerase chain reaction (qPCR) in forensically relevant biological
fluids, including venous blood, vaginal secretions, menstrual blood, semen and saliva. Accurate
quantification of miRNAs requires not only a highly sensitive and specific detection platform for
experiment operation, but also a reproducible methodology with an adequate model for data analysis. In
our study, the efficiency-calibrated model that incorporated the impact of the quantification cycle (Cq)
values and PCR efficiencies of target and reference genes was developed to calculate the relative
expression ratio of miRNAs in forensically relevant body fluids. Our results showed that venous blood
was distinguished from other body fluids according to the relative expression ratio of miR16 using as
little as 50 pg of total RNA, while the expression level of miR658 was unstable and that of miR205 was
nonspecific among different body fluids. Collectively, the findings may constitute a basis for future
miRNA-based research on body fluid identification and show miRNAs as a promising biomarker in
forensic identification of body fluids.
ß 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction in a tissue-specific manner and their expression patterns can

confirm specific body fluids, even after long periods under
In forensic casework, especially in cases of sexual assault and controlled conditions [6–8]. However, heat, humidity, UV light
child sexual abuse, to uncover the criminal nature of an event and ubiquitous ribonucleases are detrimental to mRNA stability
requires not only identification of the DNA profile, but also as a sensitive and specific biomarker for forensic applications
confirmation of the origin of DNA. One of the challenges facing the [9,10].
forensic community is how to find a reliable biomarker and MiRNAs belong to a class of small, non-coding RNA molecules
develop an accurate method for rapid identification of body fluids. that regulate gene expression at the post-transcriptional level [11–
Conventional serology-based methods for body fluid identifica- 13]. Numerous published studies have demonstrated their
tion are prone to various limitations, such as sample consump- important role in functions including embryonic development,
tion, intensive labor, time consumption, varying degrees of proliferation, hematopoiesis and apoptosis, and in the pathogene-
sensitivity and specificity, and no definitive tests for the presence sis of many human diseases such as viral infection, cancer and
of menstrual blood and vaginal secretions [1–5]. Numerous metabolic disorders [12–14]. Several studies revealed that many
published studies have reported that some mRNAs are expressed miRNAs were expressed in a tissue-specific manner [15,16]. The
intrinsically short fragment and tissue-specific expression enable
miRNA as an ideal biomarker for body fluid identification. Recently,
* Corresponding author. Tel.: +86 28 85501550; fax: +86 28 85501549. two published studies reported their preliminary application in
E-mail address: [email protected] (Y. Hou). forensic identification of body fluids [17,18]. However, the results

1872-4973/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.fsigen.2011.08.008

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2 Z. Wang et al. / Forensic Science International: Genetics xxx (2011) xxx–xxx

were inconsistent when different technology platforms and was set to 0.200 for easy comparison of results within and between
statistical methods were applied. assays. The sample with a Cq value over 40 or an amplification
In this study, we used the mirVanaTM miRNA Isolation Kit that point not reaching the threshold was considered as invalid and was
optimized extraction of small nucleic acid molecules to enrich not useful for further analysis.
miRNAs. The expression abundance was detected using stem-loop
reverse transcription (RT) followed by TaqMan quantitative real- 2.4. Data analysis
time polymerase chain reaction (qPCR) analysis. We also utilized
the gradient dilution cDNA method to test the amplification The three miRNAs assay was controlled with the snRNA (U6b)
efficiencies of target and reference genes in body fluids [19–21]. assay (Applied Biosystems, Supplemental Table 1) functioning as
The efficiency-calibrated method was used to calculate the relative reference gene to evaluate Cq values and serving as an internal
expression ratio of target genes in body fluids [19]. To further positive control if sample extraction and reverse transcription
explore its potential application in forensic cases, we used serial were performed successfully.
dilutions of total RNA for cDNA synthesis to establish the detection The relative expression ratio (R) is expressed as the target/
sensitivity of TaqMan RT-PCR assays in venous blood. reference ratio of each sample normalized by the target/reference
ratio of the normalizer [19,22].
2. Materials and methods
ðEre fx þ 1ÞCqre fx ðEre f þ 1ÞCqre f
R¼ 
2.1. Collection of body fluid samples ðEtarx þ 1Þ Cqtarx
ðEtar þ 1ÞCqtar

The body fluid samples were collected on sterile cotton swabs The above equation shows the mathematical model of relative
and dried at room temperature. Blood samples were collected by expression ratio in qPCR. The ratio of a target gene is expressed in a
venipuncture without anticoagulation treatment and 50 ml body fluid versus the normalizer (in this study, venous blood was
aliquots were spotted onto sterile cotton swabs. Semen-free designated as the normalizer) in comparison to the reference gene
vaginal secretions and menstrual blood were collected from the (U6b). Etarx is the real-time PCR efficiency of target gene in x body
vagina on sterile cotton swabs and dried at room temperature. fluids (x means vaginal secretions, menstrual blood, semen, saliva
Freshly ejaculated semen samples were provided in sealed plastic or buccal swabs); Erefx is the real-time PCR efficiency of reference
cups and dried onto sterile cotton swabs. Saliva samples were gene in x body fluid; Cqtarx is the Cq value of target gene in x body
provided in sealed plastic tubes and dried onto sterile cotton fluid; Cqrefx is the Cq value of reference gene in x body fluid; Etar is
swabs. Buccal samples were collected from donors using sterile the real-time PCR efficiency of target gene in venous blood; Eref is
swabs by swabbing the inside of mouth. Each body fluid sample the real-time PCR efficiency of reference gene in venous blood;
was collected from 10 unrelated individuals (age: 18–47 years old) Cqtar is the Cq value of target gene in venous blood; and Cqref is the
of the Chinese Han population living in Sichuan province. Written Cq value of reference gene in venous blood.
informed consent was obtained from all individuals. All the For calculation of R, the qPCR efficiencies and Cq values of
samples were stored at 80 8C for future use. A single cotton swab investigated transcripts must be known. Serial dilutions of cDNA
was used for RNA isolation. over a 100-fold range were prepared. According to linear
regression equation and E = 10[1/slope]-1, the amplification effi-
2.2. RNA isolation and quantification ciencies were calculated. R > 1 indicates higher abundance of the
target gene in x body fluid than venous blood, while R < 1 suggests
RNA was isolated using the mirVanaTM miRNA Isolation Kit otherwise.
(Ambion, Austin, TX, USA) according to the manufacturer’s
instructions. The purity and quantity of RNA were assessed using 2.5. Analytical sensitivity of miRNA TaqMan assays
the NanoDrop ND-1000 spectrophotometer (Thermo Scientific,
Wilmington, DE, USA). All the samples were diluted to a final We used serial dilutions of total RNA (10 ng to 0.01 ng) isolated
concentration of 10 ng/ml. The samples were used immediately or from venous blood as input for cDNA synthesis to establish the
stored at 80 8C for future use. detection sensitivity of TaqMan RT-PCR assays.

2.3. TaqMan RT-qPCR 3. Results

The TaqMan1 MicroRNA Reverse Transcription Kit (Applied Relative abundance of three miRNAs relative to U6b among
Biosystems, Foster City, CA, USA) was used for preparation of cDNA. body fluids were presented and average Cq values of ten unrelated
RT reactions were performed in a volume of 15 ml, and each individuals were tabulated (Supplemental Table 2). The expression
reaction contained 10 ng of total RNA. RT reactions were abundance of miR658 was lower than U6b in all body fluid
performed on a GeneAmpPCR System 9600 (Applied Biosystems) samples. MiR658 was unstably expressed in the samples,
with the following conditions: 16 8C for 30 min, 42 8C for 30 min, especially in the venous blood (not detectable in 7 of 10 blood
85 8C for 5 min, and 4 8C on hold. Reactions without addition of samples), and apparent inter-individual variation was observed in
reverse transcriptase (RT () controls) were performed alongside vaginal secretions. The expression of miR205 had much higher
with cDNA synthesis of each sample and used in subsequent abundance in buccal swabs, vaginal secretions and menstrual
procedures to control the potential genomic DNA contamination. blood compared with saliva, semen and venous blood, but it could
1 ml of RT reaction product was added in the 20 ml qPCR reaction. not be distinguished from those in vaginal secretions and
All TaqMan assays were run in triplicate on an ABI Prism 7500 menstrual blood. The expression of miR16 varied significantly
using TaqMan1 Universal PCR Master Mix II without UNG (Applied between the venous blood and other body fluids (Supplemental
Biosystems). Real-time PCR cycling conditions consisted of 95 8C Fig. 1). Obvious expression difference (dCt6) was observed,
for 10 min, followed by 50 cycles of 95 8C for 15 s and 60 8C for though the abundance of miR16 was high in both the menstrual
1 min. The Cq is defined as the PCR cycle at which the fluorescent and venous blood. The RT () controls and no template controls
signal of the reporter dye crosses an arbitrarily placed threshold in were negative in all cases. Therefore, miR16 as a potential body
the exponential phase. For our assays, the constant threshold value fluid-specific miRNA was selected for further study.

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Fig. 1. Determination of qPCR efficiencies from the slopes of the calibration curve, according to the equation: E = 10[1/slope]-1of miR16 and U6b. Cq values versus cDNA
concentration input (log scale) were plotted to calculate the slope (mean  SD; n = 3).

The Cq values of miR16 were plotted versus the log of the 4. Discussion
dilution cDNA and the linear regressions were performed from
which the mean efficiency could be derived (Fig. 1). The relative Since the discovery of miRNAs [23], many researchers have
expression ratios of miR16 in different body fluids were compared explored their tissue-specific expression patterns [15,16]. Recent-
(Fig. 2). ly, two published studies reported their preliminary application in
In sensitivity testing, miR16 and U6b were detectable in the forensic identification of body fluids [17,18]. Hanson et al. [17]
target body fluid using an amount as low as 50 pg total RNA for identified 9 body fluid-specific miRNAs using random and oligo-dT
cDNA synthesis (Supplemental Fig. 2). primers for RT and SYBR Green for qPCR, whereas Zubakov et al.

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separates the reporter dye from the quencher dye and leads to
increased fluorescence by the reporter.
The accuracy and success of qPCR analysis depend on proper
normalization of data. The purpose of normalization is to minimize
potential variation that can mask or exaggerate biologically
meaningful changes. Although Vandesompele et al. [28] proposed
to use multiple reference genes as a normalization factor for data
analysis, the study of miRNAs in forensically relevant body fluids is
at an exploratory stage and there are no generally accepted
multiple reference genes. U6b was chosen based on its high
abundance (P/N: 044972, Applied Biosystems) and apparent
stability in different body stains of forensic interest [17].
How to present the data is an essential issue in gene expression
profiling using qPCR. Several methods exist to analyze the relative
gene expression. One simple method to process qPCR, currently
known as the DDCq method, is based solely on Cq values [29].
Fig. 2. Relative expression ratio of miR16 in body fluids, i.e., venous blood (VB), However, this method assumes that all amplification efficiencies
vaginal secretions (VS), menstrual blood (MB), semen (SE), saliva (SA) and buccal
are equal to 1. Therefore it does not take into consideration
swabs (BS). Values underwent log 10 transformation.
possible variations of amplification efficiencies. Recently, Mest-
dagh et al. [30] have described an alternative approach to
normalize qPCR experiments, using the mean miRNA expression
[18] adopted the stem-loop RT primers for RT and TaqMan probe value as normalization method. Although this method generates
for qPCR in tracing another 14 body fluid-specific miRNAs. Body stable results, which performs better than using snoRNAs for
fluid-specificity of miRNAs identified by the two groups were normalization, it is applicable only when adequate miRNAs are
inconsistent and Zubakov et al. claimed that the specific markers in analyzed [31]. Soong et al. [22] proposed the efficiency-calibrated
vaginal secretions, menstrual blood and saliva suggested by mathematical method for the relative expression ratio in qPCR. The
Hanson et al. could not be replicated through their own methods. equation is, in principle, the same and the results are identical to
One possible explanation is that they used different technology those using the DDCq method under the condition of Etar = Eref = 1.
platforms, which implies different strategies for both cDNA The advantage of the efficiency-calibrated method is that the PCR
synthesis and PCR quantification. efficiencies of the target and reference genes are included in the
Accurate quantification of miRNAs requires not only a high equation, making the data analysis more accurate.
sensitive and specific detection platform for experiment operation, MiRNAs in body fluids can be expressed in three different
but also a reproducible methodology with an adequate model for patterns: differential expression, which can be detectable to
data analysis. distinguish body fluids; universal expression, which can be
The preparation of total RNA or pure miRNAs is the prerequisite potentially considered as the internal reference in the future
for accurate quantification. The amount of miRNAs in a total RNA (related research in progress in our lab); and unstable expression,
sample depends on the recovery efficiency that may be signifi- which may be affected by the physiological state. MiRNAs with
cantly affected by the different purification methods employed these three expression patterns were selected to analyze the
[24]. In this study, we used the mirVanaTM miRNA Isolation Kit that expression abundance in different body fluids using the efficiency-
is designed for purification of RNA suitable for studies of both calibrated model in this study. This research demonstrated that
siRNA and miRNA in natural populations. miR16 was venous blood-specific and was detectable using as little
The miRNA expression can be detected and quantified by as 50 pg of total RNA. However, the expressed miR658 and miR205
several methods including northern blotting, microarrays, and were not saliva-specific, which is inconsistent with the result from
real-time PCR. Northern blotting cannot discriminate mature and Hanson et al. [17], and their expression in saliva and buccal swabs
precursor miRNAs (pre-miRNA) and poses potential risks to differed. Unstable expression of miR658 in body fluids may be
researchers exposed to certain chemicals [25]. Though microarrays affected by physiological conditions, and miR205 may be
could improve the throughput of miRNA profiling, they are epithelium-specific so that other miRNAs should be considered
subjected to low sensitivity and specificity, or high expense to distinguish vaginal and oral epithelial cells.
[26]. The qPCR has become a gold standard of nucleic acid Different tissues exhibit different PCR efficiencies, caused by RT
quantification due to the specificity and sensitivity [19–21]. inhibitors, PCR inhibitors, and variations in the total RNA fraction
Detection chemistries for qPCR include SYBR Green and TaqMan. pattern extracted. Numerous published studies reported that the
Since SYBR Green binds nonspecifically to all double-stranded DNA PCR efficiency has a major impact on the accuracy of the calculated
sequences, melt curve or gel analysis is required to detect expression result [19–21,32]. Difference in PCR efficiency (DE) of
nonspecific products in case of false-positive signals. In our 3% (DE = 0.03) between target gene and reference gene generate a
research we used the TaqMan MicroRNA assay, which included falsely calculated differences in expression ratio of 47% in case of
stem-loop RT primers for RT and TaqMan MGB (minor groove Etarget < Eref and 209% in case of Etarget > Eref after 25 performed
binder) probes for qPCR. Due to the base stacking and spatial cycles [32]. We calculated the amplification efficiencies of miRNAs
constraint of the stem–loop structure, stem–loop RT primers are in different body fluids using the gradient dilution cDNA method
advantageous over conventional linear ones in terms of RT and the relative expression ratio based on the efficiency-calibrated
efficiency and specificity [27]. The TaqMan MGB probes contain model. We found that the calculation without efficiency correction
a reporter dye (FAMTM dye) linked to the 50 end of the probe and a could generate false differences in expression ratios of 30%
nonfluorescent quencher (NFQ) with a MGB at the 30 end of the (menstrual blood), 17% (semen), 6% (saliva), 6% (vaginal secretions)
probe. This MGB modification increases the melting temperature and 3% (buccal swabs), which indicates that the efficiency-
(Tm) without increasing probe length, which allows the design of calibrated model analysis is more accurate.
shorter probes [27]. During PCR, the DNA polymerase cleaves only In conclusion, this article presents a simple procedure for
probes that are specifically hybridized to the target. Cleavage miRNA-based body fluid identification and an accurate model for

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