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 Table of Contents  
Year : 2017  |  Volume : 3  |  Issue : 3  |  Page : 115-121

Cytochrome c oxidase subunit 1-based human RNA quantification to enhance mRNA profiling in forensic biology

1 Collaborative Innovation Center of Judicial Civilization, Key Laboratory of Evidence Science, Ministry of Education, China University of Political Science and Law, Beijing, China
2 National Center for Forensic Science, University of Central Florida, Orlando, Florida, United States of America

Date of Web Publication29-Sep-2017

Correspondence Address:
Jack Ballantyne
National Center for Forensic Science, University of Central Florida, 12354 Research Parkway, Orlando, Florida 32826
United States of America
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jfsm.jfsm_63_17

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RNA analysis offers many potential applications in forensic science, and molecular identification of body fluids by analysis of cell-specific RNA markers represents a new technique for use in forensic cases. However, due to the nature of forensic materials that often admixed with nonhuman cellular components, human-specific RNA quantification is required for the forensic RNA assays. Quantification assay for human RNA has been developed in the present study with respect to body fluid samples in forensic biology. The quantitative assay is based on real-time reverse transcription-polymerase chain reaction of mitochondrial RNA cytochrome c oxidase subunit I and capable of RNA quantification with high reproducibility and a wide dynamic range. The human RNA quantification improves the quality of mRNA profiling in the identification of body fluids of saliva and semen because the quantification assay can exclude the influence of nonhuman components and reduce the adverse affection from degraded RNA fragments.

Keywords: Cytochrome c oxidase subunit I, forensic science, quantitative polymerase chain reaction, RNA quantification

How to cite this article:
Zhao D, Chen X, An Z, Hanson E, Ballantyne J. Cytochrome c oxidase subunit 1-based human RNA quantification to enhance mRNA profiling in forensic biology. J Forensic Sci Med 2017;3:115-21

How to cite this URL:
Zhao D, Chen X, An Z, Hanson E, Ballantyne J. Cytochrome c oxidase subunit 1-based human RNA quantification to enhance mRNA profiling in forensic biology. J Forensic Sci Med [serial online] 2017 [cited 2017 Oct 23];3:115-21. Available from: http://www.jfsmonline.com/text.asp?2017/3/3/115/215813

  Introduction Top

Forensic RNA analysis offers many potential applications,[1],[2],[3],[4],[5],[6] such as to determine the age of wounds and injuries, and the postmortem interval, and to investigate diseases and mechanisms leading to death in forensic pathology. Furthermore, the molecular identification of body fluids by analysis of cell-specific mRNA or microRNA expression represents a new technique supplementing DNA analysis in forensic cases. Judicious use of the sample is important not only because of the limited yield of extraction but also because improper starting amount of RNA input will lead to the failure of reverse transcription and polymerase chain reaction (RT-PCR), so sample consumption must be minimized and optimized. Due to the nature of forensic materials that often admixed with nonhuman cellular components, human-specific RNA quantification is required for the forensic RNA assays, which is usually designed according to the human nucleotide sequences.

Real-time quantitative PCR (qPCR), a highly specific, sensitive, and reproducible method for quantifying nucleic acids, represents an attractive approach to quantifying RNA. Using a proper standard, absolute copy numbers of a target sequence in a sample may be determined through RT-qPCR. Several qPCR assays for the quantification of human DNA have been reported,[7],[8],[9] and a number of them have been specifically developed for forensic purposes. However, no quantification assay for RNA so far has been developed with respect to body fluid samples in forensic biology.

The quantitative assay described in the present study is based on real-time RT-qPCR of mitochondrial RNA cytochrome c oxidase subunit I; CO1; MTCO1 (COX1), which encodes a protein of stable and high expression in cell, fulfills the requirement of qPCR-based RNA quantification with high reproducibility and a wide dynamic detection range.

  Materials and Methods Top


The study was conducted in accordance with the Declaration of Helsinki and was approved by the ethics committee of authors' institution for experiments with animal and human materials. Informed written consent was obtained from the patients prior to their enrollment in this study. Body fluids were freshly collected from healthy individuals using procedures approved by the University's Institutional Review Board. Blood (human or nonhuman) was collected by venipuncture and 50 μL aliquots placed onto cotton cloth and dried at room temperature. Saliva was collected with buccal swabs and dried at room temperature. Freshly ejaculated semen was collected in a 50 mL conical tube, and then, 50 μL aliquot placed onto swabs and dried at room temperature. In general and unless otherwise indicated, a 50 μL stain or a single cotton swab was used for RNA isolation. G152A DNA samples were purchased from Promega (Madison, WI, USA).

RNA isolation and quantification

Total RNA was extracted from blood stains (human or nonhuman), saliva, semen, and vaginal swabs with acid phenol-chloroform as previously described.[10] RNA extracts of 20 μL were treated with TURBO DNA-free Kit (Life Technologies, Carlsbad, CA, USA) as previously described[10] and quantitated with RiboGreen (Quant-iT RiboGreen® RNA Kit, Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol for the high-range assay. Fluorescence was determined using a Syntax II microplate reader (BioTeck, Winooski, VT, USA).

cDNA synthesis

RT was performed with High capacity RT kit (Life Technologies, Carlsbad, CA, USA). Total RNA template and nuclease-free water were combined to a final volume of 14.2 μL, and heated at 75°C for 3 min to eliminate target mRNA secondary structure then snap-cooled on ice. To the denatured RNA, 0.8 μL of dNTP mix (100 mM), 2 μL random hexamers, 2 μL of 10X buffer, and 1.0 μL of multiscribe reverse transcriptase were added to yield a final reaction volume of 20 μL. This reaction mixture was incubated at 25°C for 10 min, 37°C for 2 h, and then at 85°C for 5 min to inactivate the RT. For the sensitivity studies, the total RNA input ranged from 1 to 500 ng was added into the 20 μL RT reaction. For Ribogreen-dependent COX1 quantification and human specificity test, 25 ng of total RNA were used in the RT reaction of blood, saliva, and semen with measuring concentration by Quant-iT RiboGreen® RNA Kit. For body fluid identification, 25 ng or Ribogreen-dependent 106 COX1 copies for blood and saliva and 25 ng or Ribogreen-dependent 5000 COX1 copies for semen were used for cDNA synthesis. Negative control was always performed for all samples without adding the multiscribe reverse transcriptase, to monitor the DNA contamination in RNA samples.

Regular reverse transcription and polymerase chain reaction and agarose gel electrophoresis for human specificity of cytochrome c oxidase subunit I amplicons

A volume of 2 μl of the RT products was amplified in a final reaction volume of 25 uL. The reaction mixture included buffer (10 mM Tris–HCl, pH 8.3, 50 mM KCl), 1.5 mM MgCl2 (Life Technologies, Carlsbad, CA, USA), 0.125 mM each dNTP (Life Technologies, Carlsbad, CA), 0.8 uM PCR primer (Invitrogen, Grand Island, NY) same with the one for COX1 quantification listed in [Table 1], and 1.25 units AmpliTaq Gold DNA Polymerase (5 U ⁄ lL) (Life Technologies, Carlsbad, CA, USA). The standard PCR conditions used for regular PCR consisted of a denaturing step (95°C, 11 min) followed by 35 cycles (94°C, 20 s; 58°C, 30 s; and 72°C, 40 s) and a final extension step (72°C, 5 min). PCR products were separated on 3.5% agarose gels and stained for 30 min with SYBR® Gold nucleic acid stain (Life Technologies, Carlsbad, CA, USA). The gels were visualized on the BioSpectrum® Imaging System (UVP, LLC Upland, CA, USA).
Table 1: Oligonucleotide sequences for primers and probe

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Cytochrome c oxidase subunit I real-time quantitative polymerase chain reaction standard preparation

Primers designed to amplify 479 bp (from base 1 to base 479) of mitochondrial COX1 sequence were listed in [Table 1], with the forward primer containing T7 promoter sequence 23 bp (in case of future use for in vitro transcription). PCR fragment is total 502 bp in length. PCR Mix was prepared with HotStar HiFidelity Polymerase Kit (QIAGEN, Hilden, Germany): 5X HotStar HiFidelity PCR buffer 5.0 μl, forward primer (40 uM) 0.5 μl, Reverse primer (40 uM) 0.5 μl, HotStar HiFidelity DNA polymerase (2.5 U/μl) 0.5 μl, and Nuclease-free water 16.5 μl. A total of 23 μl mix plus 2 μl G152A female DNA (1 ng/μl working solution) was used for PCR amplification, with 2 μl nuclease-free water as no template control (NTC). PCR program: 95°C for 5 min, 40 cycles (94°C for 20 s; 58°C for 1 min; 68°C for 2 min), and 72°C for 10 min. PCR products were stored at 4°C if to be used on the same day or overnight, otherwise stored at − 20°C. PCR products of 25 μl plus 5 μl 6X loading dye (Promega, Madison, WI, USA) were applied to 2 lanes as well as 100 bp step DNA ladder (Promega, Madison, WI, USA) in agarose gel (1%) electrophoresis in 1 × TAE buffer for 75 min at 100 Voltage. Agarose gel was stained with SYBR Gold (Life Technologies, Carlsbad, CA, USA) for 30 min on shaker. After twice rinse with deionized water, gel was put on the clean surface of UVP ultraviolet (UV) transilluminator (UVP, LLC Upland, CA, USA); DNA band was excised from the agarose gel with a clean scalpel or razor blade on the surface of UV transilluminator, and then purified by QIAEX II Agarose Gel Extraction Kit (QIAGEN, Hilden, Germany) using factory protocol. Purified DNA was measured by spectrophotometry with Hitachi U-0080D (Tokyo, JAPAN). Dilute sample to 1 ng/μl in 10 μL solution for each tube stored at −80°C. Upon use, 5 μL stock standard sample (10 ng/10 μL) was added in 13.18 μL nuclease-free water to get 0.5 × 109 COX1 copies/μL, which was diluted in 10-fold serial to generate standard curve for COX1 quantification by RT-qPCR. Formula for the calculation of copy number is (X g/μl DNA/[dsDNA length in base pairs × 660]) × 6.022 × 1023 = Y molecules/μl.

Cytochrome c oxidase subunit I quantification by real-time reverse transcription-quantitative polymerase chain reaction

Real-time PCR primer and minor-groove binding (MGB®) probe sequences were designed using Primer Express Software Version 3.0 (Life Technologies, Carlsbad, CA, USA). Real-time PCR primer pairs were designed to be 20 bases in length, with high annealing temperatures (58°C–60°C) and short mRNA/cDNA amplicons (78 bp). The real-time PCR probe was designed to be 13 bases in length and has an annealing temperature about 10°C higher than the respective primer pair. A TaqMan® MGB® probe from Life Technologies, which has a nonfluorescent quencher, was selected as the probe type. The real-time primer and probe sequences used during assay development are listed in [Table 1].

The COX1 RNA region amplified, or target sequence, corresponds to positions 6007–6084 of the revised CRS (GenBank, NC_012920). QPCR experiments were conducted with 2 μL of cDNA sample or DNA standard in a 25 μL single reaction containing 12.5 μL of TaqMan Universal Master Mix, containing UNG (Life Technologies), 400 nM forward and reverse primers, and 200 nM 6FAM-labeled MGB probe. Additional control reactions consisted of water as NTC. Reactions were amplified in duplicate, unless otherwise noted, on a 7500 Sequence Detection System (Life Technologies) in standard mode: 20 s at 50°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Data were analyzed with Sequence Detection Software (Life Technologies). Cycle threshold (Ct) values were determined at 0.2 DRn using the manual baseline algorithm (background, collected from cycle 3–6). qPCR amplification efficiencies were calculated using the slope of the standard plot regression line: efficiency = (10 [-1⁄slope]).

Body fluid identification by triplex quantitative reverse transcription and polymerase chain reaction

PCR primer sequences for SPTB, ALAS2, STATH, HTN3, PRM1, PRM2 and GAPDH were obtained from previous publication. Primers were custom synthesized by Life Technologies. A total of 3 kinds of RT products were used in the triplex qRT-PCR for efficacy comparison with respect to obtain comparable Ct values among samples: 25 ng (Ribogreen Quant-it) for cDNA dependent/or independent dependent/or independent 106 COX1 copies for blood and saliva, and 5000 COX1 copies for semen were used for cDNA synthesis. A volume of 2.67 μl from these RT products was added to 22.33 μl of mixture of TaqMan Universal Master Mix, containing UNG (Life Technologies), gene-specific primers and probe and nuclease-free water, with final concentration of primers and probes consistent to previous publication.[10]

  Results Top

Human specificity of cytochrome c oxidase subunit I polymerase chain reaction amplicon

By nature, forensic RNA evidence may contain a mixture of human RNA and contaminating RNA from nonhuman sources. Furthermore, the routinely designed qPCR primers and probe can potentially bind to and amplify nonhuman RNA, thus resulting in inaccurate yield determinations that may impact downstream analysis. Therefore, one key criterion in selecting a qPCR target sequence was low homology with nonhuman RNA. Mitochondrial COX1 gene was selected as the qPCR target site for primer and probe design for the high sequence diversity among species (used by barcode of life project) and high RNA expression level compared to nuclear genomic genes. Several potential sequence target sites in mitochondrial COX1 were amplified with variant pairs of primers (only results of the best primer pair are shown here) for human compared with 10 primates and 20 other vertebrates, and the RNA-derived amplicon was confirmed with a control treated with RNase cocktail before RT [Figure 1]. Specificity for the amplicon of target site was demonstrated by assaying 25 ng of RNA extract from human and nonhuman blood stains based on regular RT-PCR and agarose gel electrophoresis. Domesticated and wildlife species, often encountered with recovered human remains, did not show appreciable reactivity in the assay. In addition, no cross-reactivity was virtually observed between human and the bacterial species when using bacterial DNA for amplification (data not shown). Given the high human specificity of the COX1 PCR amplicon, human mtRNA quantification should be unaffected by the presence of nonhuman RNA contamination.
Figure 1: Agarose gel electrograph of cytochrome c oxidase subunit I amplicon from human RNA; (-), RT negative control; EB, extraction blank; RTB, reverse transcription blank; AmpB, amplification blank (a), both DNA and RNA can be amplified in the polymerase chain reaction system, but amplification RNA extract is derived from RNA, since Lane 4 presents no polymerase chain reaction product band after Ambion-RNase cocktail treatment (0.05 U/μl-RNase A and 2 U/μl-RNase T1) at 37°C for 30 min. (b) Polymerase chain reaction products of primate blood RNA samples, with human RNA as positive control. PM Rhesus monkey; PM2-Spider monkey; PM3-black howler monkey; PM4-brown lemur, PM5-African green monkey; PM6-baboon; PM7-Cynomologous monkey; PM8-Spot nosed guenon; PM9-pig-tailed macaque; PM10-Chipanzee; (c) Polymerase chain reaction products of nonprimate blood RNA samples, with human RNA as positive control. Mouse-AM1; Duck-Am2; turtle-AM3; Opossum-AM4; Gopher Tortoise-AM5; Rabbit-AM6; Guinea pig-AM7; Alligator-AM8; Rooster-AM9; Frog-AM10; Calf-AM11; Cow-AM12; Dog-AM13; Cat-AM14; Horse-AM15; Deer-AM16; Pig-AM17; Goat-AM18; Sheep-AM19; Patagonian Cavy-AM20

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Sensitivity and reproducibility of real-time reverse transcription and quantitative polymerase chain reaction for cytochrome c oxidase subunit I quantification

Data generated for the agarose gel-purified qPCR standard demonstrated consistently high assay sensitivity and reproducibility. Among 10 individual assays, the dilution of 107 copies of the purified COX1 standard per well exhibited an average Ct of 14.28 (range 14.04–14.54) [Figure 2]. The subsequent 10-fold dilution (106⁄well) exhibited a Ct value of 17.65 (range 17.37–17.78). The difference in Ct values between each successive dilution of the standard also averaged about 3.4 cycles. Hundred copies of standard per well were detected at an average Ct of 31.27 (range 30.76–31.70), well within the 40 cycles prescribed for the assay. These assays were conducted over a 6-month period, thereby demonstrating high stability of the qPCR standard when stored as aliquots at −80°C. To examine whether similar assay results would be obtained using different lots of the qPCR standard, five additional lots of the agarose gel-purified standard were prepared for each set. A total of 6 lots of qPCR standard, which included the original lot, were subjected to qPCR, and 107 copies of the purified COX1 standard per well exhibited an average Ct of 14.52 (standard deviation, 0.29) [Figure 3]. These results revealed consistent Ct values among all 6 lots of standard, thereby demonstrating the reliability of the qPCR standard preparation. To test the assay sensitivity for blood, saliva, semen, and vaginal secretion samples, total RNA of blood (n = 5), saliva (n = 3), semen (n = 4), and vaginal secretion (n = 3) samples were added for RT-qPCR of COX1 quantification with variant input amount of 500 ng, 100 ng, 25 ng, 5 ng, and 1 ng. The overall mean Ct value and standard deviation were calculated as 18.62 ± 2.36 (500 ng), 21.66 ± 3.26 (100 ng), 23.00 ± 3.18 (25 ng), 25.32 ± 3.03 (5 ng), and 27.56 ± 3.13 (1 ng) because these Ct values are within the range of the standard Ct of 14.28 (107 COX1 copies) and 31.27 (102 COX1 copies); therefore, as low as 1 ng total RNA for RT-qPCR is available for COX1 quantification, even for the semen samples, which shows lowest COX1 RNA abundance in body fluid samples [Table 2].
Figure 2: Standard Ct data among 10 separate quantitative PCR assays

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Figure 3: Quantitative polymerase chain reaction assay of 6 lots of quantitative polymerase chain reaction standard of 107 copies

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Table 2: Sensitivity of quantitative reverse transcription-polymerase chain reaction assays in body fluid samples

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DNA interference

Because the mitochondrial DNA is lack of introns, no available primer design can avoid the amplification of DNA if it indeed exists in RNA sample to since mitochondrial DNA lacks introns, no available primer design can avoid the amplification of DNA if it indeed exists in RNA sample. Then, the questions left are how to decrease the extent of DNA contamination when performing RNA extraction and how to monitor the extent of DNA interference when performing RNA quantification.

To decrease the extent of DNA contamination, after comparison with other similar products for DNA remove, TURBO DNA-free kit was chosen to treat the RNA samples following the acid phenol-chloroform extraction. Our data consistently show the excellent performance of the TURBO DNA-free kit in removing DNA contamination from RNA extracts.

On the other hand, negative control of RT was always performed for cDNA synthesis of all RNA samples, and COX1 RNA quantification was performed simultaneously for RT-positive and RT-negative RT products. Thereby, extent of DNA contamination in RNA extracts can be monitored quantitatively.

Efficacy in enhancing the quality of mRNA profiling in body fluid identification

To demonstrate whether the COX1 quantification improves the mRNA profiling quality for body fluid identification, two types of input calculations were used for input normalization of quantitative RT-PCR triplex for blood, saliva, and semen samples, namely, Ribogreen-25 ng and Ribogreen-dependent COX1-106 copies input for cDNA synthesis. Same volume of 2.67 μl of all these RT products was used in qPCR triplex of 25 μl system for subsequent body fluid identification. For the blood samples, no much improvement of COX1 quant was observed with Ribogreen-dependent COX1 quantification [Figure 4]. However, for saliva and semen samples [Figure 5] and [Figure 6], Ribogreen-dependent COX1 quantification showed smaller variation of Ct values, compared with Ribogreen quantification.
Figure 4: Reverse transcription-quantitative polymerase chain reaction for blood identification. Duplicate reaction for 6 individuals

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Figure 5: Reverse transcription-quantitative polymerase chain reaction for saliva identification. Duplicate reaction for 6 individuals

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Figure 6: Reverse transcription-quantitative polymerase chain reaction for semen identification. Duplicate reaction for 5 individuals

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  Discussion Top

Knowledge of the quantity and quality of RNA for a given RNA sample before commencement of forensic RNA analyses could increase the likelihood of successful analysis, improve the efficiency of analysis, and help conserve sample for potential additional analyses.

However, the routine used UV-spectrophotometry, and fluorescence (RiboGreen)-based measurement is not specific for human RNA quantitation, and heavily fragmented RNA will be counted in as well. Samples containing nonhuman or degraded RNA may affect the application of these RNA quantification to the subsequent human-specific RNA analysis.

The described qPCR assay was developed to quantify mitochondrial RNA with a high degree of sensitivity and specificity. The region targeted for real-time amplification was within the mitochondrial COX1 gene that resides in the coding region. The qPCR target region also possesses minimal sequence homology to the mtDNA of other forensically relevant species, and the small amplicon size (78 base pairs) facilitates amplification even for degraded samples. The qPCR standard was prepared from regular PCR product, which was amplified by a pair of primers as a 502 bp fragment fully covering the target sequence (78 bp) for COX1 quantification. This type of standard offers high yield, is easily obtained, and is cost-effective compared with the construction and production of traditional plasmid-based DNA standards. More importantly, a laboratory-made standard offers convenient quality control value because every factor potentially affects the preparation of standard is available for monitoring in the laboratory. The standard turns out highly consistent results and shows good reproducibility.

Considering stability and convenience of preparation and quality control, an RNA standard was not chosen for the present study. We actually use DNA standard to quantify the cDNA copy number and then extrapolate the RNA copy number in the original RNA extract, which is based on the assumption that the efficiency of the RT is the same nearly 100% for all the samples. Not much-observed improvement of the human RNA quantification in blood samples for body fluid identification might be due to the fact that venous blood contains no or little nonhuman components. Significant improvement of human RNA quantification in saliva samples for body fluid identification could be due to the expected RNA from commensal microbes being nondetectable by human-specific RNA quantification. Significant improvement of the human RNA quantification was also found in semen samples, which may be explained either by nonhuman RNA contamination or RNA degradation, considering the unique characteristics of semen samples.

In conclusion, the qPCR assay described is suitable for accurate and precise COX1 RNA quantification in RNA samples before forensic analysis and is reliable, robust, and highly sensitive, which should provide subsequent RNA profiling with a better normalization for human-specific RNA input amount.


The authors would like to thank the support from Beijing Municipal Natural Science Foundation (7,163,221), Ministry of Public Security of Material Evidence Identification Center (2017FGKFKT05), and the Program for Young Innovative Research Team in China University of Political Science and Law (2014CXTD04, 2016CXTD05).

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Hanson EK, Lubenow H, Ballantyne J. Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs. Anal Biochem 2009;387:303-14.  Back to cited text no. 1
Maeda H, Zhu BL, Ishikawa T, Quan L, Michiue T. Significance of postmortem biochemistry in determining the cause of death. Leg Med (Tokyo) 2009;11 Suppl 1:S46-9.  Back to cited text no. 2
Haas C, Klesser B, Maake C, Bär W, Kratzer A. MRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR. Forensic Sci Int Genet 2009;3:80-8.  Back to cited text no. 3
Sakurada K, Ikegaya H, Fukushima H, Akutsu T, Watanabe K, Yoshino M, et al. Evaluation of mRNA-based approach for identification of saliva and semen. Leg Med (Tokyo) 2009;11:125-8.  Back to cited text no. 4
Bauer M, Patzelt D. Identification of menstrual blood by real time RT-PCR: Technical improvements and the practical value of negative test results. Forensic Sci Int 2008;174:55-9.  Back to cited text no. 5
Bauer M. RNA in forensic science. Forensic Sci Int Genet 2007;1:69-74.  Back to cited text no. 6
Shewale JG, Schneida E, Wilson J, Walker JA, Batzer MA, Sinha SK, et al. Human genomic DNA quantitation system, H-Quant: Development and validation for use in forensic casework. J Forensic Sci 2007;52:364-70.  Back to cited text no. 7
Alonso A, Martin P, Albarrán C, Garcãa P, Primorac D, Garcãa O, et al. Specific quantification of human genomes from low copy number DNA samples in forensic and ancient DNA studies. Croat Med J 2003;44:273-80.  Back to cited text no. 8
von Wurmb-Schwark N, Higuchi R, Fenech AP, Elfstroem C, Meissner C, Oehmichen M, et al. Quantification of human mitochondrial DNA in a real time PCR. Forensic Sci Int 2002;126:34-9.  Back to cited text no. 9
Juusola J, Ballantyne J. MRNA profiling for body fluid identification by multiplex quantitative RT-PCR. J Forensic Sci 2007;52:1252-62.  Back to cited text no. 10


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2]


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