The human mitochondrial DNA (mtDNA) is a double-stranded, 16,569 base pair (bp) closed circular molecule which encodes 13 essential subunits of the oxidative phosphorylation (OXPHOS) enzymes, as well as 2 ribosomal RNA (rRNA) and 22 transfer RNA (tRNA) necessary for intramitochondrial protein synthesis (20). It is well established that mutations of the mtDNA are associated with several disorders such as Leber's hereditary optic neuropathy (LHON) (3), myoclonic epilepsy and ragged red fiber disease (MERRF) (18), and Kearns-Sayre Syndrome (KSS) (14). A new class of mitochondrial disorders has been reported which shows a severe quantitative reduction of otherwise intact mtDNA molecules, namely the mtDNA depletion. This genetic abnormality is characterized by tissue-specific loss of mtDNA molecules in the affected tissue of up to 98%(13). Diseases related to a quantitative defect of mtDNA are extremely rare and manifest in early infancy or during the first years of life and lead to early death due to reduced oxidative phosphorylation rates (19). Biochemical studies of the affected tissue show markedly decreased activities of all respiratory chain complexes containing mtDNA-encoded subunits. Several clinical phenotypes have been described like early-onset encephalomyopathy (11,15), mitochondrial myopathy (19) and fatal liver diseases (2,10). Most commonly, mtDNA depletion is detected by quantitative Southern blot measuring the ratio of the signals of mtDNA to nuclear DNA (nDNA).
Because mtDNA depletion analysis by Southern blotting relies on the isolation of high molecular weight nDNA and full-length, circular mtDNA it is essential that the ratio of proper nDNA to mtDNA is not altered during handling of the specimens. Biopsy material for genetic analysis is usually snap frozen in liquid N2 and stored at −20°C/−70°C or fixed with paraformaldehyde and embedded in paraffin for further immunologic and genetic analysis. DNA isolation from paraffin-embedded tissue is described in several studies (5,16). However, the DNA isolated is only suitable for amplification of polymerase chain reaction (PCR) products <1 kilobases (kb) (4,6,21). Therefore, either fresh or snap frozen tissue is the source for the isolation of high molecular weight DNA in clinical settings (7). Although several studies have investigated the ability to isolate mtDNA from various sources after different fixation and storage procedures suitable for PCR amplification, no information is available about the suitability to use the isolated mtDNA in quantitative Southern blot analysis.
Our study demonstrates that thawing of specimens can preferentially harm mtDNA and mimic a severe mtDNA depletion. Therefore, we postulate for the molecular diagnosis of mtDNA depletion that proper handling and storage of tissue is of most importance.
MATERIALS AND METHODS
Handling and Storage of Muscle Tissue
Thawed muscle tissue (10–20 mg musculus vastus lateralis) was incubated at room temperature (RT) for 0, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 hours and at 4°C for 0, 1, 3, 5, 9, 17, 24, 33, 48, 72, and 96 hours. Fresh tissue was incubated at RT for 0, 2, 4, 8, 24, 48, 72, 96, 120, 168, and 240 hours and at 4°C for 0, 5, 18, 24, 48, 72, 120, 168, 240, and 360 hours.
Total cellular DNA was isolated from tissue specimens according to standard procedures (17). Briefly, 10 to 20 mg of muscle tissue were chopped and incubated in approximately 10 volumes of extraction buffer (10 mmol/L TrisHCl, pH 8.0, 0.1 mol/L ethylenediaminetetraacetic acid [EDTA], 0.5% sodium dodecyl sulfate [SDS], 1 mg/mL proteinase K) in a shaking water bath at 50°C for 3 to 5 hours until the tissue was completely lysed. The DNA was isolated by two extractions with phenol-chloroform-isoamylalcohol (25:25:1) and one extraction with chloroform-isoamylalcohol (25:1). The DNA was precipitated by adding 2 volumes of 100% ethanol and 1/10 volume of 3 mol/L NaCl. The DNA precipitate was washed with 70% ethanol, collected by centrifugation, and air dried. After the DNA was completely dissolved in 10 mmol/L TrisHCl, pH 8.0, 1 mmol/L EDTA, the DNA content was measured spectrophotometrically.
Preparation of the Probes
The nuclear probe was a PCR fragment of the multicopy nuclear gene encoding for the human 18S rRNA. The PCR reaction mixture for the 18S rDNA fragment consisted of 20 mmol/L (NH4)2SO4, 75 mmol/L TrisHCl pH 9.5, 0.01% (vol/vol) Tween 20, 2.5 mmol/L MgCl2, 0.2 mmol/L of each deoxynucleotide triphosphate (dNTP), 10 pmol forward primer (3550-3571; 5`-GGT ACA GTG AAA CTG CGA ATG G-3`) and 10 pmol reverse primer (4343-4322; 5`-CAT TAT TCC TAG CTG CGG TAT C-3`), 1 U of Red Hot DNA Polymerase (Advanced Biotechnologies, Epsom, UK) and 100 ng of human DNA in a final volume of 30 μL. After an initial denaturation step of 5 minutes at 95°C, PCR amplification was performed for 35 cycles, each consisting of 30 seconds at 95°C, 45 seconds at 66°C, and 1 minute at 72°C in a thermocycler (OmniGene, Hybaid, Teddington, UK). A final extension of 5 minutes at 72°C was performed.
The mitochondrial probe was an 800 bp PCR product flanking the “common deletion,” a 4977 bp deletion (8). This probe was generated by a nested PCR with the primers A1.1 (7313-7334; 5`-CAT GAT TTG AGA AGC CTT CGC T-3`), A2 (13928-13905; 5`-CTA GGG TAG AAT CCG AGT ATG TTG-3`) and B1 (7901-7920; 5`-TGA ACC TAC GAG TAC ACC GA-3`), B2 (13650-13631; 5`-GGG GAA GCG AGG TTG ACC TG-3`) according to Gattermann et al. (8). The reaction mixtures were the same as described for the nuclear probe. The first round was performed with the following conditions: after an initial denaturation step for 5 minutes at 95°C, PCR amplification was performed for 30 cycles, each consisting of 1 minute at 95°C, 1 minute at 66°C, and 3 minutes at 72°C. A final extension of 10 minutes at 72°C was added. The second round was performed as follows: after an initial denaturation step for 5 minutes at 95°C, the cycle times consisted of 20 seconds at 95°C, 30 seconds at 64°C, and 2 minutes at 72°C for 30 cycles. A final extension of 10 minutes at 72°C was added. Because we did not use a Long Range PCR approach, only the 800 bp PCR fragment resulting from the common deletion, which can be found in several mtDNA molecules in every DNA sample, was amplified (8). Some experiments were repeated with a probe of the entire mitochondrial genome. This probe was amplified with the primers FLFor (15148-15174; 5`-GTG AGG CCA AAT ATC ATT CTG AGG GGC-3`) and FLRev (14841-14816; 5`-TTT CAT CAT GCG GAG ATG TTG GAT GG-3`) and the Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) to generate a 16,231 bp molecule of wild-type mtDNA (1,12).
The PCR products were isolated from 1.5% agarose gels with the agarose gel DNA extraction kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the instructions. A defined mixture of the mitochondrial and the nuclear probe (1:26) which was found to obtain signals of the same intensity at time 0, was prepared in advance and frozen in aliquots. The probe mixture was labeled with [α-32P] deoxyadenosine triphosphate (dATP) (3000 Ci/mmol; Amersham Pharmacia Biotech, Little Chalfont, UK) using the Prime-a-Gene Labeling System (Promega, Madison, WI, U.S.A.) and used for hybridization within two weeks.
Southern Blot Analysis
One microgram of total DNA from skeletal muscle was digested with the restriction enzyme Pvu II (Amersham Pharmacia Biotech, Little Chalfont, U.K.), separated on a 0.8% agarose gel, and transferred to nylon membranes with 400 mmol/L NaOH. After a brief wash in 2 × standard saline phosphate EDTA-buffer (SSPE) (360 mmol/L NaCl, 20 mmol/L NaH2PO4, pH 7.7, 2 mmol/L EDTA) the filters were hybridized with the mixture of the mitochondrial (0.03 ng/mL) and the nuclear (0.8 ng/mL) 32P-labeled probe. Hybridization was proceeded overnight at 65°C. After stringency washes (2 × SSPE, 0.1% SDS, and 0.1 × SSPE, 0.1% SDS) the autoradiography was proceeded for up to 20 hours. To determine the intensity of the signals, densitometry on autoradiographs was performed. The relative amount of mtDNA was expressed as the ratio of the signal of the mitochondrial probe to that of the nuclear probe (m/r ratio). At time 0, the m/r ratio was set 100%.
When frozen muscle was thawed and left at RT, a significant decrease of mtDNA was observed within the first hour. A 50% decrease in the amount of mtDNA could be already observed after 45 minutes at RT (n = 6) (Fig. 1, 2A). A similar decline was observed when thawed tissue was stored at 4°C but with a prolonged time scale. In this case, a 50% depletion of mtDNA could be detected after approximately 15 hours (n = 9) (Fig. 2B). Interestingly, the autoradiograph of the Southern blots did not indicate degradation of DNA in the samples, normally observed as a smear, even when the blot was overexposed ten times the normal period.
In contrast, the ratio of mtDNA to nDNA remained constant when fresh tissue was investigated. Incubation of fresh tissue at RT for up to 240 hours (n = 4) revealed no selective decrease in the amount of mtDNA (Fig. 3A). A similar result was obtained when fresh muscle tissue was stored at 4°C up to 360 hours (n = 7) (Fig. 3B).
The average yield of the total DNA isolated from samples kept at RT longer than 3 days decreased by approximately 60%. The inter assay variability from different DNA extractions of the same tissue ranged from 20% to 40%. The variability of the Southern blot analysis of the same DNA was up to 15%. The variability of densitometry of the same blot ranged from 5% to 15%.
The results obtained from our experiments suggest that mtDNA is degraded much faster than nDNA after a tissue was thawed and kept at RT or at 4°C. When fresh tissue was investigated under these circumstances the relative amount of mtDNA remained constant. This is in agreement with Southern blot analysis data of genomic DNA isolated from placenta or blood lymphocytes reported previously (7).
The rapid loss of mtDNA in thawed tissue may be explained by the disturbance of the double layer of mitochondrial membranes, which normally protect mtDNA from degrading enzymes. Freezing and thawing is known to disrupt cellular structures including mitochondrial membranes, exposing mtDNA and nDNA to degrading enzymes. A loss of nDNA can be seen in the lower yield of total DNA that has been isolated from the thawed samples after long-term storage at RT. However, it is obvious that mtDNA is preferentially degraded compared with nDNA. Lack of protection by histones and dense packaging probably makes mtDNA more accessible to DNAses. It is also possible that its unique structure (like the DNA-RNA hybrids in the D-loop) and maybe motifs in the sequence of the mtDNA make it more susceptible to enzymatic degradation than nDNA.
Since we compared only the stability of full length mtDNA with a 12 kb nDNA fragment, we cannot exclude that small DNA fragments found in fossils show a similar degree of degradation. Handt et al. reported DNA extraction and analysis from the Tyrolean Ice Man and stated that only multi copy sequences up to 200 bp were suitable for molecular genetic analysis (9). Therefore mtDNA seems to be more useful than nDNA in analysis of ancient specimens, such as mummies or fossil bones, most probably due to the high copy number of mtDNA.
The inter assay variability of our experiments was with 20% to 40% surprisingly high. This might be due to the fact that the muscle tissue is inhomogenously composed of muscle fibers and connective tissue. Muscle fibers contain much more mitochondria and therefore more mtDNA molecules than the cells of the connective tissue. In consequence, the isolation of DNA from two different small slices from the same tissue could result in different relative amounts of mtDNA.
The variability of the results of the Southern blot analysis from more than two blots from the same DNA revealed up to 15% variability, which is most likely due to the variation of densitometric analysis. Based on the high inter assay variability we suggest to perform two independent DNA isolations with at least two independent determinations of the m/r ratio. From our data we would propose that only results indicating more than 40% mtDNA depletion in muscle tissue should be considered as clinically relevant.
In summary, the following guidelines for sample transport, storage and handling are implicated by our stability experiments. Fresh tissue should be used whenever possible and the fresh samples should be sent to the diagnostic laboratory at 4°C within 2 to 3 days. If the tissues are snap frozen the samples have to be sent on dry ice and stored at −20°C or better at −70°C. When the samples are thawed for DNA isolation the lysis step of the tissue has to be performed immediately after thawing. In the event of a power failure of the freezer or unclear temperature conditions during storage and postage of the specimens any result indicating a mtDNA depletion is not reliable.
In conclusion, our data demonstrate that the development and observance of proper specimen storage guidelines is the key to successful diagnosis of mtDNA depletions.
The authors thank Herbert Herzog and Johann Mayr for helpful discussion during preparation of the manuscript.
This work was supported by the “Verein zur Förderung pädiatrischer Forschung Salzburg.”
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