Research Article| Volume 16, P195-202, May 2015

Comparative evaluation of different extraction and quantification methods for forensic RNA analysis

Published:January 16, 2015DOI:


      • Comparison of five different methods for forensic RNA/DNA co-extraction.
      • No method found optimal for all aspects of RNA/DNA co-analysis.
      • Best RNA-results: NucleoSpin® miRNA Kit.
      • Best DNA-results: AllPrep DNA/RNA Mini Kit.


      Since about 2005, there is increasing interest in forensic RNA analysis whose versatility may very favorably complement traditional DNA profiling in forensic casework. There is, however, no method available specifically dedicated for extraction of RNA from forensically relevant sample material.
      In this study we compared five commercially available and commonly used RNA extraction kits and methods (mirVana™ miRNA Isolation Kit Ambion; Trizol® Reagent, Invitrogen; NucleoSpin® miRNA Kit Macherey-Nagel; AllPrep DNA/RNA Mini Kit and RNeasy® Mini Kit both Qiagen) to assess their relative effectiveness of yielding RNA of good quality and their compatibility with co-extraction of DNA amenable to STR profiling.
      We set up samples of small amounts of dried blood, liquid saliva, semen and buccal mucosa that were aged for different time intervals for co-extraction of RNA and DNA. RNA quality was assessed by determination of ‘RNA integrity number’ (RIN) and quantitative PCR based expression analysis. DNA quality was assessed via monitoring STR typing success rates.
      By comparison, the different methods exhibited considerable differences between RNA and DNA yields, RNA quality values and expression levels, and STR profiling success, with the AllPrep DNA/RNA Mini Kit and the NucleoSpin® miRNA Kit excelling at DNA co-extraction and RNA results, respectively.
      Overall, there was no ‘best’ method to satisfy all demands of comprehensible co-analysis of RNA and DNA and it appears that each method has specific merits and flaws. We recommend to cautiously choose from available methods and align its characteristics with the needs of the experimental setting at hand.


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        • Oehmichen M.
        • Zilles K.
        Postmortem DNA and RNA synthesis. Preliminary studies in human cadavers.
        Z. Rechtsmed. 1984; 91: 287-294
        • Phang T.W.
        • Shi C.Y.
        • Chia J.N.
        • Ong C.N.
        Amplification of cDNA via RT-PCR using RNA extracted from postmortem tissues.
        J. Forensic Sci. 1994; 39: 1275-1279
        • Heid C.A.
        • Stevens J.
        • Livak K.J.
        • Williams P.M.
        Real time quantitative PCR.
        Genome Res. 1996; 6: 986-994
        • Bauer M.
        • Patzelt D.
        Evaluation of mRNA markers for the identification of menstrual blood.
        J. Forensic Sci. 2002; 47: 1278-1282
        • Juusola J.
        • Ballantyne J.
        Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identification.
        Forensic Sci. Int. 2003; 135: 85-96
        • Juusola J.
        • Ballantyne J.
        Multiplex mRNA profiling for the identification of body fluids.
        Forensic Sci. Int. 2005; 152: 1-12
        • Juusola J.
        • Ballantyne J.
        mRNA profiling for body fluid identification by multiplex quantitative RT-PCR.
        J. Forensic Sci. 2007; 52: 1252-1262
        • Nussbaumer C.
        • Gharehbaghi-Schnell E.
        • Korschineck I.
        Messenger RNA profiling: a novel method for body fluid identification by real-time PCR.
        Forensic Sci. Int. 2006; 157: 181-186
        • Hanson E.K.
        • Lubenow H.
        • Ballantyne J.
        Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs.
        Anal. Biochem. 2009; 387: 303-314
        • Courts C.
        • Madea B.
        Micro-RNA–a potential for forensic science?.
        Forensic Sci. Int. 2010; 203: 106-111
        • Courts C.
        • Madea B.
        Specific micro-RNA signatures for the detection of saliva and blood in forensic body-fluid identification.
        J. Forensic Sci. 2011; 56: 1464-1470
        • Wang Z.
        • Luo H.
        • Pan X.
        • Liao M.
        • Hou Y.
        A model for data analysis of microRNA expression in forensic body fluid identification.
        Forensic Sci. Int. Genet. 2012; 6: 419-423
        • Wang Z.
        • Zhang J.
        • Luo H.
        • Ye Y.
        • Yan J.
        • Hou Y.
        Screening and confirmation of microRNA markers for forensic body fluid identification.
        Forensic Sci. Int. Genet. 2013; 7: 116-123
        • Haas C.
        • Hanson E.
        • Bar W.
        • Banemann R.
        • Bento A.M.
        • Berti A.
        • et al.
        mRNA profiling for the identification of blood-results of a collaborative EDNAP exercise.
        Forensic Sci. Int. Genet. 2010;
        • Haas C.
        • Hanson E.
        • Anjos M.J.
        • Bar W.
        • Banemann R.
        • Berti A.
        • et al.
        RNA/DNA co-analysis from blood stains–results of a second collaborative EDNAP exercise.
        Forensic Sci. Int. Genet. 2012; 6: 70-80
        • Haas C.
        • Hanson E.
        • Anjos M.J.
        • Banemann R.
        • Berti A.
        • Borges E.
        • et al.
        RNA/DNA co-analysis from human saliva and semen stains–results of a third collaborative EDNAP exercise.
        Forensic Sci. Int. Genet. 2013; 7: 230-239
        • Haas C.
        • Hanson E.
        • Anjos M.J.
        • Ballantyne K.N.
        • Banemann R.
        • Bhoelai B.
        • et al.
        RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: Results of a fourth and fifth collaborative EDNAP exercise.
        Forensic Sci. Int. Genet. 2014; 8: 203-212
        • van den Berge M.
        • Carracedo A.
        • Gomes I.
        • Graham E.A.
        • Haas C.
        • Hjort B.
        • et al.
        A collaborative European exercise on mRNA-based body fluid/skin typing and interpretation of DNA and RNA results.
        Forensic Sci. Int. Genet. 2014; 10: 40-48
        • Lindenbergh A.
        • Maaskant P.
        • Sijen T.
        Implementation of RNA profiling in forensic casework.
        Forensic Sci. Int. Genet. 2013; 7: 159-166
        • Sun J.H.
        • Wang Y.Y.
        • Zhang L.
        • Gao C.R.
        • Zhang L.Z.
        • Guo Z.
        Time-dependent expression of skeletal muscle troponin I mRNA in the contused skeletal muscle of rats: a possible marker for wound age estimation.
        Int. J. Legal Med. 2010; 124: 27-33
        • Sun J.H.
        • Nan L.H.
        • Gao C.R.
        • Wang Y.Y.
        Validation of reference genes for estimating wound age in contused rat skeletal muscle by quantitative real-time PCR.
        Int. J. Legal Med. 2012; 126: 113-120
        • Takamiya M.
        • Saigusa K.
        • Kumagai R.
        • Nakayashiki N.
        • Aoki Y.
        Studies on mRNA expression of tissue-type plasminogen activator in bruises for wound age estimation.
        Int. J. Legal Med. 2005; 119: 16-21
        • Bauer M.
        • Gramlich I.
        • Polzin S.
        • Patzelt D.
        Quantification of mRNA degradation as possible indicator of postmortem interval–a pilot study.
        Legal Med. (Tokyo). 2003; 5: 220-227
        • Kimura A.
        • Ishida Y.
        • Hayashi T.
        • Nosaka M.
        • Kondo T.
        Estimating time of death based on the biological clock.
        Int. J. Legal Med. 2011; 125: 385-391
        • Sampaio-Silva F.
        • Magalhaes T.
        • Carvalho F.
        • nis-Oliveira R.J.
        • Silvestre R.
        Profiling of RNA degradation for estimation of post morterm interval.
        PLoS One. 2013; 8: e56507
        • Wang Q.
        • Ishikawa T.
        • Michiue T.
        • Zhu B.L.
        • Guan D.W.
        • Maeda H.
        Intrapulmonary aquaporin-5 expression as a possible biomarker for discriminating smothering and choking from sudden cardiac death: a pilot study.
        Forensic Sci. Int. 2012; 220: 154-157
        • Zhao D.
        • Ishikawa T.
        • Quan L.
        • Li D.R.
        • Michiue T.
        • Yoshida C.
        • et al.
        Tissue-specific differences in mRNA quantification of glucose transporter 1 and vascular endothelial growth factor with special regard to death investigations of fatal injuries.
        Forensic Sci. Int. 2008; 177: 176-183
        • Steiner G.
        • Suter L.
        • Boess F.
        • Gasser R.
        • de Vera M.C.
        • Albertini S.
        • et al.
        Discriminating different classes of toxicants by transcript profiling.
        Environ. Health Perspect. 2004; 112: 1236-1248
        • Bartosiewicz M.J.
        • Jenkins D.
        • Penn S.
        • Emery J.
        • Buckpitt A.
        Unique gene expression patterns in liver and kidney associated with exposure to chemical toxicants.
        J. Pharmacol. Exp. Ther. 2001; 297: 895-905
        • Tarone A.M.
        • Foran D.R.
        Gene expression during blow fly development: improving the precision of age estimates in forensic entomology.
        J. Forensic Sci. 2011; 56: S112-S122
        • Boehme P.
        • Spahn P.
        • Amendt J.
        • Zehner R.
        Differential gene expression during metamorphosis: a promising approach for age estimation of forensically important Calliphora vicina pupae (Diptera: Calliphoridae).
        Int. J. Legal Med. 2013; 127: 243-249
        • Gauvin J.
        • Zubakov D.
        • van Rhee-Binkhorst J.
        • Kloosterman A.
        • Steegers E.
        • Kayser M.
        Forensic pregnancy diagnostics with placental mRNA markers.
        Int. J. Legal Med. 2010; 124: 13-17
        • Schroeder A.
        • Mueller O.
        • Stocker S.
        • Salowsky R.
        • Leiber M.
        • Gassmann M.
        • et al.
        The RIN: an RNA integrity number for assigning integrity values to RNA measurements.
        BMC. Mol. Biol. 2006; 7: 3
        • Peltier H.J.
        • Latham G.J.
        Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitable reference RNA targets in normal and cancerous human solid tissues.
        RNA. 2008; 14: 844-852
      1. L. Wong, K. Lee, I. Russell, C. Chen, Endogenous Controls for Real-Time Quantitation of miRNA Using TaqMan MicroRNA Assays, Application Note (Applied Biosystems) (2010) 1–8.

        • Griffiths-Jones S.
        The microRNA registry.
        Nucleic Acids Res. 2004; 32: D109-D111
        • Bustin S.A.
        • Benes V.
        • Garson J.A.
        • Hellemans J.
        • Huggett J.
        • Kubista M.
        • et al.
        The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.
        Clin. Chem. 2009; 55: 611-622
        • Bauer M.
        • Polzin S.
        • Patzelt D.
        Quantification of RNA degradation by semi-quantitative duplex and competitive RT-PCR: a possible indicator of the age of bloodstains?.
        Forensic Sci. Int. 2003; 138: 94-103
        • Alvarez M.
        • Juusola J.
        • Ballantyne J.
        An mRNA and DNA co-isolation method for forensic casework samples.
        Anal. Biochem. 2004; 335: 289-298
        • Setzer M.
        • Juusola J.
        • Ballantyne J.
        Recovery and stability of RNA in vaginal swabs and blood semen, and saliva stains.
        J. Forensic Sci. 2008; 53: 296-305
      2. R.I. Fleming, S. Harbison, The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids, 4th ed., 2010, p. 244–256.

        • Hanson E.
        • Haas C.
        • Jucker R.
        • Ballantyne J.
        Identification of skin in touch/contact forensic samples by messenger RNA profiling.
        Forensic Sci Int. Genet. Suppl. Ser. 2011; 3: e305-e306
        • Parker C.
        • Hanson E.
        • Ballantyne J.
        Optimization of dried stain co-extraction methods for efficient recovery of high quality DNA and RNA for forensic analysis.
        Forensic Sci. Int. Genet. Suppl. Ser. 2011; 3: e309-e310
        • Hanson E.K.
        • Ballantyne J.
        Highly specific mRNA biomarkers for the identification of vaginal secretions in sexual assault investigations.
        Sci. Justice. 2013; 53: 14-22
        • Gilad S.
        • Meiri E.
        • Yogev Y.
        • Benjamin S.
        • Lebanony D.
        • Yerushalmi N.
        • et al.
        Serum microRNAs are promising novel biomarkers.
        PLoS One. 2008; 3: e3148
        • Zhang H.
        • Zhang P.
        • Ma K.J.
        • Lv Y.H.
        • Li W.C.
        • Luo C.L.
        • et al.
        The selection of endogenous genes in human postmortem tissues.
        Sci. Justice. 2013; 53: 115-120
        • Bai P.
        • Deng W.
        • Wang L.
        • Long B.
        • Liu K.
        • Liang W.
        • et al.
        Micro RNA profiling for the detection and differentiation of body fluids in forensic stain analysis.
        Forensic Sci. Int. Genet. Suppl. Ser. 2013; 4: e216-e217
        • Qi B.
        • Kong L.
        • Lu Y.
        Gender-related difference in bloodstain RNA ratio stored under uncontrolled room conditions for 28 days.
        J. Forensic Legal Med. 2013; 20: 321-325
        • Hampson C.
        • Louhelainen J.
        • McColl S.
        An RNA expression method for aging forensic hair samples.
        J. Forensic Sci. 2011; 56: 359-365
        • Lv Y.H.
        • Ma K.J.
        • Zhang H.
        • He M.
        • Zhang P.
        • Shen Y.W.
        • et al.
        A time course study demonstrating mRNA, microRNA, 18S rRNA, and U6 snRNA changes to estimate PMI in deceased rat’s spleen.
        J. Forensic Sci. 2014;
        • Turchinovich A.
        • Weiz L.
        • Langheinz A.
        • Burwinkel B.
        Characterization of extracellular circulating microRNA.
        Nucleic Acid Res. 2011; 39: 7223-7233
        • Zubakov D.
        • Hanekamp E.
        • Kokshoorn M.
        • van I.W.
        • Kayser M.
        Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples.
        Int. J. Legal Med. 2008; 122: 135-142
        • Zubakov D.
        • Kokshoorn M.
        • Kloosterman A.
        • Kayser M.
        New markers for old stains: stable mRNA markers for blood and saliva identification from up to 16-year-old stains.
        Int. J. Legal Med. 2009; 123: 71-74
        • Weber M.A.
        • Hartley J.C.
        • Brooke I.
        • Lock P.E.
        • Klein N.J.
        • Malone M.
        • et al.
        Post-mortem interval and bacteriological culture yield in sudden unexpected death in infancy (SUDI).
        Forensic Sci. Int. 2010; 198: 121-125
        • Coult N.
        • Uchimoto M.L.
        • Williams G.
        Forensic microRNA (miRNA) analysis of skin specific markers.
        in: Forensic Science Society Inaugrural Postgraduate Research Symposium, Coventry, UK2012
        • Sakurada K.
        • Akutsu T.
        • Watanabe K.
        • Yoshino M.
        Identification of nasal blood by real-time RT-PCR.
        Legal Med. (Tokyo). 2012; 14: 201-204
        • Omelia E.J.
        • Uchimoto M.L.
        • Williams G.
        Quantitative PCR analysis of blood- and saliva-specific microRNA markers following solid-phase DNA extraction.
        Anal. Biochem. 2013; 435: 120-122
        • Uchimoto M.L.
        • Beasley E.
        • Coult N.
        • Omelia E.J.
        • World D.
        • Williams G.
        Considering the effect of stem-loop reverse transcription and real-time PCR analysis of blood and saliva specific microRNA markers upon mixed body fluid stains.
        Forensic Sci. Int. Genet. 2013; 7: 418-421
        • Visser M.
        • Zubakov D.
        • Ballantyne K.N.
        • Kayser M.
        mRNA-based skin identification for forensic applications.
        Int. J. Legal Med. 2011; 125: 253-263
        • Blondal T.
        • Jensby N.S.
        • Baker A.
        • Andreasen D.
        • Mouritzen P.
        • Wrang T.M.
        • et al.
        Assessing sample and miRNA profile quality in serum and plasma or other biofluids.
        Methods. 2013; 59: S1-S6
        • van der Meer D.
        • Uchimoto M.L.
        • Williams G.
        Simultaneous analysis of micro-RNA and DNA for determining the body fluid origin of DNA profiles.
        J. Forensic Sci. 2013;
        • Nakao K.-I.
        • Shimada R.
        • Hara K.
        • Kibayahi K.
        Experimental study on age estimation of bloodstains based on biological and toxicological analysis.
        Open Forensic Sci. J. 2013; : 6-11
        • Lindenbergh A.
        • de P.M.
        • Ramdayal G.
        • Visser M.
        • Zubakov D.
        • Kayser M.
        • et al.
        A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces.
        Forensic Sci. Int. Genet. 2012; 6: 565-577
        • Courts C.
        • Grabmuller M.
        • Madea B.
        Dysregulation of heart and brain specifc micro-RNA in sudden infant death syndrome.
        Forensic Sci. Int. 2013; 228: 70-74
        • Odriozola A.
        • Riancho J.A.
        • de la Vega R.
        • Agudo G.
        • Garcia-Blanco A.
        • de C.E.
        • et al.
        miRNA analysis in vitreous humor to determine the time of death: a proof-of-concept pilot study.
        Int. J. Legal Med. 2013; 127: 573-578
        • Sauer E.
        • Madea B.
        • Courts C.
        An evidence based strategy for normalization of quantitative PCR data from miRNA expression analysis in forensically relevant body fluids.
        Forensic Sci. Int. Genet. 2014; 11C: 174-181
        • Lux C.
        • Schyma C.
        • Madea B.
        • Courts C.
        Identification of gunshots to the head by detection of RNA in backspatter primarily expressed in brain tissue.
        Forensic Sci. Int. 2014; 237: 62-69
        • Petersen C.
        • Hjort B.
        • Tvedebrink T.
        • Kielpinski L.
        • Vinther J.
        • Morling N.
        Body fluid identification of blood, saliva and semen using second generation sequencing of micro-RNA.
        Forensic Sci. Int. Genet. Suppl. Ser. 2014; 4: e204-e205
        • Lindenbergh A.
        • van den B.M.
        • Oostra R.J.
        • Cleypool C.
        • Bruggink A.
        • Kloosterman A.
        • et al.
        Development of a mRNA profiling multiplex for the inference of organ tissues.
        Int. J. Legal Med. 2013; 127: 891-900
        • Kohlmeier F.
        • Schneider P.M.
        Successful mRNA profiling of 23 years old blood stains.
        Forensic Sci. Int. Genet. 2012; 6: 274-276
        • Watanabe K.
        • Iwashima Y.
        • Akutsu T.
        • Sekiguchi K.
        • Sakurada K.
        Evaluation of a co-extraction method for real-time PCR-based body fluid identification and DNA typing.
        Legal Med. (Tokyo). 2014; 16: 56-59
        • Roeder A.D.
        • Haas C.
        mRNA profiling using a minimum of five mRNA markers per body fluid and a novel scoring method for body fluid identification.
        Int. J. Legal Med. 2013; 127: 707-721
        • Andreasson H.
        • Gyllensten U.
        • Allen M.
        Real-time DNA quantification of nuclear and mitochondrial DNA in forensic analysis.
        Biotechniques. 2002; 33: 402-411
        • Chomczynski P.
        • Sacchi N.
        Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
        Anal. Biochem. 1987; 162: 156-159
        • Bowden A.
        • Fleming R.
        • Harbison S.
        A method for DNA and RNA co-extraction for use on forensic samples using the Promega DNA IQ system.
        Forensic Sci. Int. Genet. 2011; 5: 64-68
        • Zubakov D.
        • Boersma A.W.
        • Choi Y.
        • van Kuijk P.F.
        • Wiemer E.A.
        • Kayser M.
        MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation.
        Int. J. Legal Med. 2010; 124: 217-226
        • Pena-Llopis S.
        • Brugarolas J.
        Simultaneous isolation of high-quality DNA RNA, miRNA and proteins from tissues for genomic applications.
        Nat. Protoc. 2013; 8: 2240-2255