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Efficiency evaluation of a DNA extraction and purification protocol on archival formalin-fixed and paraffin-embedded tissue
Forensic Science International 194 (2010) e25–e28
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Rapid communication
Efficiency evaluation of a DNA extraction and purification protocol on archival formalin-fixed and paraffin-embedded tissue A. Farrugia *, C. Keyser, B. Ludes Institute of Legal Medicine, EA4438, University of Strasbourg, 11 rue Human 67085, Strasbourg Cedex, France
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 February 2009 Received in revised form 29 June 2009 Accepted 2 September 2009 Available online 24 September 2009
Formalin-fixed and paraffin-embedded tissue (FF-PET) is an invaluable resource for retrospective molecular genetic studies, but the extraction of high-quality genomic DNA from FF-PET is still a problematic issue. Despite the range of DNA extraction methods currently in use, the association of phenol–chloroform extraction and silica-based purification protocols, reported in ancient DNA studies on archaeological bones, has, to our knowledge, not been used for DNA extraction from FF-PET yet. The present study compared the efficiency of three DNA extraction and purification protocols from two different FF-PET substrates, heart and liver, by using quantitative PCR and multiplex amplification. We showed that the method, using phenol–chloroform and the QIAamp DNA mini1 Kit (Qiagen), was the most effective DNA extraction and purification method and that the DNA quantity extracted from liver is statistically more important than that extracted from heart. Autosomal STR typing by multiplex amplifications gave partial allelic profiles with only small size products (less than 300 bases) amplified, suggesting that DNA extracted from FF-PET was degraded. In conclusion, the protocol presented here, previously described in studies on ancient bones, should find application in different molecular studies involving FF-PET material. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Formalin-fixed and paraffin-embedded Tissue DNA extraction Phenol–chloroform Silica based-spin columns
1. Introduction The extraction of DNA from formalin-fixed and paraffinembedded tissue (FF-PET) enables pathologists to use archival material for a variety of purposes including retrospective molecular genetic studies based on DNA amplification by polymerase chain reaction (PCR). However, there are several reasons for the failure of PCR using DNA isolated from FF-PET: (i) the generation of DNA–protein cross-linkages due to formaldehyde solution resulting in nucleic acid fragmentation [1,2], (ii) the presence of remnants of substances that inhibit the amplification reaction such as formalin [3] or inhibit the proteinase K used in the extraction procedure such as xylene [4], (iii) the risk of contamination during manipulation of samples. Because of these difficulties, number of DNA extraction methods have been tested and several studies comparing different methods have been published such as phenol–chloroform method [3–7], ammonium acetate precipitation [8,6], ethanol precipitation [9] and commercially available kits based on silica binding principle [3–11].
* Corresponding author. Tel.: +33 368853363; fax: +33 368853362. E-mail address: [email protected] (A. Farrugia). 0379-0738/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2009.09.004
The combination of phenol–chloroform extraction and silicabased purification method, developed for DNA extraction from ancient bones [12] has to your knowledge, not yet been tested on FF-PET. The aim of this work was to test the efficiency of such protocol on FF-PET. Indeed, one of the major difficulties in using FF-PET is the degradation of nucleic acids [5] like in archeological biological remains. For this purpose we compared the use of organic extraction coupled with silica-based purification method using two different commercial kits, CleanMix (Talent) [13–15] and QIAamp DNA mini1 Kit (Qiagen) to the most popular method based on direct use of the commercial QIAamp DNA mini1 Kit (Qiagen). Tests were performed from two types of substrates, heart and liver from 22 individuals. 2. Materials and methods 2.1. Samples Twenty two formalin-fixed and paraffin-embedded (FF-PE) tissue blocks from heart and liver autopsy tissue were obtained from the Forensic Institute of Strasbourg, France. The preparation of the FF-PE block was done with the same seize of fresh tissue (1.5 cm wide, 1.5 cm long, and 0.3 cm of thickness). All of them had been fixed in 10% buffered formalin. The average period of fixation was 34.27 days (min = 28 days and max = 51 days) at room temperature. This formalin fixation time usually corresponds to the time of tissue fixation before inclusion in paraffin in our Medico-legal Institute. The time elapsing between autopsy and tissue sampling was less than three days.
A. Farrugia et al. / Forensic Science International 194 (2010) e25–e28
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After the formalin fixation and paraffin inclusion steps, the surrounding paraffin was removed with a scalpel and the center of the organ was trimmed to a surface of 1 cm2 of tissue. Then using a standard microtome with disposable blade, serial sections of 50 mm thickness from each FF-PE blocks were obtained and placed in a microcentrifuge tube. Each sample weigh ranged from 38 to 57 mg. Because of the low density of paraffin wax (inferior to water’s density), we have considered that the presence of paraffin in our samples had not affected the measurements of tissue weight. Great care was taken to prevent cross-contamination between samples by using fresh blades for each specimen and by cleaning the microtome with DNAse Away (Molecular Bioproducts) between specimens. 2.2. DNA deparaffinization and rehydratation The technique for paraffin removal was based on that described by Goelz et al. [16] using two xylene washes (1 mL of xylene for 10 min at room temperature), followed by decreasing concentration of alcohol (ethanol, 100%, 95%, 70%) and then two distilled water washes [4]. Then samples were rehydrated with Tris/EDTA (Tris 100 mM, pH7.5, EDTA 5 M) for 5 min at room temperature and Tris buffered solution (100 mM, pH7.5) for 20 h at 55 8C with four changes of solution. To avoid loss of sample, centrifugation (13,500 rpm, 5 min) was performed before each change of solution [4]. 2.3. DNA extraction and purification: three protocols The material studied was submitted to one of the following DNA extraction and purification methods. 2.3.1. Protocol A: phenol–chloroform followed by the use of the CleanMix Kit [13] Tissue digestion was realized by incubation of the samples at 55 8C overnight with 300 mL of lysis-buffer (100 mM Tris–HCl (pH 8.0), 5 mM EDTA, 1% sodium dodecyl sulfate, 1 mg/mL proteinase K). After incubation, a phenol/chloroform/ isoamyl alcohol (25/24/1, v/v) extraction was performed on the supernatant. The aqueous phase was then purified with the CleanMix purification kit according to the manufacturer’s instructions (Talent, Trieste, Italy).
Fig. 1. Yield of DNA extracted from heart and liver with three different protocols: A = phenol–chloroform extraction followed by the use of the commercial CleanMix Kit, B = direct use of the commercial QIAamp DNA mini1 Kit, C = phenol– chloroform extraction followed by the use of the commercial QIAamp DNA mini1 Kit. PCR products was performed by capillary electrophoresis on an ABI PRISM1 3100 genetic analyzer (Applied Biosystems), and genotyping was made with the Gene Mapper v1.02 (Applied Biosystems). Genotypes of all persons involved in the processing of the samples were determined and compared to the results obtained from each FF-PET samples. 2.5. Concentration measurements DNA was quantified by real-time PCR using QuantifilerTM Human DNA Quantification Kit (Applied Biosystems, AB, Foster City, CA) with the ABI PRISM1 7000 Sequence Detection System (AB) following the manufacturer’s recommendations.
2.3.2. Protocol B: direct use of the QIAamp DNA mini1 Kit (Qiagen) The extraction and purification procedure was performed according to the manufacturer’s instructions (QIAamp DNA mini1 Kit, Qiagen). Briefly, samples were digested with Qiagen Proteinase K and lysis-buffer at 55 8C overnight and loaded onto spin columns. DNA was then adsorbed by short centrifugation onto the QIAamp silica membrane, washed and eluted with 100 mL water.
2.6. Statistical methods
2.3.3. Protocol C: phenol–chloroform followed by the use of the QIAamp DNA mini1 Kit (Qiagen) This method was performed as described above, but with an additional step. After proteinase K digestion, a phenol–chloroform–isoamyl alcohol purification was added as in protocol A. The supernatant was carefully removed and the next steps of the QIAamp DNA mini1 Kit (Qiagen) were performed according to the manufacturer’s instructions (QIAamp). In total 132 DNA extractions were performed, sourced from 22 blocks of heart and liver by using the three above mentioned methods.
3. Results
2.4. Polymerase chain reaction and typing
Quantitative data were submitted to a variance analysis to detect differences among the amount of DNA obtained from different extraction method and from different substrates. For statistical analysis, we used SPSS for Windows. Differences were considered statistically significant when the probability (p) was less than 0.05.
3.1. Yield of DNA The amount of DNA extracted from each FF-PET heart and liver sample by each method is shown in Fig. 1. The mean values of DNA Table 1 Mean values of DNA extracted (mg/100 mg of tissue) with three protocols without taken into account the type of substrates. Protocol
To detect potential cross-contamination and to evaluate the efficiency of the amplification, the DNA extracts were typed with AmpFlSTR1 IdentifilerTM Amplification Kit according to the manufacturer’s recommendation (Applied Biosystems). Fifteen Short Tandem Repeats (STRs) loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA) and the gender-identification locus amelogenine were simultaneously amplified using Biometra T3TM (Biometra1). The separation of the
A B C
Mean
Standard error
13.997 5.894 30.104
1.405 1.675 6.676
95% Confidence intervals (CIs) Minimum
Maximum
11.075 2.411 16.221
16.919 9.377 43.987
Table 2 Comparison of mean values differences between the three protocols (based on the estimated marginal means). Protocol (I)
Protocol (J)
A
B C A C A B
B C * a
Mean differences (I
8.103* 16.107* 8.103* 24.210* 16.107* 24.210*
J)
Standard error
Significationa
2.189 6.043 2.189 6.242 6.043 6.242
0.001* 0.014* 0.001* 0.001* 0.014* 0.001*
The difference in averages is significant for p = 0.05. Adjustment of multiple comparisons: the least significant difference (equivalent to no adjustment).
95% CIs of the mean differencesa Minimum
Maximum
3.551 28.673 12.655 37.191 3.540 11.228
12.655 3.540 3.551 11.228 28.673 37.191
A. Farrugia et al. / Forensic Science International 194 (2010) e25–e28 Table 3 Mean of DNA extracted (mg/100 mg of tissue) from heart and liver without taken into account the type of protocol used. Tissue
Heart Liver
Mean
13.645 19.685
Standard error
2.401 3.502
95% CIs Minimum
Maximum
8.652 12.402
18.638 26.969
Table 4 Comparison of mean differences between heart and liver (based on the estimated marginal means). Tissue (I)
Standard Significationa 95% CIs of the mean Tissue Mean (J) differences error differencesa (I J)
Heart Liver
Liver Heart
Minimum Maximum 6.041 6.041
2.438 2.438
0.022* 0.022*
11.111 0.971
0.971 11.111
*
The difference in averages is significant for p = 0.05. Adjustment of multiple comparisons: the least significant difference (equivalent to no adjustment). a
extracted is shown in Table 1. These values do not take the type of substrate into account. Variance analyses show significant differences between the three methods employed (p = 0.001). Each method differs from the two others: method A differs from method B (p = 0.001) and method C (p = 0.014) and method B differs from method C (p = 0.001) (Table 2). The amount of DNA obtained by method C using a combination of phenol–chloroform extraction and QIAamp DNA mini1 Kit (Qiagen) purification (p < 0.05) was higher than that obtained by methods A and B with p < 0.05. The mean values of DNA extracted from the two types of substrates is shown in Table 3. These values do not take the type of protocol used into account. Variances analyses (Table 4) show that the DNA quantity extracted from liver is more important than that extracted from the heart with statistical difference (p = 0.022). 3.2. Autosomal STR analysis The DNA profiling results were compared to the genotypes of all persons involved in processing samples. No match was observed, indicating that the precautions taken against the risk of sample contamination were efficient. The molecular and morphological sex determinations were in accordance for all samples, showing the authenticity of amplification products [17]. Autosomal STR typing gave partial allelic profiles for all subjects. Larger sized loci (more than 300 bases) always failed to amplify suggesting that DNA extracted from FF-PET was degraded. Repeated amplifications showed that the degree of reproducibility was high, indicating that despite DNA degradation, the DNA molecules present in these samples had a high quality standard. 4. Discussion Genomic DNA isolated from FF-PET has important applications in retrospective epidemiologic studies, notably in sudden unexplained death [18,19], in which most often FF-PET is the unique source of genetic material. Using paraffin-embedded material is also useful in prospective studies according to the facility to collect tissues from already existing archives in comparison to frozen fresh tissues that need a specific collection, with dedicated spaces and specific equipment. It is well known that the quality and quantity of nucleic acid extracted from archival tissue are highly dependent on the fixative procedure [11]. The samples used in this study were fixed with buffered formaldehyde during approximately 30 days that corre-
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spond to the fixative time where DNA could be detected in different tissues such as cardiac muscle, skeletal muscle, liver, kidney and brain as demonstrated previously [20,21]. According to Carturan et al. [10] different tissues show different yields of DNA. In this study, we found that DNA quantity extracted from liver is more important than DNA extracted from heart with statistical difference (p < 0.05). Such a result might be explained by the fact that the number of polyploidy cells averages 30–40% in the adult liver [22]. We could logically think that the use of two purification steps (phenol–chloroform followed by silica matrix in methods A and C) instead of one (silica matrix alone in method B) could lead to lower DNA recovery, because of DNA loss on each step. However we concluded that the method C, using phenol–chloroform and QIAamp DNA mini1 Kit (Qiagen), was the most effective DNA extraction and purification method. There are at least two different explanations of these results. The first one is that the phenol– chloroform purification probably has constituted an important step for the removal of potential contaminants from aqueous phase that could interfere with the subsequent DNA binding capacity of the silica matrix. The second one is that the phenol–chloroform and the silica matrix steps might remove potential PCR inhibitors allowing successful PCR amplification [23]. This method allowed to obtain the highest amount of DNA among the three methods tested, with statistical difference (p < 0.05). It was possible to amplify DNA from all samples but PCR amplification strength is inversely related to product size which is a typical molecular behavior of degraded DNA. Such result is typically obtained from degraded DNA [6]. Indeed, Lehmann and Kreipe [2] described that the average fragment length of DNA is 300–400 bases in biopsy tissues, but much shorter in postmortem paraffin wax embedded tissues. Despite DNA degradation, repeated amplifications showed that the degree of reproducibility was high, indicating that, the DNA molecules present in these samples were of good quality. As far as we know, this is the first report where association of phenol–chloroform and silica matrix in DNA extraction and purification from FF-PET is used. However, the use of FF-PET for retrospective studies requires the use of primers that generate small amplification products, because larger DNA fragments are more difficult to amplify [24]. In conclusion, the protocol presented in this study, previously described in articles on ancient DNA, should be widely exploited in different molecular studies involving FF-PET material. Acknowledgement We gratefully thank Jean Luc Fausser for his technical help. References [1] M.Y. Feldman, Reactions of nucleic acids and nucleoproteins with formaldehyde, Prog. Nucleic Acid Res. Mol. Biol. 13 (1973) 1–49. [2] U. Lehmann, H. Kreipe, Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies, Methods 25 (2001) 409–418. [3] W. Cao, M. Hashibe, J.Y. Rao, H. Morgenstern, Z.F. Zhang, Comparison of methods for DNA extraction from paraffin-embedded tissues and buccal cells, Cancer Detect. Prev. 27 (2003) 397–404. [4] R. Coura, J.C. Prolla, L. Meurer, P. Ashton-Prolla, An alternative protocol for DNA extraction from formalin-fixed and paraffin wax embedded tissue, J. Clin. Pathol. 58 (2005) 894–895. [5] S. Bonin, F. Petrera, B. Niccolini, G. Stanta, PCR analysis in archival postmortem tissues, Mol. Pathol. 56 (2003) 184–186. [6] E.R. Rivero, A.C. Neves, M.G. Silva-Valenzuela, S.O. Sousa, F.D. Nunes, Simple salting-out method for DNA extraction from formalin-fixed, paraffin-embedded tissues, Pathol. Res. Pract. 202 (2006) 523–529. [7] Y. Sato, R. Sugie, B. Tsuchiya, T. Kameya, M. Natori, K. Mukai, Comparison of the DNA extraction methods for polymerase chain reaction amplification from formalinfixed and paraffin-embedded tissues, Diagn. Mol. Pathol. 10 (2001) 265–271. [8] M.C. Santos, C.P. Saito, S.R. Line, Extraction of genomic DNA from paraffinembedded tissue sections of human fetuses fixed and stored in formalin for long periods, Pathol. Res. Pract. 204 (2008) 633–636.
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[9] T.S. Frank, S.M. Svoboda-Newman, E.D. Hsi, Comparison of methods for extracting DNA from formalin-fixed paraffin sections for nonisotopic PCR, Diagn. Mol. Pathol. 5 (1996) 220–224. [10] E. Carturan, D.J. Tester, B.C. Brost, C. Basso, G. Thiene, M.J. Ackerman, Postmortem genetic testing for conventional autopsy-negative sudden unexplained death: an evaluation of different DNA extraction protocols and the feasibility of mutational analysis from archival paraffin-embedded heart tissue, Am. J. Clin. Pathol. 129 (2008) 391–397. [11] M.T. Gilbert, T. Haselkorn, M. Bunce, J.J. Sanchez, S.B. Lucas, L.D. Jewell, E. Van Marck, M. Worobey, The isolation of nucleic acids from fixed, paraffin-embedded tissues—which methods are useful when? PLoS ONE 2 (2007) e537. [12] C. Keyser-Tracqui, B. Ludes, Methods for the study of ancient DNA, Methods Mol. Biol. 297 (2005) 253–264. [13] C. Keyser-Tracqui, E. Crube´zy, B. Ludes, Nuclear and mitochondrial DNA analysis of a 2,000-year-old necropolis in the Egyin Gol Valley of Mongolia, Am. J. Hum. Genet. 73 (2003) 247–260. [14] F.X. Ricaut, C. Keyser-Tracqui, E. Crube´zy, B. Ludes, STR-genotyping from human medieval tooth and bone samples, Forensic Sci. Int. 151 (2005) 31–35. [15] F.X. Ricaut, S. Kolodesnikov, C. Keyser-Tracqui, A.N. Alekseev, E. Crube´zy, B. Ludes, Molecular genetic analysis of 400-year-old human remains found in two Yakut burial sites, Am. J. Phys. Anthropol. 129 (2006) 55–63. [16] S.E. Goelz, S.R. Hamilton, B. Vogelstein, Purification of DNA from formaldehyde fixed and paraffin-embedded human tissue, Biochem. Biophys. Res. Commun. 130 (1985) 118–126.
[17] E. Meyer, M. Wiese, H. Bruchhaus, M. Claussen, A. Klein, Extraction and amplification of authentic DNA from ancient human remains, Forensic Sci. Int. 113 (2000) 87–90. [18] A. Doolan, N. Langlois, C. Chiu, J. Ingles, J.M. Lind, C. Semsarian, Postmortem molecular analysis of KCNQ1 and SCN5A genes in sudden unexplained death in young Australians, Int. J. Cardiol. 127 (2008) 138–141. [19] S.S. Chugh, O. Senashova, A. Watts, P.T. Tran, Z. Zhou, Q. Gong, J.L. Titus, S.J. Hayflick, Postmortem molecular screening in unexplained sudden death, J. Am. Coll. Cardiol. 43 (2004) 1625–1629. [20] C.E. Greer, J.K. Lund, M.M. Manos, PCR amplification from paraffin-embedded tissues: recommendations on fixatives for long-term storage and prospective studies, PCR Methods Appl. 1 (1991) 46–50. [21] F. Miething, S. Hering, B. Hanschke, J. Dressler, Effect of fixation to the degradation of nuclear and mitochondrial DNA in different tissues, J. Histochem. Cytochem. 54 (2006) 371–374. [22] B.N. Kudryavtsev, M.V. Kudryavtseva, G.A. Sakuta, G.I. Stein, Human hepatocyte polyploidization kinetics in the course of life cycle, Virchows Arch. B: Cell Pathol. Incl. Mol. Pathol. 64 (1993) 387–393. [23] D.P. Jackson, F.A. Lewis, G.R. Taylor, A.W. Boylston, P. Quirke, Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction, J. Clin. Pathol. 43 (1990) 499–504. [24] P.J. Coates, A.J. d’Ardenne, G. Khan, H.O. Kangro, G. Slavin, Simplified procedures for applying the polymerase chain reaction to routinely fixed paraffin wax sections, J. Clin. Pathol. 44 (1991) 115–118.
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Rapid communication
Efficiency evaluation of a DNA extraction and purification protocol on archival formalin-fixed and paraffin-embedded tissue A. Farrugia *, C. Keyser, B. Ludes Institute of Legal Medicine, EA4438, University of Strasbourg, 11 rue Human 67085, Strasbourg Cedex, France
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 February 2009 Received in revised form 29 June 2009 Accepted 2 September 2009 Available online 24 September 2009
Formalin-fixed and paraffin-embedded tissue (FF-PET) is an invaluable resource for retrospective molecular genetic studies, but the extraction of high-quality genomic DNA from FF-PET is still a problematic issue. Despite the range of DNA extraction methods currently in use, the association of phenol–chloroform extraction and silica-based purification protocols, reported in ancient DNA studies on archaeological bones, has, to our knowledge, not been used for DNA extraction from FF-PET yet. The present study compared the efficiency of three DNA extraction and purification protocols from two different FF-PET substrates, heart and liver, by using quantitative PCR and multiplex amplification. We showed that the method, using phenol–chloroform and the QIAamp DNA mini1 Kit (Qiagen), was the most effective DNA extraction and purification method and that the DNA quantity extracted from liver is statistically more important than that extracted from heart. Autosomal STR typing by multiplex amplifications gave partial allelic profiles with only small size products (less than 300 bases) amplified, suggesting that DNA extracted from FF-PET was degraded. In conclusion, the protocol presented here, previously described in studies on ancient bones, should find application in different molecular studies involving FF-PET material. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Formalin-fixed and paraffin-embedded Tissue DNA extraction Phenol–chloroform Silica based-spin columns
1. Introduction The extraction of DNA from formalin-fixed and paraffinembedded tissue (FF-PET) enables pathologists to use archival material for a variety of purposes including retrospective molecular genetic studies based on DNA amplification by polymerase chain reaction (PCR). However, there are several reasons for the failure of PCR using DNA isolated from FF-PET: (i) the generation of DNA–protein cross-linkages due to formaldehyde solution resulting in nucleic acid fragmentation [1,2], (ii) the presence of remnants of substances that inhibit the amplification reaction such as formalin [3] or inhibit the proteinase K used in the extraction procedure such as xylene [4], (iii) the risk of contamination during manipulation of samples. Because of these difficulties, number of DNA extraction methods have been tested and several studies comparing different methods have been published such as phenol–chloroform method [3–7], ammonium acetate precipitation [8,6], ethanol precipitation [9] and commercially available kits based on silica binding principle [3–11].
* Corresponding author. Tel.: +33 368853363; fax: +33 368853362. E-mail address: [email protected] (A. Farrugia). 0379-0738/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2009.09.004
The combination of phenol–chloroform extraction and silicabased purification method, developed for DNA extraction from ancient bones [12] has to your knowledge, not yet been tested on FF-PET. The aim of this work was to test the efficiency of such protocol on FF-PET. Indeed, one of the major difficulties in using FF-PET is the degradation of nucleic acids [5] like in archeological biological remains. For this purpose we compared the use of organic extraction coupled with silica-based purification method using two different commercial kits, CleanMix (Talent) [13–15] and QIAamp DNA mini1 Kit (Qiagen) to the most popular method based on direct use of the commercial QIAamp DNA mini1 Kit (Qiagen). Tests were performed from two types of substrates, heart and liver from 22 individuals. 2. Materials and methods 2.1. Samples Twenty two formalin-fixed and paraffin-embedded (FF-PE) tissue blocks from heart and liver autopsy tissue were obtained from the Forensic Institute of Strasbourg, France. The preparation of the FF-PE block was done with the same seize of fresh tissue (1.5 cm wide, 1.5 cm long, and 0.3 cm of thickness). All of them had been fixed in 10% buffered formalin. The average period of fixation was 34.27 days (min = 28 days and max = 51 days) at room temperature. This formalin fixation time usually corresponds to the time of tissue fixation before inclusion in paraffin in our Medico-legal Institute. The time elapsing between autopsy and tissue sampling was less than three days.
A. Farrugia et al. / Forensic Science International 194 (2010) e25–e28
e26
After the formalin fixation and paraffin inclusion steps, the surrounding paraffin was removed with a scalpel and the center of the organ was trimmed to a surface of 1 cm2 of tissue. Then using a standard microtome with disposable blade, serial sections of 50 mm thickness from each FF-PE blocks were obtained and placed in a microcentrifuge tube. Each sample weigh ranged from 38 to 57 mg. Because of the low density of paraffin wax (inferior to water’s density), we have considered that the presence of paraffin in our samples had not affected the measurements of tissue weight. Great care was taken to prevent cross-contamination between samples by using fresh blades for each specimen and by cleaning the microtome with DNAse Away (Molecular Bioproducts) between specimens. 2.2. DNA deparaffinization and rehydratation The technique for paraffin removal was based on that described by Goelz et al. [16] using two xylene washes (1 mL of xylene for 10 min at room temperature), followed by decreasing concentration of alcohol (ethanol, 100%, 95%, 70%) and then two distilled water washes [4]. Then samples were rehydrated with Tris/EDTA (Tris 100 mM, pH7.5, EDTA 5 M) for 5 min at room temperature and Tris buffered solution (100 mM, pH7.5) for 20 h at 55 8C with four changes of solution. To avoid loss of sample, centrifugation (13,500 rpm, 5 min) was performed before each change of solution [4]. 2.3. DNA extraction and purification: three protocols The material studied was submitted to one of the following DNA extraction and purification methods. 2.3.1. Protocol A: phenol–chloroform followed by the use of the CleanMix Kit [13] Tissue digestion was realized by incubation of the samples at 55 8C overnight with 300 mL of lysis-buffer (100 mM Tris–HCl (pH 8.0), 5 mM EDTA, 1% sodium dodecyl sulfate, 1 mg/mL proteinase K). After incubation, a phenol/chloroform/ isoamyl alcohol (25/24/1, v/v) extraction was performed on the supernatant. The aqueous phase was then purified with the CleanMix purification kit according to the manufacturer’s instructions (Talent, Trieste, Italy).
Fig. 1. Yield of DNA extracted from heart and liver with three different protocols: A = phenol–chloroform extraction followed by the use of the commercial CleanMix Kit, B = direct use of the commercial QIAamp DNA mini1 Kit, C = phenol– chloroform extraction followed by the use of the commercial QIAamp DNA mini1 Kit. PCR products was performed by capillary electrophoresis on an ABI PRISM1 3100 genetic analyzer (Applied Biosystems), and genotyping was made with the Gene Mapper v1.02 (Applied Biosystems). Genotypes of all persons involved in the processing of the samples were determined and compared to the results obtained from each FF-PET samples. 2.5. Concentration measurements DNA was quantified by real-time PCR using QuantifilerTM Human DNA Quantification Kit (Applied Biosystems, AB, Foster City, CA) with the ABI PRISM1 7000 Sequence Detection System (AB) following the manufacturer’s recommendations.
2.3.2. Protocol B: direct use of the QIAamp DNA mini1 Kit (Qiagen) The extraction and purification procedure was performed according to the manufacturer’s instructions (QIAamp DNA mini1 Kit, Qiagen). Briefly, samples were digested with Qiagen Proteinase K and lysis-buffer at 55 8C overnight and loaded onto spin columns. DNA was then adsorbed by short centrifugation onto the QIAamp silica membrane, washed and eluted with 100 mL water.
2.6. Statistical methods
2.3.3. Protocol C: phenol–chloroform followed by the use of the QIAamp DNA mini1 Kit (Qiagen) This method was performed as described above, but with an additional step. After proteinase K digestion, a phenol–chloroform–isoamyl alcohol purification was added as in protocol A. The supernatant was carefully removed and the next steps of the QIAamp DNA mini1 Kit (Qiagen) were performed according to the manufacturer’s instructions (QIAamp). In total 132 DNA extractions were performed, sourced from 22 blocks of heart and liver by using the three above mentioned methods.
3. Results
2.4. Polymerase chain reaction and typing
Quantitative data were submitted to a variance analysis to detect differences among the amount of DNA obtained from different extraction method and from different substrates. For statistical analysis, we used SPSS for Windows. Differences were considered statistically significant when the probability (p) was less than 0.05.
3.1. Yield of DNA The amount of DNA extracted from each FF-PET heart and liver sample by each method is shown in Fig. 1. The mean values of DNA Table 1 Mean values of DNA extracted (mg/100 mg of tissue) with three protocols without taken into account the type of substrates. Protocol
To detect potential cross-contamination and to evaluate the efficiency of the amplification, the DNA extracts were typed with AmpFlSTR1 IdentifilerTM Amplification Kit according to the manufacturer’s recommendation (Applied Biosystems). Fifteen Short Tandem Repeats (STRs) loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA) and the gender-identification locus amelogenine were simultaneously amplified using Biometra T3TM (Biometra1). The separation of the
A B C
Mean
Standard error
13.997 5.894 30.104
1.405 1.675 6.676
95% Confidence intervals (CIs) Minimum
Maximum
11.075 2.411 16.221
16.919 9.377 43.987
Table 2 Comparison of mean values differences between the three protocols (based on the estimated marginal means). Protocol (I)
Protocol (J)
A
B C A C A B
B C * a
Mean differences (I
8.103* 16.107* 8.103* 24.210* 16.107* 24.210*
J)
Standard error
Significationa
2.189 6.043 2.189 6.242 6.043 6.242
0.001* 0.014* 0.001* 0.001* 0.014* 0.001*
The difference in averages is significant for p = 0.05. Adjustment of multiple comparisons: the least significant difference (equivalent to no adjustment).
95% CIs of the mean differencesa Minimum
Maximum
3.551 28.673 12.655 37.191 3.540 11.228
12.655 3.540 3.551 11.228 28.673 37.191
A. Farrugia et al. / Forensic Science International 194 (2010) e25–e28 Table 3 Mean of DNA extracted (mg/100 mg of tissue) from heart and liver without taken into account the type of protocol used. Tissue
Heart Liver
Mean
13.645 19.685
Standard error
2.401 3.502
95% CIs Minimum
Maximum
8.652 12.402
18.638 26.969
Table 4 Comparison of mean differences between heart and liver (based on the estimated marginal means). Tissue (I)
Standard Significationa 95% CIs of the mean Tissue Mean (J) differences error differencesa (I J)
Heart Liver
Liver Heart
Minimum Maximum 6.041 6.041
2.438 2.438
0.022* 0.022*
11.111 0.971
0.971 11.111
*
The difference in averages is significant for p = 0.05. Adjustment of multiple comparisons: the least significant difference (equivalent to no adjustment). a
extracted is shown in Table 1. These values do not take the type of substrate into account. Variance analyses show significant differences between the three methods employed (p = 0.001). Each method differs from the two others: method A differs from method B (p = 0.001) and method C (p = 0.014) and method B differs from method C (p = 0.001) (Table 2). The amount of DNA obtained by method C using a combination of phenol–chloroform extraction and QIAamp DNA mini1 Kit (Qiagen) purification (p < 0.05) was higher than that obtained by methods A and B with p < 0.05. The mean values of DNA extracted from the two types of substrates is shown in Table 3. These values do not take the type of protocol used into account. Variances analyses (Table 4) show that the DNA quantity extracted from liver is more important than that extracted from the heart with statistical difference (p = 0.022). 3.2. Autosomal STR analysis The DNA profiling results were compared to the genotypes of all persons involved in processing samples. No match was observed, indicating that the precautions taken against the risk of sample contamination were efficient. The molecular and morphological sex determinations were in accordance for all samples, showing the authenticity of amplification products [17]. Autosomal STR typing gave partial allelic profiles for all subjects. Larger sized loci (more than 300 bases) always failed to amplify suggesting that DNA extracted from FF-PET was degraded. Repeated amplifications showed that the degree of reproducibility was high, indicating that despite DNA degradation, the DNA molecules present in these samples had a high quality standard. 4. Discussion Genomic DNA isolated from FF-PET has important applications in retrospective epidemiologic studies, notably in sudden unexplained death [18,19], in which most often FF-PET is the unique source of genetic material. Using paraffin-embedded material is also useful in prospective studies according to the facility to collect tissues from already existing archives in comparison to frozen fresh tissues that need a specific collection, with dedicated spaces and specific equipment. It is well known that the quality and quantity of nucleic acid extracted from archival tissue are highly dependent on the fixative procedure [11]. The samples used in this study were fixed with buffered formaldehyde during approximately 30 days that corre-
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spond to the fixative time where DNA could be detected in different tissues such as cardiac muscle, skeletal muscle, liver, kidney and brain as demonstrated previously [20,21]. According to Carturan et al. [10] different tissues show different yields of DNA. In this study, we found that DNA quantity extracted from liver is more important than DNA extracted from heart with statistical difference (p < 0.05). Such a result might be explained by the fact that the number of polyploidy cells averages 30–40% in the adult liver [22]. We could logically think that the use of two purification steps (phenol–chloroform followed by silica matrix in methods A and C) instead of one (silica matrix alone in method B) could lead to lower DNA recovery, because of DNA loss on each step. However we concluded that the method C, using phenol–chloroform and QIAamp DNA mini1 Kit (Qiagen), was the most effective DNA extraction and purification method. There are at least two different explanations of these results. The first one is that the phenol– chloroform purification probably has constituted an important step for the removal of potential contaminants from aqueous phase that could interfere with the subsequent DNA binding capacity of the silica matrix. The second one is that the phenol–chloroform and the silica matrix steps might remove potential PCR inhibitors allowing successful PCR amplification [23]. This method allowed to obtain the highest amount of DNA among the three methods tested, with statistical difference (p < 0.05). It was possible to amplify DNA from all samples but PCR amplification strength is inversely related to product size which is a typical molecular behavior of degraded DNA. Such result is typically obtained from degraded DNA [6]. Indeed, Lehmann and Kreipe [2] described that the average fragment length of DNA is 300–400 bases in biopsy tissues, but much shorter in postmortem paraffin wax embedded tissues. Despite DNA degradation, repeated amplifications showed that the degree of reproducibility was high, indicating that, the DNA molecules present in these samples were of good quality. As far as we know, this is the first report where association of phenol–chloroform and silica matrix in DNA extraction and purification from FF-PET is used. However, the use of FF-PET for retrospective studies requires the use of primers that generate small amplification products, because larger DNA fragments are more difficult to amplify [24]. In conclusion, the protocol presented in this study, previously described in articles on ancient DNA, should be widely exploited in different molecular studies involving FF-PET material. Acknowledgement We gratefully thank Jean Luc Fausser for his technical help. References [1] M.Y. Feldman, Reactions of nucleic acids and nucleoproteins with formaldehyde, Prog. Nucleic Acid Res. Mol. Biol. 13 (1973) 1–49. [2] U. Lehmann, H. Kreipe, Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies, Methods 25 (2001) 409–418. [3] W. Cao, M. Hashibe, J.Y. Rao, H. Morgenstern, Z.F. Zhang, Comparison of methods for DNA extraction from paraffin-embedded tissues and buccal cells, Cancer Detect. Prev. 27 (2003) 397–404. [4] R. Coura, J.C. Prolla, L. Meurer, P. Ashton-Prolla, An alternative protocol for DNA extraction from formalin-fixed and paraffin wax embedded tissue, J. Clin. Pathol. 58 (2005) 894–895. [5] S. Bonin, F. Petrera, B. Niccolini, G. Stanta, PCR analysis in archival postmortem tissues, Mol. Pathol. 56 (2003) 184–186. [6] E.R. Rivero, A.C. Neves, M.G. Silva-Valenzuela, S.O. Sousa, F.D. Nunes, Simple salting-out method for DNA extraction from formalin-fixed, paraffin-embedded tissues, Pathol. Res. Pract. 202 (2006) 523–529. [7] Y. Sato, R. Sugie, B. Tsuchiya, T. Kameya, M. Natori, K. Mukai, Comparison of the DNA extraction methods for polymerase chain reaction amplification from formalinfixed and paraffin-embedded tissues, Diagn. Mol. Pathol. 10 (2001) 265–271. [8] M.C. Santos, C.P. Saito, S.R. Line, Extraction of genomic DNA from paraffinembedded tissue sections of human fetuses fixed and stored in formalin for long periods, Pathol. Res. Pract. 204 (2008) 633–636.
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