Development of New Mrna Markers for the Identification of Menstrual Blood

Available online at www.annclinlabsci.org Annals of Clinical & Laboratory Science, vol. 48, no. 1, 2018 55 Development of New mRNA Markers for the Identification of Menstrual Blood

Qiong Liang1, Hongyu Sun2, Xinyao Wu2, Xueling Ou2, Guoquan Gao3, Yi Jin1, and Dayue Tong2

1Department of Pathology, the Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou, 2Forensic Biology Section, Sun Yat-Sen University, Guangzhou, and 3Department of Biochemistry and Molecular Biology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China

Abstract. Background. The aim of this study was to screen 3 mRNA markers (i.e., PAEP, LAPR3, and HOXA10) with diverse expression in different body fluids and to develop a method for the identification of menstrual blood using these mRNA markers. Methods. Body fluid (i.e., venous blood, menstrual blood, semen, and saliva) samples were collected and prepared under differing environmental conditions (temperature, humidity and time), and RNA was extracted and reverse transcribed. The expression specificity of these markers was assessed using TaqMan probe qPCR. Results. A high mean cycle threshold value cor- responds to a lower expression level. The mean cycle threshold value of the LAPR3, HOXA10, and PAEP genes are 8.37, 8.73, 4.67 in menstrual blood respectively. LAPR3 and PAEP were only expressed in menstrual blood. HOXA10 were expressed in blood, menstrual blood, and semen. No significant differences were found while the mean cycle threshold of MMP11 and PAEP were compared in the menstrual blood under common environmental conditions. There were no observed differences in the expression of the target genes in women of different ages and at different menstrual phases. The sensitivity of the expression of the 3 target genes could be examined in fluid amount range from 1 to 32 µl of body fluid. The expression of PAEP differed markedly from the expression of LAPR3 and HOXA10 in menstrual blood stains tested using mRNA-based assays (p<0.001). Conclusions. These markers, particularly PAEP,can likely be used for the identification of menstrual blood in certain forensic cases.

Introduction Thus, it is important for forensic experts to identify a biomarker that has tissue specificity and relative Human body fluids, secretions, and excrement are stability and can easily be analyzed. A new bio- commonly used as biological samples to provide marker with a suitable analytical method could evidence in forensic cases. For forensic identifica- overcome the weaknesses of DNA markers. tion, it is important to identify the sample species Menstrual blood, peripheral blood, vaginal fluid, and category and/or origin of the tissue. Previously saliva, and semen are the most commonly analyzed used identification methods could not identify the body fluid samples and have the greatest number of tissue origin of various body fluids [1,2]. Due to the identification requests. The identification of these development of molecular biological technology, fluids is also the most difficult and has the highest such as the STR locus genotyping technology, technological requirements. Currently, there are no DNA markers have been widely used in forensic satisfactory technical methods to address this identification of individuals and paternity testing; problem. this technology can also be used to identify the origin of certain body fluids [3]. However, if the sam- For this reason, forensic experts started to find new ples were old, degraded or trace specimens, or the markers such as mRNA for body fluids identifica- specimens require identifying the tissue origins tion. Although conventional RNA markers, such as from the same individual, STR markers may not MMPs (matrix metalloproteinases), SPTH ( spec- completely satisfy the identification requirements. trin beta, erythrocytic), HTN3 (Histatin 3), and PRM1 (protamine1), partially satisfy the require- Address correspondence to Dayue Tong, PhD., 74 Zhongshan ments of body fluids identification, these markers 2nd Road, Guangzhou, Guangdong, 510080, China; e mail: [email protected]; Yi Jin, Ph.D., 600 Tianhe Road, Guangzhou, are expressed in different human body fluids [4-7]. Guangdong, 510630, China; e mail: [email protected]; Guoquan MMPs are believed to catalyze the localized hydro- Gao, 74 Zhongshan 2nd Road, Guangzhou, Guangdong, 510080, China; e mail: [email protected] lysis of extracellular matrix proteins including

Figure 1. Amplification Plot. (A) indicates MMP11 in menstrual blood, (B) indicates PAEP in menstrual blood, (C) indicates SPTH in blood, (D) indicates HTN3 in saliva, (E) indicates the contra-indicator, (F) indicates LAPR3, (G) indicates PRM1in seminal fluid, and (H) indicates HOXA10. collagens, fibronectin, laminins, and proteoglycans, Chiang CL et al in 2011 [14]. HOXA10 is a mem- thereby modifying the integrity of the connective ber of the homeobox gene family and is known to tissue. Spectrin is the major constituent of the cyto- be involved in the genetic control of development; skeletal network underlying the erythrocyte plasma HOXA10 is expressed during embryonic develop- membrane. The primary protein encoded by ment and continues to play a regulatory role in the HTN3 is histatin 3. Histatins are a family of small, adult female reproductive tract [15]. The progesto- histidine-rich, salivary proteins, encoded by at least gen associated endometrial protein (PAEP or GdA) two loci (HTN3 and HTN1). Protamines substi- was reported by Ren S et al in 2011 [16]. PAEP, tute for histones in the chromatin of sperm during which has also been called GdA or placental protein the haploid phase of spermatogenesis. They com- 14 (PP14), is a major glycoprotein that is synthe- pact sperm DNA into a highly condensed, stable sized in the endometrium in response to progester- and inactive complex, belongs to the protamine P1 one and relaxin exposure [17,18]. PAEP and family. Therefore, it is very important to screen and MMP7 have similar expression patterns [18]. We develop new mRNA markers that have diverse ex- assessed the level and specificity of the expression of pression patterns in different body fluids. This three markers in human body fluid stains and the study aimed to screen and develop new mRNA sensitivity of the method to demonstrate its poten- markers with full and nil expression or with signifi- tial application in forensics. cant differential expression in different body fluids to demonstrate the optimal conditions for mRNA Materials and Methods profile analysis and provide a mRNA marker that has excellent specificity, relative stability and lower Sample collection. Ninety female volunteers were di- quality and quantity requirements for specimen vided into 3 age groups as follows: <11 years old, 22-50 analysis [8-12]. The lysophosphatidic acid receptor years old and >55 years old (i.e., preadolescence, menses, 3 (LPAR3) was reported by Chiang CL et al in and menopause). The female volunteers and 30 male 2011 [13]. The LPAR3 is unique because it is sig- volunteers were recruited in Guangdong Province in China. The participants’ ages ranged from 6 to 63 years. nificantly activated by a specific form of LPA and is Peripheral blood, saliva, semen, and menstrual blood expressed in a small number of tissues, such as the were collected from the volunteers. The samples were reproductive organs, including the uterus. stained and maintained under a range of conditions, in- Homeobox A10 (HOXA10) was reported by cluding outside under dry or humid The new markers for Identification of Menstrual Blood 57

Table 1. Sequences of the primers and probes.

Gene Primer and probe Sequence TaqMan probe Assay ID

SPTB F: 5′-GCCTTTAATGCCCTGATACACAA-3' Hs01076092_m1 R: 5′-GAGTCCTTCAGCTTATCAAAGTCGAT-3' HTN3 F: 5′-CTTGGCTCTCATGCTTTCCAT-3' Hs00173857_m1 R: 5′-TTTATACCCATGATGTCTCTTTGCA-3' PRM1 F: 5′-CAGATATTACCGCCAGAGACAAAG-3' Hs00172012_m1 R: 5′-AATTAGTGTCTTCTACATCTCGGTCTGT-3' MMP11 F: 5′-GGTGCCCTCTGAGATCGAC-3' Hs00171462_m1 R: 5′- TCACAGGGTCAAACTTCCAGT-3' LPAR3 (lysophosphatidic acid receptor 3) F: 5’-TTAGCTGCTGCCGATTTCTT-3' Hs00171829_m1 R: 5’-ATGATGAGGAAGGCCATGAG-3' HOXA10 (homeobox A 10) F: 5'-GGCAAAGAGTGGTCGGAAGAAG-3' Hs00264790_m1 R: 5'-CACTTGTCTGTCCGTGAGGTG-3' PAEP (progestogen-associated endometrial protein) F: 5'-AAGTTGGCAGGGACCTGGCACTC-3' Hs00358158_m1 R: 5'-ACGGCACGGCTCTTCCATCTGTT-3' GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) F: 5'-CAAGGTCATCCATGACAACTTTG-3' Hs99999905_m1 R: 5'-GGCCATCCACAGTCTTCTGG-3'

Table 2. Mean dCt values of SPTH, HTN3, PRM1, MMP11, LAPR3, HOXA10 and PAEP in body fluids.

SPTH HTN3 PRM1 MMP11 LAPR3 HOXA10 PAEP

Blood * -3.43 -**- 8.62 9.65 7.03 - Menstrual blood 17.32 -- -3.67 5.37 6.73 -3.03 Semen - - -2.87 -- 6.63 - Saliva - -2.58 10.21 --- -

*: Blood samples include 30 males and 90 females, Menstrual blood samples include 30 females, Semen samples include 30 males, Saliva samples include 30 males and 90 females. **: the signal was not detected when the Ct values were ≥ 45.

Table 3. Ct values of the expression of LAPR3, HOXA10, and PAEP in women of different ages.

Preadolescence (n=30) Menses female (n=30) Menopause female (n=30)

LAPR3 HOXA10 PAEP LAPR3 HOXA10 PAEP LAPR3 HOXA10 PAEP

Blood 32.32a 33.51 - 31.16 32.61 - 32.72 33.88 - MBb ×c × × 32.04 33.41 23.36 × × × Saliva ------aCt mean value. bMB: menstrual blood. c×: absence sample. conditions, for different durations (from 1 day to 1 The aliquots (50 µl) were placed on a cotton cloth and year), and at different temperatures (from room tem- dried at room temperature. Menstrual blood was collect- perature at 25°C± to >35°C). The blood samples were ed using a sanitary napkin (on the first day, third day and collected by venipuncture, and freshly ejaculated semen fifth day). All samples were stored at -20°C until use. and saliva samples were provided in sealed plastic tubes. RNA was isolated from a 1-cm2 stain or sanitary napkin. 58 Annals of Clinical & Laboratory Science, vol. 48, no. 1, 2018 sample was thoroughly mixed. The samples were then cooled on ice for 5 min and centrifuged at 12,000 g at 4°C for 10 min. The supernatants were transferred to new tubes, and 400 µl of phenol/chloroform (1:1 V) (Shantou Chemical Factory, China) were added; then, the samples were vortexed for 15 s. The mixture was in- cubated on ice for 5 min and then centrifuged at 12,000 g at 4°C for 10 min. The top (aqueous) layer was carefully transferred into new tubes. Then, 1:1 V of isopro- panol (Tianjin chemical industrial Ltd, China) was added to each tube; the samples were mixed by inversion and then centrifuged at 12,000 g at 4°C for 10 min. The supernatants were very carefully discarded. To wash the pellets with 1 ml of 75% ethanol (Shantou chemical factory, China), the samples were centrifuged at 7500 g for 5 min. The residual ethanol was removed with a pipette. The samples were air dried for 4 min. To dissolve the Figure 2. The expression level of PAEP, LAPR3 and HOXA10 in menstrual blood. The smaller the Ct values, RNA in 30 µl DEPC (Amresco Ltd, OH, USA), the the higher the expression level of PAEP, LAPR3 and samples were quantified using UV-2450 (Shimadzu, HOXA10 in menstrual blood. Japan) and diluted with H2O. The samples were then stored at -80°C. Then, 10 µl of the RNA samples were treated with 1 µl of RNase-free DNase at 37°C for 10 min. Reverse transcription (RT) was performed using high-capacity cDNA reverse transcription kits (Applied Biosystems, Foster City, USA). First, 2×master mixes were prepared as follows: 10×RT buffer 2.0 µl, 25×dNTP mix (100 mM) 0.8 µl, 10×RT random primers 2.0 µl, reverse transcriptase 1.0 µl, RNase inhibitor 1.0 µl, and nuclease-free H2O 3.2 µl; the samples were then gently mixed. Second, the cDNA reverse transcription reaction was prepared with 10 µl of the 2×master mixes in EP tubes and 10 µl of the RNA samples; then, the tubes were sealed and briefly centrifuged. The tubes were placed in a thermal cycler (GeneAmp® system 9700, Figure 3. The sensitivity was examined in different volumes of body fluid stains. The mean Ct values of each of the Applied Biosystems, Foster City, USA) with the follow- three assays tested in menstrual blood by Real-Time PCR ing conditions: 25°C, 10 min; 37°C, 120 min; 85°C, 5 assay of the sensitivity from isolated body fluids are shown. min; and 4°C forever (the amplification plot is shown in Low Ct values represent samples with a high expression level Figure 1). of the specific target gene. The samples show that the expression of the target gene PAEP is higher than that of the other 2 target genes in menstrual blood on the third day. PCR and quantitative analysis. TaqMan probes were designed to identify the peripheral blood, menstrual blood, saliva stains and semen stains. The TaqMan Ethics Statement. Written informed consent was ob- probes (SPTH, HTN3, PRM1, MMP11, LAPR3, tained from all participants who were recruited in this HOXA10, PAEP and GAPDH) were synthesized by study. In the case of children participants, their guard- Roche Molecular Systems, Inc. (Branchburg, New Jersey, ians completed the consent form. Accordingly, the ethics USA) (Assay-ID: Hs01076092_m1, Hs00173857_m1, committees of Zhongshan School of Medicine, SUN Hs00172012_m1, Hs00171462_m1, Hs00171928_ Yat-Sen University approved the study at the end of m1, Hs001264790_m1, Hs00358158_m1, and 2009. Hs99999905_m1). The sequences of the primers and probes are shown in Table 1. The housekeeping gene, RNA extraction and cDNA synthesis. RNA was extract- GAPDH, is consistently expressed in all body fluids and ed from the peripheral blood, menstrual blood, saliva was used as a control. The RT-PCR amplification was stain and semen stain using the TRIzol Reagent as rec- performed in a total volume of 20 µl, containing ommended by the manufacturer (Invitrogen Ltd, 2×Taqman universal PCR master mix 10 µl, 20×Taqman Carlsbad, CA, USA), and the RNA samples were treated gene expression assays 1.0 µl, sample cDNA 4 µl, and with DNase (Qiagen Ltd, Hilden, Germany). The 0.5 DEPE treated H2O 5 µl. Thermal cycling (GeneAmp cm2 stain samples were transferred to a 1.5 ml tube. PCR System 7500 real-time PCR system; Perkin-Elmer) Then, 1 ml of the TRIzol reagent was added, and the was performed with an initial denaturation at 50°C for 2 The new markers for Identification of Menstrual Blood 59

Table 4. Mean Ct values of the target gene expression in different menstrual phases.

First day Third day Fifth day

SPTH 31.7±0.33 33.9±0.31 31.3±0.30 HTN3 - - - PRM1 - - - MMP11 22.4±0.22 21.7±0.22 24.4±0.23 LAPR3 25.5±0.25 24.8±0.23 27.6±0.26 HOXA10 32.6±0.26 26.6±0.27 31.1±0.30 PAEP 23.9±0.23 22.5±0.22 23.2±0.23 GAPDH 24.1±0.23 23.9±0.23 23.8±0.23

min and 95°C for 10 min, followed by 50 cycles of dena- and >35°C (Supplementary Table 1). Additionally, turation at 95°C for 15s and an extension at 60°C for 1 each stain was placed outside under two conditions (sun- min. The quantitative analysis of the RT-PCR products light and rain, >1 h). The 1 cm2 stains were used for the was performed on a 7500 real-time PCR system (Applied RNA isolation from the treated samples. Biosystems, Foster City, USA). The samples and positive and negative controls were evaluated 3 times. Statistical analysis. All values are expressed as the mean ± SD. The statistical analysis software package SPSS 13.0 Comparison of the expression specificity and method (SPSS Incorporated, Chicago, USA) was used for all sensitivity. We tested the expression specificity in differ- analyses. A value of p<0.05 was considered statistically ent body fluids. The cDNA samples from each body significant. fluid type (peripheral blood, menstrual blood, saliva, and semen) were evaluated with RT-PCR in triplicate, Results and the mean Cycle threshold ( dCt ) and standard de- viation ( Std ) of these markers were compared in differ- Target gene expression and dCt value compari- ent body fluids to investigate their specificity. son. The dCt values of all target genes were examined in 4 µl of cDNA amplified products. The re- Analysis of differential expression by females of different ages and different menstrual phase. To study the sults are shown in Table 2. The dCt values were expression levels of these markers in females at different compared to those of the conventional markers ages, peripheral blood, saliva, and menstrual blood were SPTB, Prm1, and MMP11. Significant differences collected from 3 groups of females. The samples were were observed between SPTH and HOXA10 evaluated by RT-PCR in triplicate, and we compared the (p<0.001) in peripheral blood, PRM1, and mean Ct of these markers among the different age HOXA10 (p<0.001) in semen, MMP11 and groups. HOXA10 (p<0.001) in menstrual blood and MMP11 and LAPR3 (p<0.001) in menstrual Analysis of the method sensitivity. To assess the method blood. No significant difference was observed be- sensitivity, the stain and sanitary napkins, including area tween MMP11 and PAEP (p<0.241) in menstrual varying from 1, 4, 9, 25, 36, 64, 81, 100 mm2, were used to extract the RNA. Sanitary napkins collected on blood. The expression levels of PAEP, LAPR3, and the third day were chosen as menstrual samples. The RT- HOXA10 in menstrual blood are shown in Figure PCR was performed as described above. The third-day 2. menstrual blood was tested with the markers PAEP, LAPR3, and HOXA10 to compare their sensitivities. Expression of target genes in female of different ages. The expression levels of LAPR3, HOXA10, Testing of samples exposed to different conditions. To and PAEP in the peripheral blood, menstrual blood evaluate the environmental effects on the body fluids, and saliva from females in the 3 age groups (i.e., the stain samples were exposed to different temperatures preadolescence, menses, and menopause) were ob- and humidity conditions and were maintained for vari- tained by real-time PCR analysis. The Ct values are ous durations of time. Some of the samples were maintained at room temperature, protected from light, or shown in Table 3. The 3 markers were expressed in stored in envelopes and plastic bags as both wet and dry the menstrual blood. The expression levels of the stains. Each stain was stored for 1, 3, 7, 30, 90, 180 and three markers were not tested in the saliva from 365 days. Each stain was exposed to room temperature women in the 3 age groups. PAEP was not tested in 60 Annals of Clinical & Laboratory Science, vol. 48, no. 1, 2018

Forensic identification of body fluid. To deter- mine whether the screened RNA markers are suffi- ciently specific and useful for case work, we extracted RNA from evidence from two different cases. All samples were genotyped using an STR analysis. One sample originated from a raped young girl who complained of menses. RNA was extracted from the girl’s sanitary napkins 15 days after the sample was collected. Using our methods, LAPR3, HOXA10, and PAEP had positive results, and the Ct values were 27.8, 34.1 and 22.8, respectively. Another sample was collected from a bed sheet that was provided as evidence of sexual abuse. The ex- Figure 4. RQ vs Sample. The horizontal ordinate presents traction of RNA and analysis by real-time PCR us- 3 mRNA markers, and the Y-axis RQ presents the relative ing the assay with LAPR3, HOXA10, and PAEP quantification. The results of RT-PCR showed that the ex- provided Ct values of 28.3, 32.5 and 23.6, respec- pression of the target genes PAEP and GAPDH is higher than that of the LAPR3 target gene on the third menstrual tively. The sample from the bed sheet was conserved day, and HOXA10 has the lowest expression. for longer than three months. The PRM1 testing result was positive using previously reported meth- the blood samples from women in the 3 age groups. ods. The STR analysis showed the original DNA PAEP was only tested in the menstruating females profiles of the two people. The results showed that and showed the lowest Ct value. This result indi- our method is as effective as the previous method. cates tissue-specific expression. Discussion Expression on different days during the menstrual phase. To detect the expression on different days In this study, we found that HOXA10 could be de- in the menstrual phase (Table 4), the expression of tected in the menstrual blood, peripheral blood, the tested genes, except for HTN3 and PRM1, was and seminal stain. However, LAPR3 and PAEP detected in different menstrual phases. The Ct val- could only be detected in the menstrual blood, and ues of the target genes PAEP and MMP11 were the mean dCt value of PAEP was lower than that of relatively low. LAPR3. The tissue specificity of the markers HOXA10 and LAPR3 was poor in the menstrual Sensitivity of the method used to test the target blood testing. By comparing the mean dCt values markers. The method sensitivity results are shown of HOXA10 detected in peripheral blood, men- in Figure 3. The volume of menstrual blood was strual blood and seminal stain with those obtained tested from 0.1-32 µl. Samples with a high expres- using previously reported markers (SPTH for iden- sion level of a specific target gene had low Ct values. tifying peripheral blood, MMP11 for identifying Samples with lower stain volumes had higher mean menstrual blood and PRM1 for identifying seminal Ct values. The expression level of the target gene stains)[4,6-8,10,11], the mean dCt values of PAEP was higher than that of the other target genes HOXA10 in the peripheral blood, menstrual blood in the third day menstrual samples (Figure 4). and seminal stain were significantly higher than those of the corresponding markers. This result in- Te sting of samples exposed to different conditions. dicates that the effect of HOXA10 is inferior to that Signals could not be detected in samples placed of the previously reported markers for identifying outside in the rain and/or at a temperature over these body fluids. Significant differences were also 35°C with sunlight for 3 days. The expression levels found between the mean dCt values of LAPR3 and of the tested markers in blood, menstrual blood, MMP11. The expression quantity of LAPR3 was saliva, and semen are shown in Table 5; the samples lower than that of MMP11 in the menstrual blood. were stored for various times at room temperature In menstrual blood, PAEP is characterized by a away from light and heat under dry conditions. high expression, is easily detected, and has a high Detailed results are not shown because they are out- sensitivity and strong specificity. Therefore, PAEP side the scope of the current article. could be a useful marker for detecting menstrual The new markers for Identification of Menstrual Blood 61

Table 5. Stability of the target gene when samples were stored for various durations at room temperature, away from light and heat, and in dry conditions.

SPTH HTN3 PRM1 LAPR3 HOXA10 PAEP GAPDH

Blood stain 1 26.3±0.25* -** - - 30.5±0.33 - 23.4±0.23 7 26.2±0.26 - - - 30.6±0.32 - 23.3±0.23 30 27.1±0.26 - - - 32.4±0.30 - 23.7±0.22 90 30.6±0.32 - - - 33.7±0.35 - 24.6±0.23 180 35.5±0.25 - - - 38.1±0.39 - 25.5±0.24 365 38.4±0.40 - - - 42.0±0.40 - 28.1±0.27 Saliva stain 1 - 24.8±0.24 - - - - 27.5±0.28 7 - 26.3±0.25 - - - - 27.9±0.2 30 - 26.6±0.26 - - - - 29.2±0.29 90 - 27.9±0.26 - - - - 31.1±0.29 180 - 28.7±0.29 - - - - 32.6±0.31 365 - 29.5±0.29 - - - - 34.4±0.35

Semen stain 1 - - 22.7±0.22 - 30.3±0.30 - 27.4±0.27 7 - - 23.5±0.22 - 31.1±0.31 - 26.8±0.26 30 - - 22.9±0.22 - 31.6±0.31 - 28.1±0.27 90 - - 25.4±0.23 - 33.4±0.33 - 29.7±0.28 180 - - 26.9±0.25 - 32.2±0.32 - 30.0±0.30 365 - - 29.3±0.3 - - - 35.1±0.33 Menstrual stain 1 - - - 29.4±0.25 30.6±0.27 23.3±0.23 24.5±0.24 7 - - - 26.1±0.26 33.9±0.27 23.5±0.23 25.6±0.25 30 - - - 28.2±0.27 35.4±0.27 24.1±0.24 26.7±0.25 90 - - - 31.6±0.33 30.1±0.30 25.8±0.25 26.9±0.27 180 - - - 34.9±0.35 33.7±0.31 28.9±0.28 34.7±0.33 365 ------blood. Furthermore, we detected the expression of the three sensitivity was positively correlated to the amount of sample in markers in samples of blood, menstrual blood, and saliva from a certain concentration range. For instance, the dry menstrual women in 3 age groups. HOXA10 and LAPR3 could be de- blood stain was kept for 7 days at 25°C in varying amounts tected in the blood from females in all 3 age groups, but PAEP from 2 µl to 32 µl, and the testing results are shown in Figure and LAPR3 could only be detected in the menstrual blood of 3. For the samples that were treated for longer times under females during menstruation. The expression of the three certain conditions, fewer samples were tested (data not shown). markers was not detected in the saliva from women in the 3 When the samples were stored for up to 180 days, no signals age groups. Previous studies have shown that the three mark- were detected in the menstrual blood. In samples that were ers are secreted by endometrial cells, and their expression is exposed to a relatively high humidity (rain), relatively high associated with female, progestational hormones [13-15]. temperature (>35°C), and sunlight for longer than 3 days, the When the expression of these three markers was analyzed in signals could not be detected. menstrual blood during menses on the 1st, 3rd and 5th days, the expression levels of PAEP and LAPR3 were not changed. We observed that three markers selected in this study have dif- Consistently with studies investigating the impact of environ- ferent expression levels in different body fluid stains. However, mental factors on samples, under room temperature (25°C), their expression can be affected by many factors, such as dis- dry and no sunlight conditions, the testing signal was detected ease (i.e., tumor and endometrial diseases) and pregnancy [16- as the amount of sample increased. However, when the 18]. Nonetheless, this study has some limitations. We did not amount of the sample >2 µl (such as menstrual blood), the study the influence of related diseases, particularly endome- signal level increased only slightly. Thus, the detection trial diseases. Although the selected volunteers were healthy, 62 Annals of Clinical & Laboratory Science, vol. 48, no. 1, 2018 we did not test for endometrial diseases. Future re- 5. Hanson EK, Lubenow H, Ballantyne J. Identification of forensically relevant body fluids using a panel of differentially expressed mi- search should consider endometrial diseases. The croRNAs. Anal Biochem 2009;387:303-314. sample size should also be increased in future 6. Sakurada K, Ikegaya H, Fukushima H, Akutsu T, Watanabe K, research. Yoshino M. Evaluation of mRNA-based approach for identification of saliva and semen. Leg Med (Tokyo) 2009;11:125-128. 7. Juusola J, Ballantyne J. 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Capillary electrophoresis of a multiplex reverse transcription-polymerase chain reaction to target mes- and peripheral blood. These results also provide evi- senger RNA markers for body fluid identification. Methods Mol dence that new mRNA markers could be developed Biol 2012;830:169-183. for mRNA profiling in forensic science. The identifi- 11. Fleming RI, Harbison S. The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids. cation of menstrual and peripheral blood is invalu- Forensic Sci Int Genet 2010;4:244-256. able to some forensic cases, such as sexual assault. 12. Virkler K, Lednev IK. Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene. Forensic Sci Int 2009;188:1-17. Acknowledgements 13. Chiang CL, Chen SS, Lee SJ, Tsao KC, Chu PL, Wen CH, Hwang SM, Yao CL, Lee H. 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Supplementary Table 1. The sample disposed under different conditions.

Time(days) Room temperature Temperature >35°C Light Dark Place Light Dark Place

dry wet dry wet dry wet dry wet

1 day √* √ √ √ √ √ √ √ 3 days √ √ √ √ √ √ √ √ 7 days √ √ √ √ √ √ √ √ 30 days √ √ √ √ √ √ √ √ 90 days √ √ √ √ √ √ √ √ 180 days √ √ √ √ √ √ √ √ 365 days √ √ √ √ √ √ √ √

*√ The conditions have done.