- Email: [email protected]
A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle
Accepted Manuscript Notes & tips A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle Gipsy Majumdar, Santiago Vera, Marshall B. Elam, Rajendra Raghow PII: DOI: Reference:
S0003-2697(15)00003-2 http://dx.doi.org/10.1016/j.ab.2014.12.021 YABIO 11942
To appear in:
Analytical Biochemistry
Received Date: Accepted Date:
21 November 2014 30 December 2014
Please cite this article as: G. Majumdar, S. Vera, M.B. Elam, R. Raghow, A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle, Analytical Biochemistry (2015), doi: http:// dx.doi.org/10.1016/j.ab.2014.12.021
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle
Gipsy Majumdara,b*, Santiago Veraa, Marshall B Elama,b and Rajendra Raghowa,b
a
Department of Veterans Affairs Medical Center, 1030 Jefferson Avenue, Memphis, Tennessee 38104, USA and bDepartment of Pharmacology, College of Medicine, The University of Tennessee Health Science Center, 874 Union Avenue, Memphis, Tennessee 38163, USA
Short Title: Protocol for effective extraction of RNA and DNA
*Corresponding author Research Service (151), DVA Medical Center 130 Jefferson Avenue, Memphis Tennessee 38104, USA Fax: 901-522-7275 E-mail address: [email protected]
Subject Category DNA Recombinant Techniques and Nucleic Acids
Abstract We combined the TRIzolTM method of nucleic acid extraction with QIAmpTM columns to achieve co-extraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at -80oC for many months. Total RNA was recovered from the upper aqueous phase of TRIzolTM. The interphase and organic phases were precipitated with ethanol, digested with proteinase K and filtered through QIAamp MinElute ColumnTM to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues.
Key words: TRIzolTM QIAmpTM DNA/RNA extraction Peripheral blood mononuclear cells Skeletal muscle biopsy
2
Archived human blood and tissues, with parallel clinical follow-ups, represent a valuable resource for a high throughput analysis of the genome and its potential to generate phenotype [1-3]. In recent years, the use of commercial kits designed for the purpose of extraction of nucleic acids from cells and tissues is also increasing in popularity [2, 4-6]. The availability of robust methods to recover nucleic acids and proteins from fresh or archived tissues has greatly facilitated the emergence of genomic and personalized medicine [7]. A common method for simultaneous extraction of RNA, DNA and protein from blood and other tissues uses the TRIzolTM reagent (www.lifetechnologies.com), based on the original protocol described by Chomczynski and Sacchi [8, 9]. This method involves extraction of tissues in a buffered-phenol and guanidine isothiocynate solutions followed by extraction with chloroform that facilitates separation of the aqueous and organic phases demarcated by an inter-phase. RNA is recovered from the upper aqueous phase whereas genomic DNA and proteins are extracted from the lower two phases. Simultaneous extraction of tissues by TRIzolTM reagent leads to variable recovery of intact RNA and DNA; quality and quantity of recovered nucleic acids become even more problematic with archived tissues, especially if these tissues had been fixed with formaldehyde [6]. We encountered this problem firsthand in our attempts to concomitantly recover RNA and genomic DNA from human peripheral blood mononuclear cells (PBMCs) and skeletal muscle. This experience led us to devise a streamlined protocol that combines RNA extraction using TRIzol reagent followed by recovery of genomic DNA from the organic phase using QIAamp MinElute columns (www.qiagen.com). We began these studies with an aim to compare the genomes (via exome sequencing), transcriptomes (microarray and RNAseq analysis) and proteomes of PBMCs and skeletal muscle obtained from statin-tolerant and statin-intolerant patients. Since blood and skeletal muscle biopsies were collected over a period of several months these samples were processed to optimally preserve the quality and quantity of nucleic acids. The PBMCs from the blood (8-10 ml/patient) were captured on LeukoLOCK filters (Ambion, Life Technologies, CA). The filters were sequentially flushed with 3 ml each of
3
PBS (phosphate buffered saline) and RNAlater to remove red blood cells and stabilize RNA, respectively; filter-bound PBMCs were stored at -80oC. The skeletal muscle samples (10-30 mg/patient) were snap-frozen in liquid nitrogen immediately after biopsy and stored at -80oC. To extract RNA, archived PBMCs were lysed and flushed off the LeukoLOCK filters with 4 ml TRIzol reagent. The aqueous layer (2 ml) was mixed with 1 ml of nuclease-free water (0.5 volume) followed by addition of 3.75 ml of absolute ethanol (1.25 volumes) and the combined solution was filtered through a spin cartridge (Ambion, Life Technologies, CA). On hundred µl of water was used to elute RNA and it’s yield and quality were assessed. The A260/A280 and A260/A230 ratios of RNA samples were 2.07±0.1 and 2.03±0.26, respectively. Thus, both absorbance ratios of RNA recovered from LeukoLOCK filter-bound PBMCs stored >1 year at -800C were within the expected range for pure RNA. Ethidium bromide fluorescence of total RNA electrophoresed on agarose gels from 10 individual patient samples is shown (Figure 1A) to illustrate the variability seen from one sample to the other. We should emphasize that even after pipetting out the aqueous phase conservatively to avoid contamination, we routinely recovered more than sufficient amount of RNA (15-25 µg) to carry out microarray and RNAseq analyses from ~8 ml of blood. The TRIzol extraction method also yielded good quality of RNA from biopsied muscle although the total amount of RNA recovered from muscle ranged from 2.7-10.8 µg per 10 mg of skeletal muscle stored at -800C. For skeletal muscle RNA, the average A260/A280 and A260/A230 ratios of 1.9±0.14 and 1.7±0.14, respectively, were also similar to the values obtained with RNA from PBMCs. As judged by 28S and 18S ribosomal RNA bands, adequate amount of RNA was recovered from 4 out of 5 muscle samples (Figure 1A). These data are consistent with numerous published studies indicating that TRIzol extraction method works with well diverse tissues, both fresh and archived. However, we encountered serious problems when we attempted to recover genomic DNA from PBMCs or muscle after extraction of RNA by the TRIzol method. To recover genomic DNA from PBMCs that had undergone TRIzol extraction, we followed the manufacturer’s protocol. We precipitated the combined interphase and phenol phases with absolute ethanol (0.3 ml/ml of TRIzol), pelleted the precipitate by
4
centrifugation and solubilized the pellet in 8 mM NaOH. We adjusted the pH of the solution to 8.0 with 0.1 M HEPES. Based on the A260/A280 ratio of 1.01±0.08 and A260/A230 ratio of 0.16±0.03 of the solubilized “DNA”, measured using a NanoDrop 200 UV-Vis spectrometer (www.nanodrop.com), we suspected the presence of non-DNA contaminants that was corroborated by agarose gel electrophoresis (Figure 1B, lane 2). When DNA in these samples was concentrated by ammonium acetate/ethanol precipitation, we failed to recover EtBr-stainable DNA in agarose gels (Figure 1B, lane 3). Similarly, detectable amounts of DNA from these samples could not be recovered even after extraction with the “classical” phenol/chloroform/isoamyl alcohol (PCI) extraction (Figure 1B, lane 4). When PBMCs collected on the LeukoLock filters from fresh blood or cultured H9c2 cardiac myocytes (data not shown) were sequentially extracted with TRIzol followed by PCI extraction, the quality (as judged by A260/A280 ratio) and yield of the genomic DNA were very poor (Figure 1, lane 5). In contrast, DNA extracted from PBMCs (Figure 1, lane 1) or H9c2 cells (Figure 2, lane 2) by PCI method (without prior TRIzol extraction) was good both in quality and quantity. Based on these data we surmised that solubilizing the pellet in NaOH-HEPES buffer led to recovery of contaminated DNA that was not suitable for enzymatic manipulations (PCR, exome sequencing). After a number of trials, we determined that the easiest way to remove contaminants was to take up the precipitated material in 180 µl of Buffer ATL and 20 µl proteinase K, and enzymatically digest it at 56°C overnight. This was followed by sequential addition of 200 µl Buffer AL (QiagenTM) and 200 µl of absolute ethanol and the entire solution was filtered through the QIAamp MinElute columns via centrifugation at 6000xg for 1 min. The filter was washed first with 500 µl Buffer AW1 followed by a second wash with 500 µl Buffer AW2 and DNA was eluted with 30 µl of 1mM TrisEDTA, pH 8.0. Electrophoresis of DNA samples extracted from H9c2 cells (Figure 2, lane 1) and archived patient PBMCs (Figure 2, lane 3, 5) on 1% agarose gels showed a distinct DNA band that is larger than10 kb. The A260/A280 ratio of 1.7 to 1.9 suggested that good quality of genomic DNA was recovered from archived PBMCs; genomic DNA from skeletal muscle also had an acceptable A260/A280 ratio of 1.8 (Figure 2, lane 7). We used the Qiagen DNA kit alone as a control for DNA recovery from mouse blood and
5
skeletal muscle (Figure 2, lanes 4 and 6). It should be noted however, that DNA yield from frozen muscle tissue by TRIzol plus Qiagen kit was 15-20 % less than the Qiagen protocol alone for DNA (2 to 3µg/10 mg of skeletal muscle), but this method gave us intact genomic DNA (120 -150 ng/10 mg) with acceptable integrity and simultaneous recovery of high quality RNA. These findings demonstrated that the quality of genomic DNA extracted from cells or archived blood leukocytes captured on LeukoLock filters and frozen muscle tissue when subjected to TRIzol in combination with the Qiagen kit was pure and suitable for genotyping assays and other applications. This streamlined procedure is easily adaptable to either freshly isolated or archive tissues.
Acknowledgements All clinical procedures were carried out according to the protocols approved by the Institutional Review Board of the University of Tennessee Health Science Center and Memphis VA Medical Center. The experiments outlined here were partially supported by grants from the Center for Translational Research, UTHSC and the Department of Veterans Affairs (DVA) to Marshall B. Elam and Rajendra Raghow. RR is a Senior Research Career Scientist of the DVA.
6
References
[1] H.V. Chetverina, M.V. Falaleeva, A.B. Chetverin, Simultaneous assay of DNA and RNA targets in the whole blood using novel isolation procedure and molecular colony amplification, Analytical biochemistry, 334 (2004) 376-381. [2] J.H. Jansen, B.A. van der Reijden, Isolation of RNA and DNA from leukocytes and cDNA synthesis, Methods in molecular medicine, 125 (2006) 1-11. [3] R. Klopfleisch, A.T. Weiss, A.D. Gruber, Excavation of a buried treasure--DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues, Histology and histopathology, 26 (2011) 797-810. [4] M. Bauer, D. Patzelt, A method for simultaneous RNA and DNA isolation from dried blood and semen stains, Forensic science international, 136 (2003) 76-78. [5] S. Bonin, F. Hlubek, J. Benhattar, C. Denkert, M. Dietel, P.L. Fernandez, G. Hofler, H. Kothmaier, B. Kruslin, C.M. Mazzanti, A. Perren, H. Popper, A. Scarpa, P. Soares, G. Stanta, P.J. Groenen, Multicentre validation study of nucleic acids extraction from FFPE tissues, Virchows Archiv : an international journal of pathology, 457 (2010) 309-317. [6] A. Kotorashvili, A. Ramnauth, C. Liu, J. Lin, K. Ye, R. Kim, R. Hazan, T. Rohan, S. Fineberg, O. Loudig, Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens, PloS one, 7 (2012) e34683. [7] J.J. McCarthy, H.L. McLeod, G.S. Ginsburg, Genomic medicine: a decade of successes, challenges, and opportunities, Science translational medicine, 5 (2013) 189sr184. [8] P. Chomczynski, A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples, BioTechniques, 15 (1993) 532-534, 536-537. [9] P. Chomczynski, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Analytical biochemistry, 162 (1987) 156-159.
7
Figure Legends Figure 1. Extraction of RNA and DNA from PBMCs and skeletal muscle by the TRIzol method. A. RNA extraction. Agarose gel electrophoresis and ethidium bromide staining is shown to demonstrate the variability of recovery and integrity of RNA from 10 individual PBMC samples (Top) and 5 individual samples of muscle tissue (Bottom). B. Poor recovery of DNA from patient PBMCs after TRIzol extraction of RNA. DNA from the lower two phases was recovered, solubilized in 8 mM NaOH and run on a 1% agarose gel. Lane 2 shows DNA from patient PBMCs stored at -80oC on LeukoLOCK filters. Lane 3 demonstrates ammonium acetate/ethanol precipitated DNA recovered from 8 mM NaOH-solubilized DNA from lane 2. Lane 4 shows PCI precipitation of soluble DNA in 8mM NaOH from lane 2. DNA from fresh PBMCs solubilized in 8 mM NaOH-HEPES buffer and subjected to PCI (Lane 5). DNA from fresh PBMCs directly subjected to PCI extraction (Lane 1). Figure 2. Extraction of DNA from PBMCs and skeletal muscle with combined TRIzol and QIAmp method. The agarose gel depicts DNA recovered from fresh H9c2 cells by TRIzol and QIAmp kit (Lane 1) or PCI extraction (Lane 2). Lane 3 shows purified DNA from archived patient PBMCs using TRIzol and QIAmp kit. Lane 4 from a parallel experiment shows DNA from mouse blood using mini QIAmp blood kit as control and Lane 5 depicts DNA from a patient blood using TRIzol and QIAmp kit. Lane 6 from another parallel experiment depicts DNA from fresh mouse skeletal muscle samples (12 mg) using only QIAmp kit as control and Lane 7 demonstrates DNA recovered from a patient frozen muscle sample (<10 mg) by TRIzol and QIAmp kit.
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Figure 2
S0003-2697(15)00003-2 http://dx.doi.org/10.1016/j.ab.2014.12.021 YABIO 11942
To appear in:
Analytical Biochemistry
Received Date: Accepted Date:
21 November 2014 30 December 2014
Please cite this article as: G. Majumdar, S. Vera, M.B. Elam, R. Raghow, A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle, Analytical Biochemistry (2015), doi: http:// dx.doi.org/10.1016/j.ab.2014.12.021
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle
Gipsy Majumdara,b*, Santiago Veraa, Marshall B Elama,b and Rajendra Raghowa,b
a
Department of Veterans Affairs Medical Center, 1030 Jefferson Avenue, Memphis, Tennessee 38104, USA and bDepartment of Pharmacology, College of Medicine, The University of Tennessee Health Science Center, 874 Union Avenue, Memphis, Tennessee 38163, USA
Short Title: Protocol for effective extraction of RNA and DNA
*Corresponding author Research Service (151), DVA Medical Center 130 Jefferson Avenue, Memphis Tennessee 38104, USA Fax: 901-522-7275 E-mail address: [email protected]
Subject Category DNA Recombinant Techniques and Nucleic Acids
Abstract We combined the TRIzolTM method of nucleic acid extraction with QIAmpTM columns to achieve co-extraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at -80oC for many months. Total RNA was recovered from the upper aqueous phase of TRIzolTM. The interphase and organic phases were precipitated with ethanol, digested with proteinase K and filtered through QIAamp MinElute ColumnTM to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues.
Key words: TRIzolTM QIAmpTM DNA/RNA extraction Peripheral blood mononuclear cells Skeletal muscle biopsy
2
Archived human blood and tissues, with parallel clinical follow-ups, represent a valuable resource for a high throughput analysis of the genome and its potential to generate phenotype [1-3]. In recent years, the use of commercial kits designed for the purpose of extraction of nucleic acids from cells and tissues is also increasing in popularity [2, 4-6]. The availability of robust methods to recover nucleic acids and proteins from fresh or archived tissues has greatly facilitated the emergence of genomic and personalized medicine [7]. A common method for simultaneous extraction of RNA, DNA and protein from blood and other tissues uses the TRIzolTM reagent (www.lifetechnologies.com), based on the original protocol described by Chomczynski and Sacchi [8, 9]. This method involves extraction of tissues in a buffered-phenol and guanidine isothiocynate solutions followed by extraction with chloroform that facilitates separation of the aqueous and organic phases demarcated by an inter-phase. RNA is recovered from the upper aqueous phase whereas genomic DNA and proteins are extracted from the lower two phases. Simultaneous extraction of tissues by TRIzolTM reagent leads to variable recovery of intact RNA and DNA; quality and quantity of recovered nucleic acids become even more problematic with archived tissues, especially if these tissues had been fixed with formaldehyde [6]. We encountered this problem firsthand in our attempts to concomitantly recover RNA and genomic DNA from human peripheral blood mononuclear cells (PBMCs) and skeletal muscle. This experience led us to devise a streamlined protocol that combines RNA extraction using TRIzol reagent followed by recovery of genomic DNA from the organic phase using QIAamp MinElute columns (www.qiagen.com). We began these studies with an aim to compare the genomes (via exome sequencing), transcriptomes (microarray and RNAseq analysis) and proteomes of PBMCs and skeletal muscle obtained from statin-tolerant and statin-intolerant patients. Since blood and skeletal muscle biopsies were collected over a period of several months these samples were processed to optimally preserve the quality and quantity of nucleic acids. The PBMCs from the blood (8-10 ml/patient) were captured on LeukoLOCK filters (Ambion, Life Technologies, CA). The filters were sequentially flushed with 3 ml each of
3
PBS (phosphate buffered saline) and RNAlater to remove red blood cells and stabilize RNA, respectively; filter-bound PBMCs were stored at -80oC. The skeletal muscle samples (10-30 mg/patient) were snap-frozen in liquid nitrogen immediately after biopsy and stored at -80oC. To extract RNA, archived PBMCs were lysed and flushed off the LeukoLOCK filters with 4 ml TRIzol reagent. The aqueous layer (2 ml) was mixed with 1 ml of nuclease-free water (0.5 volume) followed by addition of 3.75 ml of absolute ethanol (1.25 volumes) and the combined solution was filtered through a spin cartridge (Ambion, Life Technologies, CA). On hundred µl of water was used to elute RNA and it’s yield and quality were assessed. The A260/A280 and A260/A230 ratios of RNA samples were 2.07±0.1 and 2.03±0.26, respectively. Thus, both absorbance ratios of RNA recovered from LeukoLOCK filter-bound PBMCs stored >1 year at -800C were within the expected range for pure RNA. Ethidium bromide fluorescence of total RNA electrophoresed on agarose gels from 10 individual patient samples is shown (Figure 1A) to illustrate the variability seen from one sample to the other. We should emphasize that even after pipetting out the aqueous phase conservatively to avoid contamination, we routinely recovered more than sufficient amount of RNA (15-25 µg) to carry out microarray and RNAseq analyses from ~8 ml of blood. The TRIzol extraction method also yielded good quality of RNA from biopsied muscle although the total amount of RNA recovered from muscle ranged from 2.7-10.8 µg per 10 mg of skeletal muscle stored at -800C. For skeletal muscle RNA, the average A260/A280 and A260/A230 ratios of 1.9±0.14 and 1.7±0.14, respectively, were also similar to the values obtained with RNA from PBMCs. As judged by 28S and 18S ribosomal RNA bands, adequate amount of RNA was recovered from 4 out of 5 muscle samples (Figure 1A). These data are consistent with numerous published studies indicating that TRIzol extraction method works with well diverse tissues, both fresh and archived. However, we encountered serious problems when we attempted to recover genomic DNA from PBMCs or muscle after extraction of RNA by the TRIzol method. To recover genomic DNA from PBMCs that had undergone TRIzol extraction, we followed the manufacturer’s protocol. We precipitated the combined interphase and phenol phases with absolute ethanol (0.3 ml/ml of TRIzol), pelleted the precipitate by
4
centrifugation and solubilized the pellet in 8 mM NaOH. We adjusted the pH of the solution to 8.0 with 0.1 M HEPES. Based on the A260/A280 ratio of 1.01±0.08 and A260/A230 ratio of 0.16±0.03 of the solubilized “DNA”, measured using a NanoDrop 200 UV-Vis spectrometer (www.nanodrop.com), we suspected the presence of non-DNA contaminants that was corroborated by agarose gel electrophoresis (Figure 1B, lane 2). When DNA in these samples was concentrated by ammonium acetate/ethanol precipitation, we failed to recover EtBr-stainable DNA in agarose gels (Figure 1B, lane 3). Similarly, detectable amounts of DNA from these samples could not be recovered even after extraction with the “classical” phenol/chloroform/isoamyl alcohol (PCI) extraction (Figure 1B, lane 4). When PBMCs collected on the LeukoLock filters from fresh blood or cultured H9c2 cardiac myocytes (data not shown) were sequentially extracted with TRIzol followed by PCI extraction, the quality (as judged by A260/A280 ratio) and yield of the genomic DNA were very poor (Figure 1, lane 5). In contrast, DNA extracted from PBMCs (Figure 1, lane 1) or H9c2 cells (Figure 2, lane 2) by PCI method (without prior TRIzol extraction) was good both in quality and quantity. Based on these data we surmised that solubilizing the pellet in NaOH-HEPES buffer led to recovery of contaminated DNA that was not suitable for enzymatic manipulations (PCR, exome sequencing). After a number of trials, we determined that the easiest way to remove contaminants was to take up the precipitated material in 180 µl of Buffer ATL and 20 µl proteinase K, and enzymatically digest it at 56°C overnight. This was followed by sequential addition of 200 µl Buffer AL (QiagenTM) and 200 µl of absolute ethanol and the entire solution was filtered through the QIAamp MinElute columns via centrifugation at 6000xg for 1 min. The filter was washed first with 500 µl Buffer AW1 followed by a second wash with 500 µl Buffer AW2 and DNA was eluted with 30 µl of 1mM TrisEDTA, pH 8.0. Electrophoresis of DNA samples extracted from H9c2 cells (Figure 2, lane 1) and archived patient PBMCs (Figure 2, lane 3, 5) on 1% agarose gels showed a distinct DNA band that is larger than10 kb. The A260/A280 ratio of 1.7 to 1.9 suggested that good quality of genomic DNA was recovered from archived PBMCs; genomic DNA from skeletal muscle also had an acceptable A260/A280 ratio of 1.8 (Figure 2, lane 7). We used the Qiagen DNA kit alone as a control for DNA recovery from mouse blood and
5
skeletal muscle (Figure 2, lanes 4 and 6). It should be noted however, that DNA yield from frozen muscle tissue by TRIzol plus Qiagen kit was 15-20 % less than the Qiagen protocol alone for DNA (2 to 3µg/10 mg of skeletal muscle), but this method gave us intact genomic DNA (120 -150 ng/10 mg) with acceptable integrity and simultaneous recovery of high quality RNA. These findings demonstrated that the quality of genomic DNA extracted from cells or archived blood leukocytes captured on LeukoLock filters and frozen muscle tissue when subjected to TRIzol in combination with the Qiagen kit was pure and suitable for genotyping assays and other applications. This streamlined procedure is easily adaptable to either freshly isolated or archive tissues.
Acknowledgements All clinical procedures were carried out according to the protocols approved by the Institutional Review Board of the University of Tennessee Health Science Center and Memphis VA Medical Center. The experiments outlined here were partially supported by grants from the Center for Translational Research, UTHSC and the Department of Veterans Affairs (DVA) to Marshall B. Elam and Rajendra Raghow. RR is a Senior Research Career Scientist of the DVA.
6
References
[1] H.V. Chetverina, M.V. Falaleeva, A.B. Chetverin, Simultaneous assay of DNA and RNA targets in the whole blood using novel isolation procedure and molecular colony amplification, Analytical biochemistry, 334 (2004) 376-381. [2] J.H. Jansen, B.A. van der Reijden, Isolation of RNA and DNA from leukocytes and cDNA synthesis, Methods in molecular medicine, 125 (2006) 1-11. [3] R. Klopfleisch, A.T. Weiss, A.D. Gruber, Excavation of a buried treasure--DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues, Histology and histopathology, 26 (2011) 797-810. [4] M. Bauer, D. Patzelt, A method for simultaneous RNA and DNA isolation from dried blood and semen stains, Forensic science international, 136 (2003) 76-78. [5] S. Bonin, F. Hlubek, J. Benhattar, C. Denkert, M. Dietel, P.L. Fernandez, G. Hofler, H. Kothmaier, B. Kruslin, C.M. Mazzanti, A. Perren, H. Popper, A. Scarpa, P. Soares, G. Stanta, P.J. Groenen, Multicentre validation study of nucleic acids extraction from FFPE tissues, Virchows Archiv : an international journal of pathology, 457 (2010) 309-317. [6] A. Kotorashvili, A. Ramnauth, C. Liu, J. Lin, K. Ye, R. Kim, R. Hazan, T. Rohan, S. Fineberg, O. Loudig, Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens, PloS one, 7 (2012) e34683. [7] J.J. McCarthy, H.L. McLeod, G.S. Ginsburg, Genomic medicine: a decade of successes, challenges, and opportunities, Science translational medicine, 5 (2013) 189sr184. [8] P. Chomczynski, A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples, BioTechniques, 15 (1993) 532-534, 536-537. [9] P. Chomczynski, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Analytical biochemistry, 162 (1987) 156-159.
7
Figure Legends Figure 1. Extraction of RNA and DNA from PBMCs and skeletal muscle by the TRIzol method. A. RNA extraction. Agarose gel electrophoresis and ethidium bromide staining is shown to demonstrate the variability of recovery and integrity of RNA from 10 individual PBMC samples (Top) and 5 individual samples of muscle tissue (Bottom). B. Poor recovery of DNA from patient PBMCs after TRIzol extraction of RNA. DNA from the lower two phases was recovered, solubilized in 8 mM NaOH and run on a 1% agarose gel. Lane 2 shows DNA from patient PBMCs stored at -80oC on LeukoLOCK filters. Lane 3 demonstrates ammonium acetate/ethanol precipitated DNA recovered from 8 mM NaOH-solubilized DNA from lane 2. Lane 4 shows PCI precipitation of soluble DNA in 8mM NaOH from lane 2. DNA from fresh PBMCs solubilized in 8 mM NaOH-HEPES buffer and subjected to PCI (Lane 5). DNA from fresh PBMCs directly subjected to PCI extraction (Lane 1). Figure 2. Extraction of DNA from PBMCs and skeletal muscle with combined TRIzol and QIAmp method. The agarose gel depicts DNA recovered from fresh H9c2 cells by TRIzol and QIAmp kit (Lane 1) or PCI extraction (Lane 2). Lane 3 shows purified DNA from archived patient PBMCs using TRIzol and QIAmp kit. Lane 4 from a parallel experiment shows DNA from mouse blood using mini QIAmp blood kit as control and Lane 5 depicts DNA from a patient blood using TRIzol and QIAmp kit. Lane 6 from another parallel experiment depicts DNA from fresh mouse skeletal muscle samples (12 mg) using only QIAmp kit as control and Lane 7 demonstrates DNA recovered from a patient frozen muscle sample (<10 mg) by TRIzol and QIAmp kit.
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