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 (tem- perature, 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 men- strual 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 tar- get 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 ori- gin 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 0091-7370/18/0100-055. © 2018 by the Association of Clinical Scientists, Inc. 56 Annals of Clinical & Laboratory Science, vol. 48, no. 1, 2018 Figure 1. Amplification Plot. (A) indicates MMP11 in menstrual blood, (B) indicates PAEP in menstrual blood, (C) in- dicates SPTH in blood, (D) indicates HTN3 in saliva, (E) indicates the contra-indicator, (F) indicates LAPR3, (G) indi- cates 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 in- clude 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.