THE RELATIVE RECOVERABILITY OF DNA AND RNA PROFILES FROM FORENSICALLY RELEVANT BODY FLUID STAINS. CHARLY PARKER B.S. Berry College, 2008

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1 THE RELATIVE RECOVERABILITY OF AND RNA PROFILES FROM FORENSICALLY RELEVANT BODY FLUID STAINS by CHARLY PARKER B.S. Berry College, 2008 A thesis submitted in partial fulfillment of the requirements for the degree of Master of Forensic Science in the Department of Chemistry in the College of Sciences at the University of Central Florida Orlando, Florida Spring Term 2011

2 2011 Charly Parker ii

3 ABSTRACT Biological material (fluids or tissues) whether from the victim or suspect is often collected as forensic evidence, and methods to obtain and analyze the found in that material have been well established. The type of body fluid (i.e. blood, saliva, semen, vaginal secretions, and menstrual blood) from which the originated is also of interest, and messenger RNA typing provides a specific and sensitive means of body fluid identification. In order for mrna profiling to be utilized in routine forensic casework, RNA of sufficient quantity and quality must be obtained from biological fluid stains and the methods used for RNA analysis must be fully compatible with current analysis methodologies. Several /RNA co-extraction methods were evaluated based on the quantity and quality of and RNA recovered and were also compared to standard non-co-extraction methods. The two most promising methods, the in-house developed NCFS co-extraction and the commercially available AllPrep /RNA Mini kit, were then optimized by improving nucleic acid recovery and consistency of CE (capillary electrophoresis) detection results. The sensitivity of the two methods was also evaluated, and and RNA profiles could be obtained for the lowest amount of blood (0.2 µl) and saliva and semen (1 µl) tested. Both extraction methods were found to be acceptable for use with forensic samples, and the ability to obtain full profiles was not hindered by the co-extraction of RNA. It is generally believed that RNA is less stable than which may prevent its use in forensic casework. However, the degradation rates of and RNA in the same biological fluid stain have not been directly compared. To determine the relative stability of and RNA, the optimized NCFS co-extraction protocol was used to isolate and RNA from iii

4 environmentally compromised stains. Dried blood, saliva, and semen stains and vaginal secretions swabs were incubated at set temperatures and outside for up to 1 year. Even at 56 C, and RNA were both stable out to 1 year in the blood and semen stains, out to 3 months () and 1 year (RNA) in the saliva stains, and out to 6 months () and 3 months (RNA) in the vaginal secretions swabs. The recoverability of both nucleic acids was reduced when the samples were exposed to increased humidity, sunlight, and rain. In general, and RNA stability was found to be similar with a loss in ability to obtain a or RNA profile occurring at the same time point; however, there were instances where RNA body fluid markers were detected when a poor/no profile was obtained, indicating that RNA in dried stains is sufficiently stable for mrna body fluid typing to be used in forensic casework. iv

5 To my mother and late father. Thank you for your constant love and support. v

6 ACKNOWLEDGMENTS I would first and foremost like to thank Dr. Jack Ballantyne for giving me the opportunity to be a part of his laboratory and learn more than I could have hoped. I feel lucky to have worked with someone who has as much expertise and knowledge as you do. I would also like to thank Dr. Erin Hanson for all of her guidance and assistance during the course of this project. To my other committee members, Dr. Jingdong Ye and Dr. Dmitry Kolpashchikov, thank you for taking the time to be a part of this process. Additionally, I would like to acknowledge those who donated one or more body fluids for this project. vi

7 TABLE OF CONTENTS LIST OF FIGURES... ix LIST OF TABLES... xiii LIST OF ACRONYMS/ABBREVIATIONS... xv CHAPTER ONE: INTRODUCTION... 1 CHAPTER TWO: METHODOLOGY... 6 Sample Preparation... 6 Degradation Samples... 6 Standard RNA Extractions... 7 Standard Organic RNA Extraction... 7 RNeasy Micro Kit... 8 Standard Extractions... 9 Standard Organic Extraction... 9 Investigator Kit /RNA Co-Extraction Methods AllPrep /RNA Mini kit ToTALLY RNA Kit/ Back Extraction TRIzol Extraction/ Back Extraction Chaos Buffer/Spin Columns NCFS Co-extraction DNase I Digestion Quantitation of Isolated and RNA c Synthesis Polymerase Chain Reaction Amplification Detection of Amplified Products Post-PCR Purification Optimization of Co-extraction Methods Testing of Optimized Protocols and RNA Stability CHAPTER THREE: RESULTS (CO-EXTRACTION) Evaluation of Co-extraction Methods vii

8 Standard and RNA Extractions Co-extraction Methods Optimization of Extraction Protocols RNeasy Micro Kit AllPrep /RNA Mini Kit NCFS Co-extraction RNA Detection Testing of Optimized Protocols Sensitivity CHAPTER FOUR: RESULTS ( AND RNA STABILITY) Indoor Samples Room Temperature C C Substrates Carpet Denim Environmental Samples Outside Covered and Uncovered Other Conditions CHAPTER FIVE: DISCUSSION /RNA Co-extraction and RNA Stability CHAPTER SIX: CONCLUSION APPENDIX A: FIGURES APPENDIX B: TABLES REFERENCES viii

9 LIST OF FIGURES Figure 1: Evaluation of and RNA Recovery Using the Five Co-extraction Methods and Standard and RNA Extraction Methods Figure 2: RNA Detection Results for Blood and Saliva Samples Extracted Using the Five Coextraction Methods Figure 3: Profiles Obtained from Blood Samples Using the Five Co-extraction Methods. 69 Figure 4: Profiles Obtained from Saliva Samples Using the Five Co-extraction Methods 70 Figure 5: Effect of Lysis Incubation Time and Temperature on RNA Recovery Figure 6: Comparison of and RNA Recovery Using Standard and Optimized Conditions with the AllPrep /RNA Mini Kit Figure 7: Comparison of and RNA Recovery Using Standard and Optimized Conditions with the NCFS Co-extraction Figure 8: RNA Detection Results Using the Optimized Protocols of the NCFS Co-extraction and AllPrep /RNA Mini Extraction Figure 9: Profiles Obtained Using the Optimized NCFS Co-extraction and AllPrep /RNA Mini Protocols Figure 10: Comparison of and RNA Recovery Using the Optimized NCFS Co-extraction and AllPrep Extractions Figure 11: Profiling Success Rate of Optimized Protocols Figure 12: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Blood (RNA) Figure 13: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Saliva (RNA) Figure 14: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Semen (RNA) ix

10 Figure 15: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Blood () Figure 16: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Saliva () Figure 17: Sensitivity of the Optimized AllPrep and NCFS Co-extractions: Semen () Figure 18: and RNA Recovery from Blood Stains (Swab) Exposed to Various Temperatures Figure 19: and RNA Recovery from Saliva Stains (Swab) Exposed to Various Temperatures Figure 20: and RNA Recovery from Semen Stains (Swab) Exposed to Various Temperatures Figure 21: and RNA Recovery from Vaginal Secretion Swabs Exposed to Various Temperatures Figure 22: and RNA Stability in Blood Stains (Swab) Incubated at 37 C and 56 C Figure 23: and RNA Stability in Saliva Stains (Swab) Incubated at 37 C and 56 C Figure 24: and RNA Stability in Semen Stains (Swab) Incubated at 56 C Figure 25: and RNA Stability in Vaginal Secretion Swabs Incubated at 37 C and 56 C.. 91 Figure 26: and RNA Stability in Blood Stains Made on Different Substrates, 37 C Figure 27: and RNA Stability in Blood Stains Made on Different Substrates, 56 C Figure 28: and RNA Profiles (Blood Stains on Different Substrates) Figure 29: and RNA Stability in Saliva Stains Made on Different Substrates, 37 C Figure 30: and RNA Stability in Saliva Stains Made on Different Substrates, 56 C Figure 31: and RNA Profiles (Saliva Stains on Different Substrates) Figure 32: and RNA Stability in Semen Stains Made on Different Substrates, 37 C x

11 Figure 33: and RNA Stability in Semen Stains Made on Different Substrates, 56 C Figure 34: and RNA Profiles (Semen Stains on Different Substrates) Figure 35: Recovery of and RNA from Blood Stains (Outside) Figure 36: Average HBB and ALAS2 Peak Heights: Blood (Outside) Figure 37: Average Allele Peak Heights: Blood (Outside) Figure 38: and RNA Stability in Blood Stains Exposed to Different Environmental Conditions Figure 39: and RNA Profiles Obtained from Blood Stains Exposed to the Environment (Outside Covered) Figure 40: and RNA Profiles Obtained from Blood Stains Exposed to the Environment (Outside Uncovered) Figure 41: Recovery of and RNA from Saliva Stains (Outside) Figure 42: Average HTN3 Peak Height: Saliva (Outside) Figure 43: Average Allele Peak Heights: Saliva (Outside) Figure 44: and RNA Stability in Saliva Stains Exposed to Different Environmental Conditions Figure 45: and RNA Profiles Obtained from Saliva Stains Exposed to the Environment (Outside Covered) Figure 46: and RNA Profiles Obtained from Saliva Stains Exposed to the Environment (Outside Uncovered) Figure 47: Recovery of and RNA from Semen Stains (Outside) Figure 48: Average PRM2 and TGM4 Peak Heights: Semen (Outside) xi

12 Figure 49: Average Allele Peak Heights: Semen (Outside) Figure 50: and RNA Stability in Semen Stains Exposed to Different Environmental Conditions Figure 51: and RNA Profiles Obtained from Semen Stains Exposed to the Environment (Outside Covered) Figure 52: and RNA Profiles Obtained from Semen Stains Exposed to the Environment (Outside Uncovered) Figure 53: Recovery of and RNA from Vaginal Secretion Swabs (Outside) Figure 54: Average MUC4 Peak Heights: Vaginal Secretion (Outside) Figure 55: Allele Peak Heights: Vaginal Secretions (Outside) Figure 56: and RNA Stability in Vaginal Secretion Swabs Exposed to Different Environmental Conditions Figure 57: and RNA Profiles Obtained from Vaginal Secretion Swabs Exposed to the Environment (Outside Covered) Figure 58: and RNA Profiles Obtained from Vaginal Secretion Swabs Exposed to the Environment (Outside Uncovered) Figure 59: and RNA Recovery from Blood Stains (Shade, Sun, Patio) Figure 60: and RNA Recovery from Saliva Stains (Shade, Sun, Patio) Figure 61: and RNA Recovery from Semen Stains (Shade, Sun, Patio) Figure 62: and RNA Stability in Blood Stains (Shade, Sun, Patio) Figure 63: and RNA Stability in Saliva Stains (Shade, Sun, Patio) Figure 64: and RNA Stability in Semen Stains (Shade, Sun, Patio) xii

13 LIST OF TABLES Table 1: Co-extraction Methods Evaluated Table 2: Body Fluid Markers Table 3: Co-extraction Methods Evaluation: and RNA Detection Results Table 4: RNA Results Obtained Using Optimized and Standard Co-extraction Protocols Table 5: Sensitivity of the Optimized AllPrep and Nucleospin Extractions Using Whole, 1/2, and 1/4 Stains or Swabs Table 6: Sensitivity of the Optimized AllPrep and Nucleospin Co-extraction Methods: Stains 147 Table 7: and RNA Stability in Blood Stains (Swab), 37 C Table 8: and RNA Stability in Blood Stains (Swab), 56 C Table 9: and RNA Stability in Saliva Stains (Swab), 37 C Table 10: and RNA Stability in Saliva Stains (Swab), 56 C Table 11: and RNA Stability in Semen Stains (Swab), 56 C Table 12: and RNA Stability in Vaginal Secretion Swabs, 37 C Table 13: and RNA Stability in Vaginal Secretion Swabs, 56 C Table 14: Substrate s Effect on and RNA Stability in Blood Stains, 37 C Table 15: Substrate s Effect on and RNA Stability in Blood Stains, 56 C Table 16: Substrate s Effect on and RNA Stability in Semen Stains, 56 C Table 17: and RNA Stability in Blood Stains (Outside Covered) Table 18: and RNA Stability in Blood Stains (Outside Uncovered) Table 19: Average Allele Peak Heights (Blood) Table 20: and RNA Stability in Saliva Stains (Outside Covered) xiii

14 Table 21: and RNA Stability in Saliva Stains (Outside Uncovered) Table 22: Average Allele Peak Heights (Saliva) Table 23: and RNA Stability in Semen Stains (Outside Covered) Table 24: and RNA Stability in Semen Stains (Outside Uncovered) Table 25: Average Allele Peak Heights (Semen) Table 26: and RNA Stability in Vaginal Secretion Swabs (Outside Covered) Table 27: and RNA Stability in Vaginal Secretion Swabs (Outside Uncovered) Table 28: Average Allele Peak Heights (Vaginal Secretions) xiv

15 LIST OF ACRONYMS/ABBREVIATIONS ALAS2 aminolevulinate synthase 2 BL blood CE capillary electrophoresis c complementary Ct cycle threshold DEPC diethylpyrocarbonate deoxyribonucleic acid DNase deoxyribonuclease DTT dithiothreitol EDTA ethylenediaminetetraacetic acid HBB hemoglobin-beta chain HTN3 histatin 3 IPC internal positive control VAG1 vaginal secretion primer MMP-10 matrix metalloproteinase-10 mrna messenger RNA MB menstrual blood MUC4 mucin 4 MUC7 mucin 7 NCFS National Center for Forensic Science OS-C outside covered OS-UC outside uncovered PBGD porphobilinogen deaminase PCR polymerase chain reaction P/C/IAA phenol/chloroform/isoamyl alcohol PRM2 protamine 2 RFU relative fluorescence unit RNA ribonucleic acid RNase ribonuclease RT room temperature/reverse transcription RT-PCR reverse transcription-polymerase chain reaction SA saliva SDS sodium dodecyl sulfate SE semen STATH statherin STR short tandem repeat TE tris-edta TGM4 transglutaminase 4 VAG1 vaginal marker VS vaginal secretions xv

16 CHAPTER ONE: INTRODUCTION Biological material (fluids or tissues) whether from the victim or suspect is often collected as forensic evidence, and methods to obtain and analyze the (deoxyribonucleic acid) found in that material have been well established [1]. STR (short tandem repeat) profiles that are obtained can then be used for making suspect inclusions and exclusions [1]. Initially, evidence gathered during the investigation of a crime is screened for the presence of biological fluids so that potential sources of can be identified [1;2]. The type of body fluid (i.e. blood, saliva, semen, vaginal secretions, and menstrual blood) from which the originated is also of interest as it can provide information about the crime that took place. The conventional serological methods that are used for body fluid identification can be costly, must be performed sequentially, and can consume the often limited amount of available sample, leaving less for analysis [2]. Therefore, there is a need for advances in body fluid identification strategies to reduce the amount of time and sample required to perform both body fluid and analysis. In the past few years, numerous studies have demonstrated the ability to use mrna (messenger ribonucleic acid) expression profiling for the identification of forensic biological stains [2-12]. Cells are differentiated to form various body fluids and tissues, and the cell types contained within those fluids or tissues have a unique set of active genes from which mrna is transcribed, making it possible to identify a fluid or tissue based on the type and abundance of mrna transcripts present [13]. There is not yet widespread use of mrna profiling in forensic casework, but this molecular based approach offers the advantages of improved sensitivity and specificity while making it possible to simultaneously test for multiple body fluids at once [2]. In order for this body fluid typing technique to be utilized in routine forensic casework, RNA of 1

17 sufficient quantity and quality must be obtained from biological fluid stains and the methods used for RNA analysis must be fully compatible with current analysis methodologies. While several methods that describe the simultaneous isolation of and RNA from the same sample have been reported, few of these studies have performed thorough optimization and validation experiments focused on the often reduced quantity and compromised quality of samples encountered in forensic casework [14-23]. To determine what extraction method would allow for and RNA of high quality to be obtained from forensic samples, several /RNA co-extraction methods were compared (Table 1). The methods evaluated included the AllPrep /RNA Mini kit (Qiagen, Valencia, CA), the ToTALLY RNA kit (Ambion, Inc., Austin, TX), the TRIzol reagent (Invitrogen, Grand Island, NY), an organic based coextraction method developed in-house at the National Center for Forensic Science (NCFS coextraction), and a method which utilizes an in-house chaos buffer and spin columns. The AllPrep /RNA Mini kit is a spin column-based method that allows for the purification of genomic and total RNA consisting of 200 nucleotides (mostly mrna) from animal cells and tissues in a short amount of time [23]. This method was chosen because it is a kit specifically designed for the co-extraction of and RNA and can be automated. Some of the previously described co-extraction methods involve isolating RNA from the aqueous phase and from the organic phase during phenol/chloroform purification which is possible because a lower ph allows for this differential separation of the nucleic acids to occur [15;22;24]. The ToTALLY RNA kit was designed for the isolation of pure RNA by using two separate phenol/chloroform purification steps, but a modified protocol allows for the from the normally discarded organic phases to be isolated using a back extraction buffer [24]. In the same way, the 2

18 TRIzol reagent is normally used for the isolation of RNA from cells and tissues, but protocols are also available for the back extraction of from the organic phase [18;22]. In 2004, an organic /RNA co-extraction method was developed in-house for use with forensic casework samples [14]. Alvarez, et al. showed that this method could be used to successfully isolate and RNA from samples of reduced quality and quantity often encountered in forensic casework; however, it is an organic extraction and requires several hours to complete, so it was not necessarily expected to be the best available co-extraction method. In a recent article, the authors compared several co-extraction methods using fish embryos [22]. They looked at both organic and spin column methods including TRIzol and back extractions, the AllPrep /RNA Micro kit for small samples, and a guanidinium-based chaos buffer used for sample lysis followed by spin column purification. They found that the highest quantity of and RNA obtained without sacrificing quality could be achieved when the chaos buffer was used to lyse the sample and the lysate was then split in half and applied to separate DNeasy and RNeasy Qiagen spin columns. In order to determine which of the above methods would be most suitable for forensic samples, the quantity and quality of and RNA recovered using each of the co-extraction methods were compared. Standard non-co-extraction methods were also performed to establish a basis for comparison. The most promising methods were then chosen for optimization in order to maximize extraction efficiency and nucleic acid recovery. After optimization of a chosen co-extraction protocol, the method could then be used as a means to compare the relative stability of and RNA in the same environmentally compromised biological fluid stain. Messenger RNA has a limited lifetime in the cell and is often 3

19 quickly degraded because of its role as a control for protein synthesis [25-27]. Additionally, in alkaline conditions the 2 OH group of the ribose sugar which deoxyribose does not have makes it more susceptible to hydrolysis resulting in strand cleavage [28]. For these reasons and because of the abundant endogenous and exogenous RNases present that can degrade RNA, it is often assumed that in forensically relevant stains would remain stable for a longer period of time [25;29]. This concern about RNA stability has in some ways prevented the acceptance of mrna profiling in forensic casework [30]. Unfavorable conditions such as UV light, humidity, bacterial growth, and high temperatures have all been shown to accelerate the degradation of both and RNA in dried stains [31-35], but a thorough investigation of the relative degradation rates of and RNA in the same sample has not been performed. Hydrolysis is a major cause of degradation, and it results in base loss and strand scission which can then impede amplification of a target sequence [36-38], but the dehydrated state of a body fluid stain greatly reduces the occurrence of hydrolysis as well as other potentially harmful chemical reactions [34]. in dried stains has been shown to remain stable for years, but even under proper storage conditions some degradation can still occur [31;32]. It was thought that RNA in dried stains would not remain stable for as long, but studies have shown that RNA can also be obtained and analyzed after several years [29;31]. The effect that storage conditions can have on RNA recoverability and stability has also been evaluated, and RNA suitable for body fluid identification by RT-PCR (reverse transcription-polymerase chain reaction) analysis was obtained from body fluid stains stored under various conditions at room temperature after 547 days [33]. 4

20 The studies showing the stability of RNA are promising, but it is important to know if one can expect RNA body fluid markers to be detected in a degraded sample when a profile can still be obtained, if RNA will not be recoverable for as long as, or if RNA can be successfully analyzed beyond the point that a profile can be obtained. In order to determine this relationship, we incubated forensically relevant body fluid stains (blood, saliva, semen, and vaginal secretions) at different set temperatures for increasing lengths of time as well as outdoors to determine the relative effects of environmental conditions (i.e. sunlight, rain, humidity) on and RNA stability. Stains were also made on different substrates that biological fluid stains could be found on to determine if the substrate itself affects the relative level of and RNA degradation. 5

21 CHAPTER TWO: METHODOLOGY Sample Preparation Body fluids were collected from volunteers using procedures approved by the university s institutional review board. Blood was collected by venipuncture, and 50 µl aliquots were deposited onto sterile cotton cloth (t-shirt). For sensitivity testing of the optimized coextraction protocols, 10 µl, 5 µl, 1 µl, and 0.2 µl blood stains were prepared on cotton. Saliva was obtained by swabbing the inside of the cheek with a cotton-tipped swab. For sensitivity testing, liquid saliva was collected in sterile 15 ml centrifuge tubes and 50 µl, 25 µl, 10 µl, 5 µl, and 1 µl stains were then made on sterile cotton cloth. Semen was collected in 50 ml centrifuge tubes, and swabs of the sample were taken or 50 µl, 25 µl, 10 µl, 5 µl, and 1 µl stains were made for sensitivity testing. Semen-free vaginal secretion and menstrual blood swabs were collected by swabbing the vaginal cavity with sterile cotton-tipped swabs. During optimization and sensitivity testing, ½ and ¼ buccal, semen, vaginal, and menstrual blood swabs and ½ and ¼ of 50 µl blood stains were also extracted. Stains or swabs were allowed to air dry at room temperature and were then stored at -20 C until needed. Degradation Samples For the comparison of and RNA degradation rates, body fluid stains or swabs were made for blood, saliva, semen, and vaginal secretions. The body fluids were obtained from the donors as described above except that non-edta tubes were used to collect blood. The substrates upon which stains were made included t-shirt cotton (100% cotton, white), denim 6

22 (100% cotton, medium blue), carpet (nylon, tan, short fibers; used), and swabs (sterile cotton tipped applicators). Stains/swabs prepared for three donors per body fluid were then incubated protected from light at room temperature (~22ºC, 53% humidity), 37ºC (incubator), or 56ºC (3 incubators to accommodate all samples) with the ambient humidity of the room/incubator for 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, and 1 year, and Time 0 control samples were also prepared. Blood, saliva, semen, and vaginal secretion samples (previously prepared) from four (blood and saliva) or two donors (semen and vaginal secretions) were also incubated outside exposed to light, heat (average high of 36 C), humidity, and rain (uncovered) or light, heat, and humidity (covered) for 1 day, 3 days, 1 week, 4 weeks, 3 months, 6 months, and 1 year. A few additional blood, saliva, and semen stains (1 donor per fluid, previously prepared) were incubated in the shade, sun, and on a patio for various lengths of time. Standard RNA Extractions To establish what results would be expected using standard, non-co-extraction methods, both organic and spin column-based RNA extractions were completed using 50 μl dried blood stains and saliva, semen, vaginal secretion, and menstrual blood swabs. Extractions were performed twice to verify that results could be duplicated. Standard Organic RNA Extraction A standard organic RNA extraction using guanidine isothiocyanate-phenol:chloroform was performed [39]. A mixture of 500 µl denaturing solution (guanidine isothiocyanate, 0.02 M 7

23 sodium citrate dihydrate, 0.5% sarkosyl) and 3.6 µl of β-mercaptoethanol was preheated for 10 minutes at 56 C. The sample (stain or swab) was placed into a safe-lock microcentrifuge tube (Eppendorf, Hauppauge, NY) and incubated in the denaturing solution mixture for 30 minutes at 56 C. The swab or cloth pieces were placed into a spin basket (Promega, Madison, WI) which was then inserted back into the extraction tube, and the sample was centrifuged at 8,160 x g for 10 minutes. The spin basket and its contents were discarded. Fifty microliters of 2 M sodium acetate and 600 µl of acid-phenol:chloroform 5:1 (ph 4.5, Ambion, Inc.) were added to the lysate to create a phase separation, maintaining the RNA in the aqueous phase. The sample was incubated at 4 C for 30 minutes and then centrifuged at full speed (16,000 x g) for 20 minutes to separate the phases. The aqueous phase was transferred to a new 1.5 ml microcentrifuge tube, and the RNA was precipitated at -20 C for at least 1 hour in a mixture of 500 µl isopropanol and 2 µl GlycoBlue glycogen carrier (Ambion, Inc.). The sample was centrifuged at full speed for 20 minutes, and the resulting pellet was washed with 75% ethanol/25% DEPC-treated water, dried in a vacuum centrifuge for 5 minutes, and incubated at 60 C in 12 µl of RNA Secure Resuspension Solution (Ambion, Inc.) for 10 minutes to resolubilize the pellet. RNeasy Micro Kit The RNeasy Micro kit (Qiagen) was used as the standard spin column method. The extraction was completed either by hand or using the QIAcube robot from Qiagen following the protocol provided by the manufacturer. The sample was first lysed in the provided Buffer RLT containing β-mercaptoethanol. One volume of 70% ethanol was added to the lysate, and the entire sample was transferred to the RNeasy MinElute spin column. The column was washed 8

24 with Buffer RW1, and an on-column DNase I digestion was then performed. A mixture of 10 µl DNase I stock and 70 µl Buffer RDD was applied to the column which was then incubated at room temperature for 15 minutes. Buffers RWI and RPE and 80% ethanol were used to wash the column, and the RNA was eluted into a new 1.5 ml tube with 14 µl of RNase-free water (provided). Standard Extractions As with the RNA extractions, two different standard, non-co-extraction methods, both organic and spin column-based, were used. Standard Organic Extraction A standard organic extraction, as previously described, was performed [40]. The sample was incubated at 56 C overnight in 400 µl of stain extraction buffer (10 mm Tris-HCl (ph 8.0), 0.1 M NaCl, 0.5 M EDTA, 20% SDS), 20 µg/µl proteinase K, and µg/µl dithiothreitol (DTT, semen samples only). The cut sample pieces were placed into a spin basket which was then inserted back into the extraction tube, and the sample was centrifuged at full speed for 5 minutes. Four hundred microliters of phenol/chloroform/isoamyl alcohol (P/C/IAA, 25:24:1, ph 6.6) was added to create a separation of the organic material and. The sample was centrifuged at full speed to separate the layers, and the aqueous phase was removed to a new 1.5 ml tube. was precipitated at -20 C for at least 1 hour using cold 100% ethanol. The sample was centrifuged at full speed for 15 minutes to pellet the, and the pellet was washed 9

25 with 70% ethanol, dried in a vacuum centrifuge for 5 minutes, and then resolubilized in 100 μl of TE -4 (10 mm Tris, 0.1 mm EDTA, ph 7.5) at 56ºC overnight. Investigator Kit The QIAamp Investigator kit (Qiagen) was chosen as the standard spin-column extraction method, and it was performed following the manufacturer s instructions. The provided Buffer ATL and proteinase K were added to the sample which was then incubated at 56ºC for 1 hour. The sample was vortexed every 10 minutes to facilitate lysis. Buffer AL was added, and the sample was incubated at 70ºC an additional 10 minutes for complete lysis. Ethanol (100%) was added to the lysate to create proper binding conditions, and the sample was transferred to the QIAamp MinElute spin column which selectively binds. Contaminants were washed from the column using the provided Buffers AW1 and AW2 and 70% ethanol. The column was incubated at room temperature for 10 minutes to dry the membrane, and the was then eluted in 50 µl of Buffer ATE. /RNA Co-Extraction Methods Each of the co-extraction methods evaluated (Table 1) was performed twice using a 50 µl blood stain (two different donors) and a buccal swab (the same donor). AllPrep /RNA Mini kit The AllPrep /RNA Mini kit extraction (Qiagen) was performed either manually or using the QIAcube robot following the manufacturer s instructions. The sample was lysed 10

26 briefly using 350 µl of the provided Buffer RLT and a 0.01 volume of β-mercaptoethanol. The lysate was applied to the AllPrep Mini spin column, and the flow-through was then mixed with 70% ethanol to create proper conditions for the selective binding of RNA to the RNeasy spin column. Contaminants were washed from the column using the Buffers AW1 and AW2 and from the RNA column using the Buffers RW1 and RPE. The was eluted in 100 µl of Buffer EB, and RNA was eluted in 30 µl of RNase-free water. ToTALLY RNA Kit/ Back Extraction The ToTALLY RNA kit (Ambion, Inc.) was designed for RNA extractions, but the protocol can be adapted to allow for isolation as well [24]. The manufacturer s protocol was followed with a few adjustments. The sample was lysed in 300 µl of the provided guanidinium based Denaturation Solution for minutes at room temperature. The cut sample pieces were placed into a spin basket which was then inserted back into the extraction tube, and the sample was centrifuged at full speed for 5 minutes. The RNA was then purified by first using 1 volume of P/C/IAA (ph 6.6). The sample was vortexed for 1 minute, and then incubated on ice for 5 minutes. After centrifugation for 5 minutes at 8,160 x g, the aqueous phase was transferred to a new tube, and the organic phase was set aside. Sodium acetate (1/10 volume) and acid-phenol:chloroform (ph 4.5, 1 volume) were added to the aqueous phase, and the same steps were followed as with the P/C/IAA purification. The aqueous phase was transferred to a new tube, and the organic phase was set aside. The RNA was precipitated from the aqueous phases using an equal volume of isopropanol at -20 C for at least 30 minutes, and the resulting pellet was washed with 70% ethanol and then resuspended in 12 µl of RNA Secure 11

27 Resuspension Solution as used with the standard RNA organic extraction. The organic phase from both the P/C/IAA and acid-phenol:chloroform purifications were combined, and a back extraction buffer (0.1 M NaCl, 10 mm Tris-HCl, ph 8.0, 1 mm EDTA, 1% SDS, ph 12) was added to isolate the remaining in the organic phases. The sample was incubated at -20 C for 10 minutes and centrifuged at 8,160 x g for 20 minutes. The aqueous layer was transferred to a new 1.5 ml tube, and the was precipitated with 2 volumes of cold 100% ethanol at -20 C for at least 1 hour. The pellet was washed with 70% ethanol and then resuspended in 50 µl TE -4 for 5 minutes at 56 C. TRIzol Extraction/ Back Extraction The TRIzol LS reagent (Invitrogen) is used for RNA extractions, but can also be isolated by adding a back extraction buffer to the saved organic phase [18;22]. Eight hundred microliters of the TRIzol reagent which contains both guanidine isothiocyanate and phenol was used to lyse the sample, and the cut sample pieces were then placed into a spin basket which was inserted back into the extraction tube and centrifuged at full speed for 5 minutes. Two hundred microliters of chloroform was added to create a phase separation, and the sample was centrifuged at 8,160 x g for 15 minutes. The aqueous phase was transferred to a new 1.5 ml tube, and the organic phase was set aside. The RNA was precipitated with 500 µl isopropanol and 2 µl GlycoBlue glycogen carrier at room temperature for 10 minutes, and the sample was then centrifuged at full speed for 10 minutes. The resulting pellet was washed with 1 ml of 75% ETOH/25% DEPC water, air dried for 10 minutes, and resuspended in 12 µl of RNA Secure Resuspension Solution. A back extraction buffer (4 M guanidine thiocyanate; 50 mm sodium 12

28 citrate; 1 M Tris, ph 8.0) was added to the saved organic phase to isolate the remaining. The sample was incubated at room temperature for 10 minutes and centrifuged at full speed for 15 minutes. The aqueous phase was transferred to a new 1.5 ml tube, and the was precipitated at -20 C for 1 hour in an equal volume of isopropanol. The pellet was washed with 70% ethanol, dried in a vacuum centrifuge, and then resuspended in 50 µl TE -4 at 56 C for 10 minutes. Chaos Buffer/Spin Columns In an article by Triant and Whitehead (2008), various /RNA co-extraction methods were tested, and their results showed that using a Chaos Buffer (4.5 M guanidine isothiocyanate, 2% sarkosyl, 50 mm EDTA, 0.1 M β-mercaptoethanol) and spin columns allowed them to isolate and RNA of high quality and quantity from the same sample [22]. Four hundred microliters of the chaos buffer was added to the sample which was incubated at 56 C for 30 minutes, and the lysate was then split in half, one portion (~200 µl) being applied to an RNeasy spin column and the other being applied to a DNeasy Blood and Tissue spin column (Qiagen). The manufacturer s protocols were followed for the RNeasy Mini and DNeasy Blood and Tissue kits. The RNA was bound to the RNeasy column, contaminants were washed from the column with the Buffers RW1 and RPE, and the RNA was then eluted in 30 µl of RNase-free water. The portion was mixed with Buffer AL and 100% ethanol and then applied to the DNeasy column, the column was washed with the Buffers AW1 and AW2, and the was eluted in 100 µl of Buffer AE. 13

29 NCFS Co-extraction The in-house NCFS co-extraction method used was developed by Alvarez, et al. in 2004 [14]. The sample was first lysed in 500 µl of the same stain extraction buffer used with the standard organic extraction and proteinase K. DTT (10% of extraction volume) was added to semen samples only. The cut sample pieces were placed into a spin basket which was then inserted back into the extraction tube, and the sample was centrifuged at full speed for 5 minutes. Fifty microliters of sodium acetate (2 M) and 600 µl of acid-phenol:chloroform (ph 4.5) were added to the sample to create a phase separation. The sample was incubated at 4 C for 20 minutes and then centrifuged at full speed for 20 minutes. The aqueous phase (~500 µl) was transferred to a new 1.5 ml tube labeled, and half of it was then transferred to another tube labeled RNA. and RNA were then precipitated following the same procedures used with the standard organic and RNA extractions. was precipitated in ethanol and then resolubilized in 50 μl TE -4 at 56 C for 45 minutes, while RNA was precipitated in isopropanol and resolubilized in 12 μl of RNA Secure Resuspension Solution. DNase I Digestion All RNA extracts were treated with DNase to remove any contaminating that might have remained in the sample. Six units of TURBO DNase I (2 U/µL, Ambion, Inc.) and 10X DNase Buffer were added, and the sample was incubated at 37 C for 1 hour. The enzyme was inactivated by incubating the sample at 75 C for 10 minutes [41-43]. A slightly different DNase digestion method was used for the degradation study: TURBO -free DNase (Ambion) and 0.1 volume of 10X TURBO DNase buffer were added to the RNA extract, and the sample 14

30 was then incubated at 37 C for 20 minutes. To inactivate the enzyme, a 0.1 volume of DNase Inactivation Reagent was added. The samples were stored at -20 C until needed. Quantitation of Isolated and RNA The Quantifiler Human Quantification kit (Applied Biosystems, Foster City, CA) and the ABI 7500 Fast Real Time PCR system were used for quantitation. Fast 96-well plates were prepared with 0.8 µl of standard or sample and 9.2 µl of PCR reaction/primer mix. The cycling conditions were 10 minutes at 95ºC followed by 45 cycles of 10 sec at 95ºC and 1 minute at 60ºC. concentrations were calculated based on Ct values of the quantitation standards. The Quant-iT Ribogreen assay (Molecular Probes, Eugene, OR) was used for RNA quantitation using the high-range standard concentrations (20 ng/ml to 1 µg/ml) [44]. The 20X TE buffer provided with the kit was diluted to 1X and mixed with 2 µl of sample in a 96-well plate. The Ribogreen reagent was diluted in the 1X TE and 100 µl of the solution was added to the sample wells for a total assay volume of 200 µl. The plate was incubated in the dark for 3 minutes, and the fluorescence emission at 535 nm (485 nm excitation) was measured using a Wallac Victor 2 microplate reader (Perkin Elmer Life Sciences, Boston, MA). Total RNA concentrations of the samples were calculated based on a standard curve created using the fluorescence measurements of the quantitation standards. 15

31 c Synthesis Reverse transcription (RT) was used to synthesize complementary (c) strands from the isolated mrna transcripts [45;46]. Three different RT reactions were used over the course of this project. The reactions performed included using reagents from Ambion (10X firststrand buffer, random decamer primers, 10 mm dntp mix (Applied Biosystems), SUPERase- In RNase Inhibitor, and Moloney Murine Leukemia Virus-Reverse Transcriptase), the SuperScript III First-Strand Synthesis System (Invitrogen), and the High-Capacity c Reverse Transcription kit (Applied Biosystems). The target amount of RNA used in the reaction was 50 ng. For samples which contained less than 50 ng, the entire extract was used. For the first RT reaction listed, sample extracts were preheated at 75 C for 3 minutes, the RT reagents mix was added for a total volume of 30 µl, and the samples were then incubated for 1 hour at 42 C and for 10 minutes at 95 C to inactivate the enzyme. For the SuperScript III RT reaction, samples were preheated with dntp mix and random primers at 65 C for 5 minutes, the RT mix (0.1 M DTT, 25 mm MgCl 2, 10X RT buffer, RNaseOUT RNase inhibitor, and SuperScript III RT) was added for a total volume of 20 µl, and the samples were incubated at 25 C for 10 minutes, at 50 C for 50 minutes, and at 85 C for 5 minutes to inactivate the enzyme. For the ABI High Capacity RT reaction, sample extracts were preheated at 75 C for 3 minutes, the RT mix (10X RT buffer, 100 mm dntp mix, 10X random primers, and MultiScribe RT) was added for a total volume of 20 µl, and the samples were incubated at 25 C for 10 minutes, at 37 C for 120 minutes, and at 85 C for 5 minutes to inactivate the enzyme. An RT+ reagent blank containing the RT reagents and water and RT- controls containing sample extract but no reverse transcriptase enzyme were included with each reaction. The RT- controls were used to show that 16

32 no or pseudogenes were being amplified and that only peaks from true mrna transcripts were observed. The first RT method (Ambion) was used during the co-extraction methods evaluation and the Superscript III First-Strand kit was used with the optimized methods. For the degradation study, only the ABI High Capacity RT kit was used. Polymerase Chain Reaction Amplification For all of the co-extraction evaluation and optimization samples, the AmpFlSTR SGM Plus PCR Amplification Kit (Applied Biosystems) was used for STR amplification [47]. The target amount of used in the reaction was 1-2 ng or pg for a half reaction. The cycling conditions were 11 minutes at 95 C, followed by 28 cycles of 1 minute at 94 C, 1 minute at 59 C, and 1 minute 72 C, and a final extension of 45 minutes at 60 C. The SGM Plus kit amplifies 10 STR loci plus Amelogenin for sex determination with 2.5 U/1.25 U AmpliTaq Gold Polymerase (5 U/µL, Applied Biosystems) in a 25 µl reaction or 12.5 µl half reaction volume. For the samples of the degradation study, the AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems) was used [48]. The target amount of was the same as used with the SGM Plus kit. The Identifiler kit amplifies 15 STR loci plus Amelogenin. The cycling conditions used were also the same except that the final extension was 60 minutes at 60 C. For RNA body fluid testing, 3 µl of c product was amplified using an mrna body fluid typing multiplex. A mix of 10X PCR reaction buffer (Applied Biosystems), 10 mm dntps (Applied Biosystems), 25 mm MgCl 2 (Applied Biosystems), and 1.25 or 1.5 U AmpliTaq Gold was prepared and a combination of the following primers (Applied Biosystems, Invitrogen) was 17

33 added: PBGD or ALAS2 for blood, HTN3 or STATH for saliva, PRM2 for semen, MUC4 and/or VAG1 (unpublished) for vaginal secretion, and MMP-10 for menstrual blood. Table 2 lists the primer sequences of the body fluid markers used. The primer sequences of PBGD, PRM2, and MUC4 were obtained from published sources [9]. Different PRM2 primers (F: 5 -VIC- GGCGCAAAAGACGCTCC, R: 5 -GCCCAGGAAGCTTAGTGCC) were used during optimization to amplify smaller c fragments for better amplification efficiency [10]. The sequences and sizes for the HTN3, STATH, ALAS2, and MMP-10 markers differed from published sequences and were designed using Primer3 Online primer design software. The cycling conditions were 11 minutes at 95 C, 35 cycles of 94 C for 20 sec, 58 C for 30 sec, and 72 C for 40 sec, and a final extension of 5 minutes at 72 C. During the optimization of the coextraction methods, the multiplex used employed different primers and therefore, slightly different cycling conditions: 11 minutes at 95ºC, 35 cycles of 94ºC for 20 sec, 60ºC for 30 sec, and 72ºC for 40 sec, and a final extension of 80 minutes at 72ºC. For the degradation samples, we wanted to maximize the possibility of RNA being detected (i.e. amplification with highest sensitivity) so singleplexes or duplexes were used instead of a multiplex. HBB [8] and ALAS2 singleplexes were used for blood, an HTN3 singleplex was used for saliva, a PRM2 and TGM4 [6] duplex was used for semen to account for the possibility of a vasectomized donor, and a MUC4 singleplex (FAM labeled) was used for vaginal secretions. The same amplification conditions as used with the multiplex (during optimization) were used for the singleplex/duplex reactions except for the HBB singleplex: 11 minutes at 95ºC, 30 cycles of 94ºC for 20 sec, 57ºC for 30 sec C per cycle, and 72ºC for 40 sec, and a final extension of 30 minutes at 72ºC. 18

34 Detection of Amplified Products One microliter or 1.5 µl of amplified RNA and products was denatured in Hi-Di formamide (Applied Biosystems) containing either the GeneScan 500 LIZ or ROX size standard (Applied Biosystems). Samples were analyzed by capillary electrophoresis (CE) using either the ABI 3130 or 310 Genetic Analyzer, and results were analyzed using GeneMapper and GeneScan software, respectively. Post-PCR Purification For some RT-PCR amplification reactions, the presence of dye blobs interfered with interpretation of results, so the MinElute PCR Purification kit (Qiagen) was used to clean-up amplified samples using the QIAcube robot. The MinElute kit was also used to increase peak heights in other instances. The entire volume (25 µl) of PCR product was combined with 75 µl Buffer EB, and then 5 volumes of Buffer PB were added. The sample was applied to the MinElute column, Buffer PE was added, and the purified c was then eluted in 15 µl of Buffer EB. One microliter of sample was still used for CE analysis. Optimization of Co-extraction Methods An organic (NCFS co-extraction) and spin column (AllPrep /RNA Mini extraction) extraction method were chosen for optimization based on the initial evaluation of the coextraction methods. Due to low RNA yields obtained with the spin column extractions, attempts were first made to increase the amount of RNA obtained using the standard RNeasy Micro kit 19

35 extraction. The extraction was performed using various lysis incubation times (1 hour, 3 hours, overnight) and temperatures (room temperature, 37ºC, and 56ºC) with duplicate 50 µl blood stains on cotton (whole stain used) and buccal swabs (whole swab used). Additional extractions were also completed using carrier RNA provided with the kit, ½ and ¼ of 50 µl blood stains to determine if the column was being clogged when using a whole stain, and the lysis step used for the standard organic RNA extraction (Denaturing solution, β-mercaptoethanol; 30 minutes at 56ºC). The RNeasy Micro kit extraction was then repeated with duplicate semen, vaginal secretion, and menstrual blood swab samples using the standard conditions and the altered conditions which resulted in the most RNA obtained from the blood and saliva samples. Optimization of the AllPrep /RNA Mini kit extraction began with using the same lysis incubation times (1 hour, 3 hours, overnight) and temperatures (RT, 37ºC, and 56ºC) that were used with the RNeasy Micro kit. These extractions were first performed with blood and saliva samples only, and then later the standard conditions (no incubation) and the altered conditions which resulted in improved and RNA recovery were used with semen, vaginal secretions, and menstrual blood swab samples. Further attempts to optimize the AllPrep protocol included substituting the RNeasy MinElute columns provided with the RNeasy Micro kit for the RNeasy Mini columns normally used with the extraction, substituting DTT for β- mercaptoethanol, adding carrier RNA, and omitting β-mercaptoethanol. Attempts were then made to optimize the NCFS co-extraction. To decrease the time required to perform the extraction and reduce the presence of PCR inhibitors, Nucleospin Clean-up XS and Nucleospin RNA Clean-up XS columns (Macherey-Nagel, Bethlehem, PA) were used to replace the precipitation step of the NCFS co-extraction. The aqueous phase was 20

36 split in half as normal, but then an equal volume (~250 µl) of Buffer RCU was added to the RNA fraction, and the sample was then applied to the Nucleospin RNA column while the fraction was adjusted to 800 µl with TE -4 and 200 µl NT binding buffer was added before then applying the sample to the Nucleospin column [49]. Centrifugation steps were carried out at 11,000 x g. The RNA and columns were washed with the provided Buffer RA3 and B5, respectively. RNA was then eluted in 10 µl of RNase-free H 2 O, and was eluted in 20 µl of Buffer BE. The NCFS co-extraction was also performed using the standard RNA lysis step (Denaturing solution, β-mercaptoethanol; 30 minutes at 56ºC) with and without Nucleospin columns and substituting P/C/IAA for acid-phenol:chloroform. The lysis incubation length was also varied using 15 minute, 30 minute, and 3 hour incubations at 56 C. Testing of Optimized Protocols Larger sample sets were used to test the optimized co-extraction protocols, and the sensitivity of the methods was also evaluated. Samples from a total of 16 donors were used for blood and ½ and ¼ stains were also extracted for 10 of these donors. For saliva, buccal swabs from 10 donors were used, for semen and vaginal secretion, swabs from 8 donors were used, and for menstrual blood, swabs from 5 donors were used. In some cases additional testing was necessary to obtain a positive RNA result (different RT method, altered PCR cycling conditions, RNA singleplex, etc.), and for any RNA samples that were still negative after additional testing, a new swab or stain from that donor was extracted. 21

37 and RNA Stability The NCFS co-extraction with Nucleospin columns was used for the extraction of the samples prepared for the /RNA degradation study. As mentioned previously, the Identifiler kit was used for STR typing and a duplex or singleplex was used for mrna body fluid typing. A standard RNA extraction using Nucleospin columns was also performed with pristine blood, saliva, semen, and vaginal secretion samples to provide non-co-extraction controls to amplify with the degraded samples. 22

38 CHAPTER THREE: RESULTS (CO-EXTRACTION) Evaluation of Co-extraction Methods Standard and RNA Extractions Standard non-co-extractions, both organic and spin column-based, were performed using blood and saliva stains to provide a basis for comparison for the co-extraction methods. For the standard extractions, on average about 4.5 times and 2 times more was recovered using the organic extraction method as compared to the spin column method for blood and saliva, respectively (Figures 1A and 1B). Both organic and spin column extractions produced enough product for downstream reactions, and full STR profiles (SGM Plus ) could be obtained upon CE analysis. For the standard RNA extractions, on average about 8.5 times more RNA was obtained from the blood samples and 18.5 times more was obtained from saliva using the organic extraction compared to the spin column method (Figures 1A and 1B). The RNeasy Micro kit protocol is not adapted for use with forensic samples like the Investigator kit which may explain why the difference in yield between the organic and spin column methods was so much greater for RNA. Additionally, only RNA >200 bases is isolated using spin columns, so the extracts would not contain small RNAs that would be isolated with an organic extraction. Despite the low yields, the HTN3 saliva marker was still detected in the saliva samples from each extraction, and either the PBGD or ALAS2 blood marker was detected in the blood samples. 23