1
Electrophoresis 2014, 00, 1–7
Jong-Lyul Park1,2 Seong-Min Park1,2 Oh-Hyung Kwon1 Han-chul Lee3 Jin-young Kim3 Hyun Ha Seok4 Woo Sik Lee4 Seung-Hwan Lee3 Yong Sung Kim1,2 Kwang-Man Woo3 ∗ Seon-Young Kim1,2 1 Medical
Genomics Research Center, KRIBB, Daejeon, Republic of Korea 2 Department of Functional Genomics, University of Science of Technology, Daejeon, Republic of Korea 3 DNA Forensic Division, Supreme Prosecutor’s Office, Seoul, Republic of Korea 4 Department of Obstetrics and Gynecology, Fertility Center of CHA Gangnam Medical Center, College of Medicine, CHA University, Seoul, Republic of Korea
Received February 12, 2014 Revised May 27, 2014 Accepted May 28, 2014
Research Article
Microarray screening and qRT-PCR evaluation of microRNA markers for forensic body fluid identification MicroRNAs (miRNA) are a class of small (22 nucleotides) noncoding RNAs that regulate diverse biological processes at the post-transcriptional level. MiRNAs have great potential for forensic body fluid identification because they are expressed in a tissue specific manner and are less prone to degradation. Previous studies reported several miRNAs as body fluid specific, but there are few overlaps among them. Here, we used a genome-wide miRNA microarray containing over 1700 miRNAs to assay 20 body fluid samples and identify novel miRNAs useful for forensic body fluid identification. Based on Shannon Entropy and Q-statistics, 203 miRNAs specifically expressed in each body fluid were first selected. Eight miRNAs were then selected as novel forensically relevant miRNA markers: miR484 and miR-182 for blood, miR-223 and miR-145 for saliva, miR-2392 and miR-3197 for semen, and miR-1260b and miR-654–5p for vaginal secretions. When the eight selected miRNAs were evaluated in 40 additional body fluid samples by qRT-PCR, they showed high sensitivity and specificity for the identification of the target body fluid. We suggest that the eight miRNAs may be candidates for developing an effective molecular assay for forensic body fluid identification. Keywords: Body fluid identification / Forensic / MicroRNA
DOI 10.1002/elps.201400075
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Forensic body fluid identification is important for crime scene reconstruction. Traditionally, enzymatic tests [1–5] have mainly been used, but recent developments in genomics have expanded the repertoire of molecules that may be used for forensic body fluid identification to mRNA, microRNA (miRNA), and even DNA [6–8]. A number of studies have established that many mRNAs are specifically expressed in each forensic body fluid and have high sensitivity and specificity in forensic body fluid identification [9–17]. MiRNAs belong to a class of small noncoding RNA molecules containing 18–25 nucleotides that regulate various physiological processes by post-transcriptional mechanisms [18]. Due to their small size, miRNAs are known to be
Correspondence: Dr. Seon-Youn Kim, Medical Genomics Research Center, KRIBB, 125 Gwahang-no, Yuseong-gu, Daejeon, Republic of Korea E-mail: [email protected] Fax: +82-042-879-8119
Abbreviations: AUC, area under the ROC curve; miRNA, microRNA; RMA, robust multichip average C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
highly stable in forensic body fluids even in a cell-free state because of their short hairpin structures, encapsulation by microvesicles, and association with protein complexes [19–23]. In contrast, most RNAs are not stable enough to endure detrimental conditions such as heat, humidity, UV light, and ubiquitous ribonucleases. In this regard, miRNA has a major advantage over mRNA in the development of markers for forensic body fluid identification. As with mRNA, the simultaneous extraction and analysis of miRNA and DNA is possible. For example, van der Meer et al. extracted DNA and total RNA in a single process and successfully identified blood and saliva [24]. Omelia et al. successfully extracted miRNA from previously extracted DNA samples used for blood and saliva detection [25]. Similar to mRNAs, some miRNAs are expressed in a tissue specific manner [26–29]. Recently, four studies have reported the identification and evaluation of body fluid-specific miRNAs for forensic use. While each study reported several body fluid-specific miRNAs, only four miRNAs were supported in two or more studies: miR-16 and miR-451 for
∗
Additional corresponding author: Dr. Kwang-Man Woo. E-mail: [email protected]
www.electrophoresis-journal.com
2
J.-L. Park et al.
blood [30, 31], miR-891a for semen [31, 32], and miR-205 for saliva [30, 33]. Most of the suggested miRNAs were not independently evaluated in other studies, suggesting that more studies are needed before establishing reliable miRNA markers for forensic body fluid identification. Additionally, previous studies are limited because they investigated less than 800 miRNAs, while there are more than 1700 currently known human miRNAs. In this study, we performed genome-wide miRNA expression profiling of 20 body fluid samples using the Affymetrix 3.0 miRNA array containing 1733 known mature miRNAs and 2216 human small RNAs to identify potential body fluid-specific miRNA markers. We first identified 203 body fluid-specific miRNA markers and then evaluated eight novel miRNAs in 40 additional samples using qRT-PCR.
2 Materials and methods 2.1 Sample collection and RNA preparation Each body fluid sample (blood, saliva, semen, and vaginal secretion) was collected from 60 healthy volunteers after obtaining informed consent from the participants. The study was approved by the Institutional Review Board of Cha University College of Medicine. Blood samples were drawn from a peripheral vein using an EDTA-containing tube (BD Vacutainer), and a 100 L aliquot was stored at −20°C until needed. Saliva and freshly ejaculated semen were collected in a 10 mL falcon tube, and 100 L aliquot was stored at −20°C. Vaginal fluids were collected using sterile cotton swabs and stored at −20°C. For total RNA extraction, swabs were placed into 10 mL falcon tubes with lysis buffer (3 M guanidine HCL, 0.3 M sodium acetate, and 0.2 M EDTA). Total RNA including miRNA was isolated from 100 L of each body fluid using the miRNeasy Mini kit (Qiagen, Carlsbad, CA) according to the manufacturer’s instructions. The quality and quantity of the extracted total RNA was analyzed with ExperionTM RNA StdSens (Bio-Rad, Hercules, CA). We note that most of the RNAs are from body-fluid specific cells because we did not spin down the cells during sample collection.
2.2 MiRNA microarray experiment and data analysis To identify body fluid-specific miRNA markers, we used the Affymetrix Gene Chip miRNA 3.0 array, which comprises a total of 19 724 miRNA probe sets including 1733 human mature miRNAs, 1658 human pre-miRNAs based on miRBase v.17, and 2216 human small RNAs such as small nucleolar RNAs (snoRNAs) and small cajal body-specific RNAs (sca RNAs). Briefly, 500 ng of total RNA was polyA tailed, ligated using the Affymetrix FlashTagTM Biotin HSR RNA labeling kit, and then hybridized to the miRNA 3.0 array according to the manufacturer’s instructions. After hybridization, the miRNA 3.0 array was scanned with the Affymetrix GeneChip Scanner 3000. Scanned images were converted into inten C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2014, 00, 1–7
sity values using the AffymetrixTM Expression Console 1.3 program. The robust multichip average (RMA) method was used for data normalization [34]. All primary miRNA microarray data were deposited in Gene Expression Omnibus (GEO) under accession number GSE49630.
2.3 qRT-PCR miRNA-specific qRT-PCR was performed to evaluate eight body fluid-specific miRNA candidates. Briefly, 500 ng of total RNA was used for first-strand cDNA synthesis (20 L) using the NCode VILO miRNA cDNA Synthesis kit (Life Technologies, Carlsbard, CA), and 1 L of synthesized cDNA was used in a qRT-PCR reaction (20 L) containing primer sets and the 2X SYBR Premix (Bio-Rad) using a CFX96 Real-Time PCR (Bio-Rad) machine. The specificity of the PCR amplicon and absence of primer-dimers was verified by melt-curve analysis using Bio-Rad CFX manager software v1.5 (Bio-Rad). The PCR conditions were as follows: 95°C for 3 min followed by 40 cycles of 95°C for 10 s, 60°C for 20 s and 72°C for 20 s. The expression level of each miRNA was normalized to noncoding small nuclear U6 RNA expression [35]. U6 was used as a reference control as previously described by Wang et al. [31]. Expression levels were determined using the delta Ct (⌬ Ct ) method. Information for the synthesized forward primers (Bioneer, Daejeon, Korea) and conditions are provided in Supporting Information Table 1. To evaluate the detection sensitivity of Real-Time PCR assays for the eight selected miRNAs, serial dilutions of cDNA were used as input for PCR reaction.
2.4 Statistical analysis Shannon Entropy (H) and Q-statistics (Q) were used to select body fluid-specific markers as previously described [36]. Student’s t-test was used to infer the significance of differences in miRNA expression between one type of body fluid and the remaining body fluid types. Receiver operating characteristic (ROC) and the respective area under the ROC curve (AUC) values were calculated for each miRNA marker using the ROCR package with R (version 2.6.1). Results with a p-value ⬍ 0.05 were considered significant.
3 Results 3.1 MiRNA expression profiling and selection of body fluid-specific miRNAs One of the difficulties in using RNA for forensic body fluid identification is that most RNAs from body fluids are highly degraded. Not surprisingly, total RNA from body fluids, with the exception of blood, is severely degraded (Supporting Information Fig. 1). However, after our miRNA microarray experiment, all samples passed the QC test and produced good www.electrophoresis-journal.com
Electrophoresis 2014, 00, 1–7
Nucleic acids
3
Figure 1. Unsupervised hierarchical clustering of 20 body fluid samples using 203 selected body fluid-specific miRNAs. MiRNA expression profiling was performed for 20 body fluid samples (five samples for each body fluid) using the Affymetrix Gene Chip miRNA 3.0 array. Two hundred and three miRNAs were selected as body fluid specific based on Q-value (Q < 3.3).
results (Supporting Information Table 2). For quality check of miRNA 3.0 arrays, we checked the signal of spike-in controls (2, 23, 29, 31, and 36) and BioB, BioC, BioD and cre as ligation, poly (A) tailing, and hybridization controls. If the labeling protocol was successful, the following spike-in control probe sets should have signal greater than or equal to 9.96 for log2 signal and the hybridization control probe sets should have signal commensurate with concentration: BioB3 ⬍ BioC-3 ⬍ BioD-3 ⬍ cre-3. All of array metrics for QC is given in Supporting Information Table 2. Raw intensity data from the microarray were normalized by the RMA method and used in subsequent analyses (Supporting Information Fig. 2). Each body fluid sample demonstrated good withingroup correlation (Supporting Information Fig. 3). Shannon entropy (E) and Q-statistics (Q) were calculated for each miRNA to select body fluid-specific miRNAs as previously described [36]. Expression differences among the four body fluids were also considered. Thus, the following two criteria were applied: (i) Q value ⬍ 3.3 for each body fluid and (ii) miRNA expression difference ⬎ 2 (log2 scale) between a target and remaining body fluids. As a result, 203 body fluid-specific miRNAs were initially selected (Supporting Information Table 3). These miRNAs included 97 from blood, 13 from saliva, 76 from semen, and 17 from vaginal secretions (Fig. 1).
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.2 Evaluation of body fluid-specific miRNAs by qRT-PCR Of the 203 body fluid-specific miRNAs, eight were selected for independent evaluation by qRT-PCR. Four criteria were used for further filtering: (i) the Q-value rank in each body fluid, (ii) specific expression in one body fluid, (iii) high expression level, and (iv) previously unreported miRNA. Two miRNAs were selected for each body fluid (Table 1). The eight selected miRNAs showed a good body fluidspecific expression pattern in our microarray data as expected (Fig. 2). Additionally, we evaluated previously known miRNA markers in our microarray data. We found that nine of the previously reported miRNAs showed a good body fluidspecific expression pattern (Table 2). These miRNAs include six blood-specific miRNAs (hsa-miR-126, hsa-miR-106a, hsamiR-451, hsa-miR-185, hsa-miR-486, and hsa-miR-20a), two saliva-specific miRNAs (hsa-miR-203 and hsa-miR-205), and one semen-specific miRNA (hsa-miR-891a) [30–33]. One blood-specific miRNAs (hsa-miR-16) was highly expressed in the blood, but it was not blood specific as it was also highly expressed in the saliva and vaginal secretions. Two saliva-specific miRNAs (hsa-miR-203 and hsa-miR-205) were highly expressed in the saliva, but they were also expressed in the semen.
www.electrophoresis-journal.com
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J.-L. Park et al.
Electrophoresis 2014, 00, 1–7
Table 1. List of the eight selected body fluid-specific miRNAs
MicroRNA
hsa-miR-484 hsa-miR-182 hsa-miR223 hsa-miR-145 hsa-miR-2392 hsa-miR-3197 hsa-miR-1260b hsa-miR-654–5q
Type
Blood Blood Saliva Saliva Semen Semen Vaginal Vaginal
Expression (RMA)
Entropy
Blood
Saliva
Semen
Vaginal
5.55 10.42 3.48 3.23 4.60 4.65 2.99 1.08
1.91 3.73 9.75 9.60 3.44 4.30 3.24 1.08
1.54 2.80 0.96 0.64 10.45 10.72 2.43 1.84
1.61 2.94 4.13 4.80 4.40 7.90 6.95 3.90
1.75 1.75 1.65 1.61 1.85 1.90 1.86 1.78
Q-value Blood
Saliva
Semen
Vaginal
2.69 2.68 4.05 4.11 4.17 4.47 4.25 4.65
4.23 4.16 2.55 2.53 4.58 4.58 4.13 4.65
4.53 4.58 5.89 6.43 2.98 3.26 4.55 3.88
4.47 4.50 3.79 3.54 4.23 3.70 3.03 2.79
a) AUC
b) P-value
1.00 1.00 1.00 0.97 1.00 0.93 1.00 0.81
Electrophoresis 2014, 00, 1–7
Jong-Lyul Park1,2 Seong-Min Park1,2 Oh-Hyung Kwon1 Han-chul Lee3 Jin-young Kim3 Hyun Ha Seok4 Woo Sik Lee4 Seung-Hwan Lee3 Yong Sung Kim1,2 Kwang-Man Woo3 ∗ Seon-Young Kim1,2 1 Medical
Genomics Research Center, KRIBB, Daejeon, Republic of Korea 2 Department of Functional Genomics, University of Science of Technology, Daejeon, Republic of Korea 3 DNA Forensic Division, Supreme Prosecutor’s Office, Seoul, Republic of Korea 4 Department of Obstetrics and Gynecology, Fertility Center of CHA Gangnam Medical Center, College of Medicine, CHA University, Seoul, Republic of Korea
Received February 12, 2014 Revised May 27, 2014 Accepted May 28, 2014
Research Article
Microarray screening and qRT-PCR evaluation of microRNA markers for forensic body fluid identification MicroRNAs (miRNA) are a class of small (22 nucleotides) noncoding RNAs that regulate diverse biological processes at the post-transcriptional level. MiRNAs have great potential for forensic body fluid identification because they are expressed in a tissue specific manner and are less prone to degradation. Previous studies reported several miRNAs as body fluid specific, but there are few overlaps among them. Here, we used a genome-wide miRNA microarray containing over 1700 miRNAs to assay 20 body fluid samples and identify novel miRNAs useful for forensic body fluid identification. Based on Shannon Entropy and Q-statistics, 203 miRNAs specifically expressed in each body fluid were first selected. Eight miRNAs were then selected as novel forensically relevant miRNA markers: miR484 and miR-182 for blood, miR-223 and miR-145 for saliva, miR-2392 and miR-3197 for semen, and miR-1260b and miR-654–5p for vaginal secretions. When the eight selected miRNAs were evaluated in 40 additional body fluid samples by qRT-PCR, they showed high sensitivity and specificity for the identification of the target body fluid. We suggest that the eight miRNAs may be candidates for developing an effective molecular assay for forensic body fluid identification. Keywords: Body fluid identification / Forensic / MicroRNA
DOI 10.1002/elps.201400075
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Forensic body fluid identification is important for crime scene reconstruction. Traditionally, enzymatic tests [1–5] have mainly been used, but recent developments in genomics have expanded the repertoire of molecules that may be used for forensic body fluid identification to mRNA, microRNA (miRNA), and even DNA [6–8]. A number of studies have established that many mRNAs are specifically expressed in each forensic body fluid and have high sensitivity and specificity in forensic body fluid identification [9–17]. MiRNAs belong to a class of small noncoding RNA molecules containing 18–25 nucleotides that regulate various physiological processes by post-transcriptional mechanisms [18]. Due to their small size, miRNAs are known to be
Correspondence: Dr. Seon-Youn Kim, Medical Genomics Research Center, KRIBB, 125 Gwahang-no, Yuseong-gu, Daejeon, Republic of Korea E-mail: [email protected] Fax: +82-042-879-8119
Abbreviations: AUC, area under the ROC curve; miRNA, microRNA; RMA, robust multichip average C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
highly stable in forensic body fluids even in a cell-free state because of their short hairpin structures, encapsulation by microvesicles, and association with protein complexes [19–23]. In contrast, most RNAs are not stable enough to endure detrimental conditions such as heat, humidity, UV light, and ubiquitous ribonucleases. In this regard, miRNA has a major advantage over mRNA in the development of markers for forensic body fluid identification. As with mRNA, the simultaneous extraction and analysis of miRNA and DNA is possible. For example, van der Meer et al. extracted DNA and total RNA in a single process and successfully identified blood and saliva [24]. Omelia et al. successfully extracted miRNA from previously extracted DNA samples used for blood and saliva detection [25]. Similar to mRNAs, some miRNAs are expressed in a tissue specific manner [26–29]. Recently, four studies have reported the identification and evaluation of body fluid-specific miRNAs for forensic use. While each study reported several body fluid-specific miRNAs, only four miRNAs were supported in two or more studies: miR-16 and miR-451 for
∗
Additional corresponding author: Dr. Kwang-Man Woo. E-mail: [email protected]
www.electrophoresis-journal.com
2
J.-L. Park et al.
blood [30, 31], miR-891a for semen [31, 32], and miR-205 for saliva [30, 33]. Most of the suggested miRNAs were not independently evaluated in other studies, suggesting that more studies are needed before establishing reliable miRNA markers for forensic body fluid identification. Additionally, previous studies are limited because they investigated less than 800 miRNAs, while there are more than 1700 currently known human miRNAs. In this study, we performed genome-wide miRNA expression profiling of 20 body fluid samples using the Affymetrix 3.0 miRNA array containing 1733 known mature miRNAs and 2216 human small RNAs to identify potential body fluid-specific miRNA markers. We first identified 203 body fluid-specific miRNA markers and then evaluated eight novel miRNAs in 40 additional samples using qRT-PCR.
2 Materials and methods 2.1 Sample collection and RNA preparation Each body fluid sample (blood, saliva, semen, and vaginal secretion) was collected from 60 healthy volunteers after obtaining informed consent from the participants. The study was approved by the Institutional Review Board of Cha University College of Medicine. Blood samples were drawn from a peripheral vein using an EDTA-containing tube (BD Vacutainer), and a 100 L aliquot was stored at −20°C until needed. Saliva and freshly ejaculated semen were collected in a 10 mL falcon tube, and 100 L aliquot was stored at −20°C. Vaginal fluids were collected using sterile cotton swabs and stored at −20°C. For total RNA extraction, swabs were placed into 10 mL falcon tubes with lysis buffer (3 M guanidine HCL, 0.3 M sodium acetate, and 0.2 M EDTA). Total RNA including miRNA was isolated from 100 L of each body fluid using the miRNeasy Mini kit (Qiagen, Carlsbad, CA) according to the manufacturer’s instructions. The quality and quantity of the extracted total RNA was analyzed with ExperionTM RNA StdSens (Bio-Rad, Hercules, CA). We note that most of the RNAs are from body-fluid specific cells because we did not spin down the cells during sample collection.
2.2 MiRNA microarray experiment and data analysis To identify body fluid-specific miRNA markers, we used the Affymetrix Gene Chip miRNA 3.0 array, which comprises a total of 19 724 miRNA probe sets including 1733 human mature miRNAs, 1658 human pre-miRNAs based on miRBase v.17, and 2216 human small RNAs such as small nucleolar RNAs (snoRNAs) and small cajal body-specific RNAs (sca RNAs). Briefly, 500 ng of total RNA was polyA tailed, ligated using the Affymetrix FlashTagTM Biotin HSR RNA labeling kit, and then hybridized to the miRNA 3.0 array according to the manufacturer’s instructions. After hybridization, the miRNA 3.0 array was scanned with the Affymetrix GeneChip Scanner 3000. Scanned images were converted into inten C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2014, 00, 1–7
sity values using the AffymetrixTM Expression Console 1.3 program. The robust multichip average (RMA) method was used for data normalization [34]. All primary miRNA microarray data were deposited in Gene Expression Omnibus (GEO) under accession number GSE49630.
2.3 qRT-PCR miRNA-specific qRT-PCR was performed to evaluate eight body fluid-specific miRNA candidates. Briefly, 500 ng of total RNA was used for first-strand cDNA synthesis (20 L) using the NCode VILO miRNA cDNA Synthesis kit (Life Technologies, Carlsbard, CA), and 1 L of synthesized cDNA was used in a qRT-PCR reaction (20 L) containing primer sets and the 2X SYBR Premix (Bio-Rad) using a CFX96 Real-Time PCR (Bio-Rad) machine. The specificity of the PCR amplicon and absence of primer-dimers was verified by melt-curve analysis using Bio-Rad CFX manager software v1.5 (Bio-Rad). The PCR conditions were as follows: 95°C for 3 min followed by 40 cycles of 95°C for 10 s, 60°C for 20 s and 72°C for 20 s. The expression level of each miRNA was normalized to noncoding small nuclear U6 RNA expression [35]. U6 was used as a reference control as previously described by Wang et al. [31]. Expression levels were determined using the delta Ct (⌬ Ct ) method. Information for the synthesized forward primers (Bioneer, Daejeon, Korea) and conditions are provided in Supporting Information Table 1. To evaluate the detection sensitivity of Real-Time PCR assays for the eight selected miRNAs, serial dilutions of cDNA were used as input for PCR reaction.
2.4 Statistical analysis Shannon Entropy (H) and Q-statistics (Q) were used to select body fluid-specific markers as previously described [36]. Student’s t-test was used to infer the significance of differences in miRNA expression between one type of body fluid and the remaining body fluid types. Receiver operating characteristic (ROC) and the respective area under the ROC curve (AUC) values were calculated for each miRNA marker using the ROCR package with R (version 2.6.1). Results with a p-value ⬍ 0.05 were considered significant.
3 Results 3.1 MiRNA expression profiling and selection of body fluid-specific miRNAs One of the difficulties in using RNA for forensic body fluid identification is that most RNAs from body fluids are highly degraded. Not surprisingly, total RNA from body fluids, with the exception of blood, is severely degraded (Supporting Information Fig. 1). However, after our miRNA microarray experiment, all samples passed the QC test and produced good www.electrophoresis-journal.com
Electrophoresis 2014, 00, 1–7
Nucleic acids
3
Figure 1. Unsupervised hierarchical clustering of 20 body fluid samples using 203 selected body fluid-specific miRNAs. MiRNA expression profiling was performed for 20 body fluid samples (five samples for each body fluid) using the Affymetrix Gene Chip miRNA 3.0 array. Two hundred and three miRNAs were selected as body fluid specific based on Q-value (Q < 3.3).
results (Supporting Information Table 2). For quality check of miRNA 3.0 arrays, we checked the signal of spike-in controls (2, 23, 29, 31, and 36) and BioB, BioC, BioD and cre as ligation, poly (A) tailing, and hybridization controls. If the labeling protocol was successful, the following spike-in control probe sets should have signal greater than or equal to 9.96 for log2 signal and the hybridization control probe sets should have signal commensurate with concentration: BioB3 ⬍ BioC-3 ⬍ BioD-3 ⬍ cre-3. All of array metrics for QC is given in Supporting Information Table 2. Raw intensity data from the microarray were normalized by the RMA method and used in subsequent analyses (Supporting Information Fig. 2). Each body fluid sample demonstrated good withingroup correlation (Supporting Information Fig. 3). Shannon entropy (E) and Q-statistics (Q) were calculated for each miRNA to select body fluid-specific miRNAs as previously described [36]. Expression differences among the four body fluids were also considered. Thus, the following two criteria were applied: (i) Q value ⬍ 3.3 for each body fluid and (ii) miRNA expression difference ⬎ 2 (log2 scale) between a target and remaining body fluids. As a result, 203 body fluid-specific miRNAs were initially selected (Supporting Information Table 3). These miRNAs included 97 from blood, 13 from saliva, 76 from semen, and 17 from vaginal secretions (Fig. 1).
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.2 Evaluation of body fluid-specific miRNAs by qRT-PCR Of the 203 body fluid-specific miRNAs, eight were selected for independent evaluation by qRT-PCR. Four criteria were used for further filtering: (i) the Q-value rank in each body fluid, (ii) specific expression in one body fluid, (iii) high expression level, and (iv) previously unreported miRNA. Two miRNAs were selected for each body fluid (Table 1). The eight selected miRNAs showed a good body fluidspecific expression pattern in our microarray data as expected (Fig. 2). Additionally, we evaluated previously known miRNA markers in our microarray data. We found that nine of the previously reported miRNAs showed a good body fluidspecific expression pattern (Table 2). These miRNAs include six blood-specific miRNAs (hsa-miR-126, hsa-miR-106a, hsamiR-451, hsa-miR-185, hsa-miR-486, and hsa-miR-20a), two saliva-specific miRNAs (hsa-miR-203 and hsa-miR-205), and one semen-specific miRNA (hsa-miR-891a) [30–33]. One blood-specific miRNAs (hsa-miR-16) was highly expressed in the blood, but it was not blood specific as it was also highly expressed in the saliva and vaginal secretions. Two saliva-specific miRNAs (hsa-miR-203 and hsa-miR-205) were highly expressed in the saliva, but they were also expressed in the semen.
www.electrophoresis-journal.com
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J.-L. Park et al.
Electrophoresis 2014, 00, 1–7
Table 1. List of the eight selected body fluid-specific miRNAs
MicroRNA
hsa-miR-484 hsa-miR-182 hsa-miR223 hsa-miR-145 hsa-miR-2392 hsa-miR-3197 hsa-miR-1260b hsa-miR-654–5q
Type
Blood Blood Saliva Saliva Semen Semen Vaginal Vaginal
Expression (RMA)
Entropy
Blood
Saliva
Semen
Vaginal
5.55 10.42 3.48 3.23 4.60 4.65 2.99 1.08
1.91 3.73 9.75 9.60 3.44 4.30 3.24 1.08
1.54 2.80 0.96 0.64 10.45 10.72 2.43 1.84
1.61 2.94 4.13 4.80 4.40 7.90 6.95 3.90
1.75 1.75 1.65 1.61 1.85 1.90 1.86 1.78
Q-value Blood
Saliva
Semen
Vaginal
2.69 2.68 4.05 4.11 4.17 4.47 4.25 4.65
4.23 4.16 2.55 2.53 4.58 4.58 4.13 4.65
4.53 4.58 5.89 6.43 2.98 3.26 4.55 3.88
4.47 4.50 3.79 3.54 4.23 3.70 3.03 2.79
a) AUC
b) P-value
1.00 1.00 1.00 0.97 1.00 0.93 1.00 0.81
