An mRNA and DNA co-isolation method for forensic casework samples

ANALYTICALBIOCHEMISTRY

Analytical Biochemistry 335 (2004) 289–298

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An mRNA and DNA co-isolation method for forensic casework samples

Michelle Alvareza,c, Jane Juusolaa,c, Jack Ballantynea,b,c,¤

a Graduate Program in Biomolecular Science, University of Central Florida, P.O. Box 162366, Orlando, FL 32816, USAb Department of Chemistry, University of Central Florida, P.O. Box 162366, Orlando, FL 32816, USA

c National Center for Forensic Science, P.O. Box 162367, Orlando, FL 32816, USA

Received 4 August 2004Available online 28 October 2004

Abstract

RNA analysis is expected to play an increasingly important role in the area of biomolecular forensic analysis. For example,mRNA expression analysis performed on a total RNA sample isolated from a biological stain may be used to identify the nature ofthe tissue(s) comprising the stain. Many of the physiological stains encountered at crime scenes involve heterogeneous mixtures ofdiVerent body Xuids (e.g., semen and saliva, semen and vaginal secretions). Separate sampling of these mixed stains from diVerent“geographical” locations of the stains to isolate DNA and RNA could result in a misleading estimate of the ratio of the body Xuidspresent and, in extreme cases, even fail to detect one of the contributors. Thus, a prerequisite for the use of mRNA expression proWl-ing in routine forensic analysis is the ability to co-extract DNA and RNA from the same stain. This article describes an optimizedmethod that was speciWcally developed to co-extract mRNA and DNA from the same physiological stain and that appears to besuYciently sensitive and robust for routine forensic use. 2004 Elsevier Inc. All rights reserved.

Keywords: mRNA; DNA; Co-extraction; Body Xuid identiWcation

During the commission of violent crimes, biologicalmaterial (e.g., blood, semen, saliva, and hair) is oftentransferred between the victim, the perpetrator, and thecrime scene. Routinely, DNA is isolated from this shedphysiological material and is subjected to DNA analysisusing polymorphic nuclear DNA markers such as shorttandem repeats (STRs)1 or, in certain cases, mitochon-drial DNA. A number of robust standard DNA isola-tion methods that have been modiWed speciWcally forforensic use are employed in forensic laboratories world-

* Corresponding author. Fax: +1 407 823 2252.E-mail address: [email protected] (J. Ballantyne).

1 Abbreviations used: STR, short tandem repeat; DT, dithiothreitol;EDTA, ethylenediamine tetraacetic acid; SDS, sodium dodecyl sulfate;DEPC, diethyl pyrocarbonate; dNTP, deoxyribonucleoside triphos-phate; PCR, polymerase chain reaction; RT, reverse transcriptase;BSA, bovine serum albumin.

0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2004.09.002

wide [1–3]. It is apparent, however, that RNA will alsoplay an increasingly important role in the area of biomo-lecular forensic analysis. For example, mRNA expres-sion analysis performed on a total RNA sample isolatedfrom a biological stain may be used to identify thenature of the tissue(s) comprising the stain [4–8]. Manyof the physiological stains encountered at crime scenesinvolve heterogeneous mixtures of diVerent body Xuids(e.g., semen and saliva, semen and vaginal secretions).Separate sampling of these mixed stains from diVerent“geographical” locations of the stains to isolate DNAand RNA could result in a misleading estimate of theratio of the body Xuids present and, in extreme cases,even fail to detect one of the contributors. Thus, a pre-requisite to the use of mRNA expression proWling inroutine forensic analysis is the ability to co-extract DNAand RNA from the same stain.

mailto: [email protected]

mailto: [email protected]

290 M. Alvarez et al. / Analytical Biochemistry 335 (2004) 289–298

A number of methods that describe the simultaneousisolation of DNA and RNA from the same sample havebeen reported [9–16]. However, most of these have notbeen optimized to deal with the reduced quantity andcompromised quality of samples encountered in forensiccasework. Because we had limited success in attemptingto adapt these systems to forensic stains due to theinability to obtain reproducible analyte yields of suY-cient quantity and quality, we embarked on the develop-ment of a method that is more suitable for this purpose.This article describes an optimized method that was spe-ciWcally developed to co-extract RNA and DNA fromthe same physiological stain and that appears to be suY-ciently sensitive and robust for routine forensic use.

Materials and methods

Sample preparation

Body Xuids were collected from apparently healthyhuman volunteers in accordance with proceduresapproved by the university’s institutional review board.Liquid whole blood was collected by venipuncture, andblood stains were made by dispensing 50-�l aliquotsonto sterile cotton gauze. Diluted blood stains were pre-pared by appropriate dilution of whole blood in 1£ PBSbuVer (0.05 M, pH 7.4), and 50-�l aliquots subsequentlywere dispensed onto sterile swabs. Saliva (without priorfood consumption) and freshly ejaculated semen werecollected in sterile plastic cups and dispensed in 50-�laliquots onto sterile cotton gauze. For sensitivity testing,fresh liquid saliva and semen samples were diluted in 1£PBS buVer (0.05 M, pH 7.4), and 50-�l stains were dis-pensed onto sterile cotton swabs. Semen-free vaginalsecretions were obtained from volunteers who hadabstained from intercourse for 3 days by swabbing thevaginal cavity with sterile cotton swabs. All stains wereallowed to air-dry overnight at room temperature andwere stored at ¡45 °C until needed.

Samples for aging studies were prepared by deposit-ing preservative-free liquid blood or semen (50 �l) onsterile cotton gauze and stored at room temperature(20 °C) for varying times. Preservative-free blood wascollected from alcohol-swabbed Wngers using lancets,and stains were prepared as described and evaluated forthe presence of DNA and RNA after 3, 6, 9, and 14months. Semen stains were aged for 6, 9, and 19 monthsbefore testing for the presence of DNA and RNA.

Simultaneous DNA and RNA isolation

The sample (stain or swab) was placed into a Spin-Ease extraction tube (Gibco-BRL, Life Technologies,Gaithersburg, MD, USA) and incubated at 56 °C for 1 hin 500 �l of extraction solution [0.1 M NaCl, 10 mM

Tris–HCl (pH 8.0), 40 mM dithiothreitol (DTT), 10 mMethylenediamine tetraacetic acid (EDTA, pH 8.0),70 mM sodium dodecyl sulfate [SDS], 0.65 mg/ml pro-teinase K] prepared in nuclease-free water (Ambion,Austin, TX, USA). In the case of semen stains, an addi-tional 0.39 M DTT was included in the extraction solu-tion. The fabric was removed from the extraction buVerand placed into a Spin-Ease extraction tube Wlter insert,placed back inside the extraction tube, and centrifugedfor 5 min at 16,000g, after which the Wlter and the fabricremnants were discarded. Next, 50 �l of 2 M sodium ace-tate and 600 �l of acid phenol:chloroform (5:1, pH 4.5,Ambion) were added to the extract, vortexed vigorously,incubated at 4 °C until two phases were resolved (at least20 min), and centrifuged at 16,000g for 20 min. The aque-ous phase containing both DNA and RNA (»400 �l)was transferred to a sterile 1.5-ml microcentrifuge tubeand labeled “DNA,” and 200 �l of this aqueous phasewas transferred to another sterile 1.5-ml microcentrifugetube and labeled “RNA.”

DNA was precipitated for at least 1 h or overnight, at¡20 °C with 500 �l ice-cold 100% ethanol and then cen-trifuged at 16,000g for 15 min. The supernatant was care-fully removed and discarded, and the (invisible) pelletwas washed once with 1 ml 70% ethanol (room tempera-ture), centrifuged at 16,000g for 5 min, vacuum-dried (4–7 min), and resolubilized in 50–100 �l of TE¡4 buVer(10 mM Tris, 0.1 mM EDTA) at 56 °C for 45 min. DNAsamples were used immediately or stored at 4 °C untilneeded.

After the addition of 2 �l GlycoBlue glycogen carrier(Ambion), the RNA was precipitated by the addition of250 �l isopropanol for at least 1 h or overnight at ¡20 °Cand then centrifuged at 16,000g for 20 min. The superna-tant was carefully removed and discarded, and the pelletwas washed once with 1 ml 75% ethanol/25% diethylpyrocarbonate (DEPC)-treated water and then recentri-fuged at 16,000g for 10 min. The supernatant was dis-carded, and the pellet was dried in a vacuum centrifugefor 3–5 min. The RNA pellet was resolubilized in 12–20 �l of RNAsecure Resuspension Solution (Ambion) at60 °C for 10 min. RNA samples were used immediatelyor stored at ¡20 °C until needed.

Standard RNA isolation

A standard guanidine isothiocyanate–phenol:chloro-form extraction was performed [4,17]. BrieXy, 500 �ldenaturing solution (4 M guanidine isothiocyanate,0.02 M sodium citrate, 0.5% sarkosyl, 0.1 M �-mercap-toethanol) was preheated in a Spin-Ease extraction tubeat 56 °C for 10 min. The stain was then added and incu-bated at 56 °C for 30 min. Samples were processed asdescribed above for the RNA component of the simulta-neous isolation procedure with the following modiWca-tions. The Spin-Ease extraction tube Wlter insert

M. Alvarez et al. / Analytical Biochemistry 335 (2004) 289–298 291

containing the sample gauze was centrifuged at 8160gfor 10 min. After separation by centrifugation of theaqueous/organic phases at 16,000g for 20 min, the entireaqueous phase was transferred to a sterile microcentri-fuge tube and precipitated with 500 �l of isopropanol.

Standard DNA isolation

For the standard DNA isolation, an organic solventextraction method was performed [1]. BrieXy, sampleswere incubated overnight at 56 °C in stain extractionbuVer [0.1 M NaCl, 10 mM Tris–HCl (pH 8.0), 25 mMEDTA (pH 8.0), 20 mM SDS] supplemented with 0.5 mg/ml proteinase K and 0.39 M DTT (semen only). Anequal volume of phenol:chloroform:isoamyl alcohol(25:24:1, pH 6.6) was added to the extract, mixed gentlyby inversion, and centrifuged for 5 min at 16,000g to sep-arate the phases. The entire aqueous layer containing theDNA was transferred to a sterile 1.5-ml microcentrifugetube, precipitated with 1 ml of ice-cold 100% ethanol for1 h or overnight, centrifuged at 16,000g for 15 min,washed once with 1 ml 70% ethanol, and solubilized in100 �l TE¡4 buVer at 56 °C overnight.

DNase I digestion

Total RNA was treated with six units of TURBODNase (RNase-Free, 2 U/�l, Ambion) at 37 °C for 2–3 h.The TURBO DNase was inactivated at 75 °C for 10 min,and the samples were chilled on ice and then stored at¡20 °C until needed [18,19].

Quantitation of nucleic acids

RNA was quantiWed using a sensitive Xuorescenceassay based on the binding of the unsymmetrical cyaninedye RiboGreen (Molecular Probes, Eugene, OR, USA)[20]. The manufacturer’s instructions were followed forthe high-range assay, which detects from 20 ng/ml to1 �g/ml. BrieXy, 200-�l assays consisted of 2 �l TURBODNase-treated RNA extract, 98 �l TE buVer [10 mMTris–HCl (pH 7.5), 1 mM EDTA, in nuclease-free water],and 100 �l of 750 nM RiboGreen reagent in a 96-wellplate format. After the addition of RiboGreen and a 3-min incubation at room temperature protected fromlight, Xuorescence emission at 535 nm (excited at 485 nm)was determined using a Wallac Victor2 microplatereader (Perkin–Elmer, Boston, MA, USA). RNA con-centration was calculated using an appropriate standardcurve as described by the manufacturer [20].

DNA was electrophoresed in a 1% agarose gel inTAE buVer (0.04 M Tris–acetate, 0.001 M EDTA) at200 V for 15 min and stained using 1% ethidium bromide(Fisher ScientiWc, Foster City, CA, USA). Samples werevisualized on a shortwave UV transilluminator. Quanti-tation was accomplished by a comparison of the inten-

sity of the samples against known standards and wasphotographed under UV transillumination.

cDNA synthesis

RNA (80 ng) was heated at 75 °C for 3 min (for sam-ples containing less than 80 ng total RNA, the entireextract was added to the reaction). To the RNA, 4 �l of a10-mM deoxyribonucleoside triphosphate (dNTP) mix(Applied Biosystems, Foster City, CA, USA), 2�l of 10£Wrst-strand buVer [500 mM Tris–HCl (pH 8.3), 750 mMKCl, 30 mM MgCl2, 50 mM DTT], 2 �l of RandomDecamer primers (50 �M) (Ambion), 1 �l of SUPERase-In RNase Inhibitor (20 U/�l, Ambion), 1 �l of MoloneyMurine Leukemia Virus–Reverse Transcriptase (100 U/�l, Ambion), and nuclease-free water (Ambion) wereadded to yield a Wnal reaction volume of 20 �l. The reac-tion mixture was incubated at 42 °C for 1 h and at 95 °Cfor 10 min to inactivate the reverse transcriptase (RT)[4,21].

Polymerase chain reaction ampliWcation

DNA from blood, saliva, semen (1–10 ng), and vagi-nal secretions (45 ng) or cDNA from blood, saliva,semen (2–6 ng), and vaginal secretions (66 ng) was ampli-Wed in a total reaction volume of 25 �l. The reaction mixcontained 1£ polymerase chain reaction (PCR) buVer[10 mM Tris–HCl (pH 8.3), 50 mM KCl], 1.5 mM MgCl2,0.125 mM each dNTP, 0.8 �M primers, and 1.25 U Amp-liTaq Gold DNA polymerase (5 U/�l, Roche MolecularSystems, Branchburg, NJ, USA). Primer sequences wereas follows: for �-spectrin (blood), F 5�-AGG-ATG-GCT-TGG-CCT-TTA-AT-3� and R 5�-ACT-GCC-AGC-ACC-TTC-ATC-TT-3�; for histatin 3 (saliva), F5�-GCA-AAG-AGA-CAT-CAT-GGG-TA-3� and R 5�-GCC-AGT-CAA-ACC-TCC-ATA-ATC-3� [4]; forprotamine 1 (semen), F 5�-GTC-CGA-TAC-CGC-GTG-AGG-AGC-CTG-3� and R 5�-GCC-TTC-TGC-ATG-TTC-TCT-TCC-TGG-3� [6]; and for mucin 4(vaginal secretions), F 5�-GGA-CCA-CAT-TTT-ATC-AGG-AA-3� and R 5�-TAG-AGA-AAC-AGG-GCA-TAG-GA-3�. PCR conditions consisted of a 5-min dena-turing step at 95 °C followed by 35 cycles (94 °C for 20 s,55 °C for 30 s, and 72 °C for 40 s) and a Wnal extensionstep (72 °C for 5 min) [4,21–23].

Post-PCR electrophoresis

PCR and RT-PCR ampliWed products were visual-ized on 2.5% NuSieve GTG agarose gels (Cambrex BioScience Rockland, Rockland, ME, USA). Electrophore-sis was carried out at 100 V for 60 min in TAE buVer(0.04 M Tris–acetate, 0.001 M EDTA). Gels were stainedwith SYBR Gold nucleic acid stain (Molecular Probes),visualized on the Omega10 Chemiluminescence Imaging


System (LTRA-LUM, Claremont, CA, USA), and ana-lyzed with ONE-Dscan 2.05 one-dimensional gel analy-sis software for Windows (Scanalytics, Fairfax, VA,USA).

STR analysis

An autosomal STR multiplex system was used toamplify 9 STR loci in a 25-�l reaction volume consistingof 2 ng template DNA, 1£ PCR buVer [10 mM Tris–HCl(pH 8.3), 50 mM KCl], 1.5 mM MgCl2, 10 �g bovineserum albumin (BSA, nonacetylated, Sigma–Aldrich, St.Louis, MO, USA), 0.25 mM each dNTP, 2.5 U Amp-liTaq Gold DNA polymerase (5 U/�l, Roche MolecularSystems) with primer sequences [24] at the followingconcentrations: D5S818 (0.175 �M), D13S317 (0.12 �M),D7S820 (0.2375�M), D16S539 (0.1875 �M), vWA(0.36 �M), TH01 (0.36 �M), AMELOGENIN(0.225 �M), TPOX (0.3725 �M), and CSF1PO (0.36 �M).PCR conditions were as follows: denaturing at 95 °C for11 min and at 96 °C for 1 min, followed by 10 cycles(ramp 100% to 94 °C for 30 s, ramp 29% to 60 °C for 30 s,ramp 23% to 70 °C for 45 s), 20 cycles (ramp 100% to90 °C for 30 s, ramp 29% to 60 °C for 30 s, ramp 23% to70 °C for 45 s), and Wnal extension 60 °C for 30 min.AmpliWed DNA product (0.75 �l) was combined with12 �l of deionized formamide (Amresco, Solon, OH,USA) and 0.5 �l of Xuorescent ladder (CXR, Promega,Madison, WI, USA), heated at 95 °C for 3 min, andsnap-cooled on ice for 3 min. Samples were detected with

the ABI Prism 310 Genetic Analyzer capillary electro-phoresis system (Applied Biosystems) and analyzed withGeneScan analysis software (version 2.1) using ModuleGS STR POP4 (1 ml) A (Applied Biosystems).

Results

Description of the method

The method described herein (Fig. 1) employs a stan-dard extraction buVer that is commonly used by forensiclaboratories during the initial stages of DNA puriWca-tion to release both RNA and DNA into solution by celllysis [1]. A subsequent acid phenol:chloroform extrac-tion, used in traditional RNA isolation methodologies[17], deproteinizes the sample and eVectively partitionsboth nucleic acids into the aqueous phase. In this appli-cation, the standard extraction buVer incorporates apotent RNase inhibitor to protect the RNA from degra-dation by contaminating ribonucleases. After samplesplitting, the DNA and RNA are precipitated and resus-pended in EDTA-containing and ribonuclease-freebuVers, respectively (Fig. 1).

Co-isolation of high-quality RNA and DNA from body Xuid stains

To ascertain whether the simultaneous nucleic acidextraction procedure was capable of isolating DNA and

Fig. 1. Summary of the simultaneous DNA and RNA co-extraction method for body Xuid stains.


RNA of suYcient quality and quantity for analysis fromforensic specimens, stains from the most commonlyencountered body Xuids in forensic casework were pre-pared and processed by this method. SpeciWcally, DNAand RNA were simultaneously isolated from dried vagi-nal secretion swabs and 50-�l-sized blood, saliva, andsemen stains. The RNA and DNA were tested usingappropriately designed RT-PCR and PCR assays,respectively, for the presence of genes that are expressedin a body Xuid-speciWc manner. �-Spectrin, histatin 3,protamine 1, and mucin 4 comprised the blood-, saliva-,semen-, and vaginal secretion-speciWc genes, respectively.The RT-PCR and PCR assays for each body Xuid-spe-ciWc gene were designed to use the same set of primersthat possessed binding sites in separate exons to permitdiscrimination between RNA and DNA amplicons fromthe same gene. Care was also taken to ensure that thegenes used did not possess processed pseudogenes orintronless paralogs that could confound the RNA/DNAamplimer discrimination [4].

The gel electrophoresis results (Fig. 2A) indicatedthat the co-isolation method produced an eYcient sepa-ration of DNA and RNA, as demonstrated by the lackof cross-contamination in the RNA (by DNA) andDNA (by RNA) extracts. The amplimer sizes were thoseexpected for the DNA and RNA products of all thegenes tested. It was important to demonstrate that thequality of the RNA and DNA isolated by the simulta-neous method was of suYcient quality for analysis. ThemRNA component of the total RNA isolated from allfour body Xuid stains yielded single gene-speciWc prod-ucts of the expected sizes after RT-PCR (Fig. 2A). TheDNA was subjected to genetic analysis using a set ofeight standard autosomal STRs plus the gender-speciWclocus amelogenin in a multiplex format [24]. The result-ing electropherograms (Fig. 2B) indicate that the DNAwas of high quality and yielded complete, high-qualityproWles that were consistent with the known DNA pro-Wles of the donors of the body Xuid stains.

Sensitivity

Because many specimens recovered from crime scenesfor forensic analysis are limited in size, it is important toevaluate the sensitivity of any new DNA or RNA analy-sis method. The sensitivity of the simultaneous DNA/RNA extraction method was tested using typically sizedblood, saliva, semen, and vaginal secretion stains.

A typical liquid blood drop volume is approximately50 �l, but this may be reduced signiWcantly in blood spat-ter as a result of low- or high-velocity impact on liquidblood by an object or a person [25]. RNA and DNAfrom a series of dried blood stains, which contained theequivalents of 50, 25, 12.5, 6.25, 3.1, and 1.6 �l of wholeblood from a single individual, were isolated by thesimultaneous extraction procedure and subjected to test-

ing using appropriately designed RT-PCR (for mRNA)and PCR (for DNA) assays for the presence of theblood-speciWc gene �-spectrin. �-Spectrin mRNA wasdetectable with as little as 3.1 �l of blood, whereas the�-spectrin DNA was detectable at the lowest volumeevaluated (1.6 �l) (Fig. 3A) and yielded complete, high-quality STR proWles (Supplementary Fig. 1A).

An analysis of dried diluted saliva and semen stainsproduced similar results in that the body Xuid-speciWcmRNA and DNA (histatin 3 for saliva and protamine 1for semen) were detectable with the lowest volumestested (1.6 and 0.2 �l for saliva and semen, respectively)(Figs. 3B and C), whereas the corresponding DNA fromthe same samples yielded complete, high-quality STRproWles (Supplementary Figs. 1B and C).

Vaginal secretions were tested by subjecting semen-free vaginal swabs, or portions thereof, to a similar RNAand DNA analysis using the vaginal Xuid-speciWc genemucin 4. With the lowest amount tested, namely one-fourth of a swab, gene-speciWc mRNA and DNA wasdetected (Fig. 3D) and a high-quality STR proWle wasobtained from the latter (Supplementary Fig. 1D).

Aged samples

Because biological stains are often recovered fromcrime scenes weeks or months after deposition, it wasimportant to evaluate the eYcacy of the co-isolationmethod using aged samples. Blood and semen stains(50�l) were stored at room temperature (»20 °C) pro-tected from light for varying time periods, ranging from12 weeks to 19 months, prior to being processed bythe simultaneous DNA and RNA extraction method(Fig. 4).

Body Xuid-speciWc mRNA and DNA (�-spectrin forblood and protamine 1 for semen) was detectable withthe oldest samples tested, 14-month blood stains (Fig.4A) and 19-month semen stains (Fig. 4B), with the DNAsubsequently yielding complete and accurate STR pro-Wles (Supplementary Figs. 2A and B).

Comparison with standard DNA and RNA isolation methods

The above results demonstrate that the simultaneousextraction method can isolate nucleic acids of suYcientquality and quantity for use in identifying both the tissuesource of an unknown body Xuid stain (mRNA) and thegenetic proWle of the individual from whom the bodyXuid originated (DNA). Next, we compared the recoveryeYciencies of the new method directly with standardDNA and RNA extraction procedures.

Blood, saliva, and semen stains (50 �l) were subjectedto RNA and DNA isolation procedures using the simul-taneous method presented herein, a guanidine isothiocy-anate–phenol:chloroform extraction for RNA isolation


secretion stains using the simultaneous nucleic acid extraction procedure.

Fig. 2. (A) Isolation of DNA and RNA from body Xuid stains using the co-extraction method. Blood, saliva, and semen stains (50�l) and singlevaginal swabs were processed for both DNA and RNA by the simultaneous isolation procedure. The extracts were analyzed by PCR (DNA) andRT-PCR (RNA) (using the same gene-speciWc primer sets) for genes that have a restricted pattern of gene expression. �-Spectrin, histatin 3, prot-amine 1, and mucin 4 were examined in blood, saliva, semen, and vaginal secretions, respectively. Products were visualized on 2.5% agarose gelsstained with SYBR Gold nucleic acid stain. D, DNA product; R, RNA product; L, 100-bp DNA ladder with sizes (bp) indicated. An asterisk (*) indi-cates the presence of an amplimer that is diYcult to visualize. (B) STR proWles from DNA (2 ng) isolated from blood, saliva, semen, and vaginal


Fig. 3. Sensitivity of the co-extraction method. Various-sized blood (A), saliva (B), and semen (C) stains, and portions of vaginal swabs (D), were pro-cessed for both DNA and RNA by the simultaneous isolation procedure. The extracts were analyzed by PCR (DNA) and RT-PCR (RNA) (usingthe same gene-speciWc primer sets) for genes that have a restricted pattern of gene expression. �-Spectrin, histatin 3, protamine 1, and mucin 4 wereexamined in blood, saliva, semen, and vaginal secretions, respectively. Products were visualized on 2.5% agarose gels stained with SYBR Gold nucleicacid stain. Each sample pair shows the DNA (D) and RNA (R) products in adjacent lanes and the stain volumes (or swab proportions) tested. L, 100-bp DNA ladder with sizes (bp) indicated. An asterisk (¤) indicates the presence of an amplimer that is diYcult to visualize.


[4,17], and an organic solvent extraction method to iso-late DNA [1]. Results of a comparison of the total recov-ered RNA and DNA using the diVerent methods aresummarized in Fig. 5, which displays the mean recover-ies from four separate extractions. In general, the recov-ery of DNA and RNA from the simultaneous extractionmethod compared favorably with the recoveriesobtained by standard methods.

DNA recovery from saliva and semen stains using thesimultaneous extraction protocol was comparable tothat obtained by the standard method, whereas therecovery from blood stains was reduced by approxi-mately two-thirds (Fig. 5A). Despite the reduced recov-ery of DNA from blood using the co-extraction method(»100 vs. »300 ng), this would be more than suYcientfor genetic analysis, which typically requires approxi-mately 1–2 ng for DNA proWling using autosomal STRmarkers. RNA recovery from blood and saliva with theco-extraction method was similar to that with the stan-dard method, whereas the recovery of RNA from semenwas signiWcantly improved (P D 0.007) (Fig. 5B).

Discussion

For mRNA methods for body Xuid identiWcation togain widespread acceptance in forensic genetics case-work, RNA isolation methodologies that are compatiblewith current DNA typing methodologies, and conse-quently do not compromise the ability to obtain DNAproWles from a variety of degraded and low-copy numbertemplates, need to be adopted. Ideally, RNA and DNAshould also be co-extracted from the same commonaqueous extract obtained from the dried body Xuid stainrather than from separate samplings of diVerent “geo-graphical” locations from the stain. To accomplish thesegoals, we evaluated a number of published simultaneousDNA and RNA isolation methods [9–16]. In attemptingto adapt these methods for forensic use, we found themto be nonoptimal with respect to isolation eYciency and,consequently, with respect to the ability to yield DNAand RNA of suYcient quality and quantity for analysisfrom dried physiological stains of low quantity and sam-ple integrity. Thus, we embarked on the development of a

Fig. 4. Co-extraction of DNA and RNA from aged blood and semen stains. Blood and semen stains (50 �l) deposited at various times prior to analy-sis were processed for both DNA and RNA by the simultaneous isolation procedure. The extracts were analyzed by PCR (DNA) and RT-PCR(RNA) (using the same gene-speciWc primer sets) for blood- (�-spectrin-) and semen- (protamine 1-) speciWc genes. Products were visualized on 2.5%agarose gels stained with SYBR Gold nucleic acid stain. D, DNA product; R, RNA product; L, 100-bp DNA ladder with sizes (bp) indicated. (A)Blood stains that are 3, 6, 9, and 14 months old. (B) Semen stains that are 6, 9, and 19 months old.


method more suitable for forensic samples. The resultwas the development of an optimized method that spe-ciWcally co-extracts mRNA and DNA from the samephysiological stain and appears to be suYciently sensitiveand robust for routine casework use.

Acknowledgments

The authors thank Ashley Hall for providing thereagents, formulations, and protocols for the multiplexautosomal STR analysis. The project was funded by theState of Florida through its contribution to the UCFCenter for Forensic Science.

Appendix A. Supplementary data

Supplementary data associated with this article canbe found, in the online version, at doi:10.1016/j.ab.2004.09.002.

Fig. 5. Recovery of DNA and RNA from blood, saliva, and semenstains. Comparison of the yields of DNA (A) and RNA (B) isolatedfrom 50 �l-sized blood, saliva, and semen stains using the DNA andRNA simultaneous isolation method with standard DNA [1] and RNAperformed [4,17] extraction procedures. The vertical error bars repre-sent the variations in yield quantity observed (length of bar D 1 SD).

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