High-Throughput Sample Preparation for Mitochondrial Diagnostics
Mitochondrial diagnostics in research and clinics are performed using various techniques such as sequencing, PCR, and biochemical assays. These methods are used to identify DNA mutations and measure mitochondrial function. Sequencing helps detect genetic mutations, while PCR can quantify specific DNA sequences. Biochemical assays evaluate the functionality of mitochondrial proteins and enzymes.
The diagnostic of mitochondrial diseases is especially difficult due to the pronounced clinical variability of these diseases as well as due to the complex interaction between two differently inherited genomes: mitochondrial DNA (mtDNA) and the nuclear genome.
DNA Extraction and Fragmentation using Sonication
DNA extraction from muscle tissue is particularly useful when tissue-specific changes in mtDNA are suspected. These changes might include deletions of mtDNA in chronic progressive external ophthalmoplegia (CPEO), point mutations of mtDNA in mitochondrial myopathies, or depletion of mtDNA in Alpers syndrome. The DNA extracted from muscle tissue can then be used for various genetic tests, such as Southern blot and long-range PCR for deletions, real-time PCR for depletion, or sequencing for point mutations.
Sonication, using intense ultrasound waves, is applied for several applications in mitochondrial diagnostic. It is used to lyse cells to extract intracellular contents such as mitochondria and DNA, to shear DNA such as mtDNA and nDNA for sequencing, and to homogenize samples.
UIP400MTP Plate Sonicator for high-throughput sample preparation uniformly sonicates samples in multi-well and 96-well plates
How to Isolate Mitochondria from Cells and Tissues
Mitochondrial isolation comprises two essential steps: disrupting cells to release their contents, and employing differential centrifugation to separate and recover mitochondrial fractions.
Sonication
Hielscher sonicators are compatible with standard lysis buffers and kits, making them suitable for the isolation of mitochondria. Sonication serves two main purposes:
- Cell Lysis: The ultrasonic waves disrupt cell membranes, releasing intracellular contents.
- Mitochondrial Disruption: Sonication in a subsequent step can break open the mitochondria to release mitochondrial proteins or mitochondrial DNA.
- mtDNA Fragmentation: Sonication is a reliable technique to shear mitochondrial DNA for sequencing.
The multi-well plate sonicator UIP400MTP allows for high-throughput sample preparation of mitochondrial samples. In conjunction with differential centrifugation, sonication enhances the efficiency of mitochondrial isolation, ensuring high yields of intact mitochondria suitable for various downstream applications.
Hielscher UIP400MTP multiwell plate sonicator works with any standard plate
The multi-well plate sonicator UIP400MTP offers numerous benefits for high-throughput sample preparation, e.g. in mitochondrial diagnostic.
UIP400MTP Multiwell Plate Sonicator for high-throughput sample preparation in biomarker diagnostic.
Exemplary Instructions for Ultrasonic mtDNA Fragmentation
Preparation and Extraction:
- C57BL/6 mice were killed by cervical dislocation.
- Livers were quickly extracted and washed in ice-cold sterile PBS.
Mitochondria Isolation:
- Mitochondria were isolated using a 2-mL Dounce tissue grinder and a Mitochondria Isolation Kit for Tissue.
- Initial centrifugation at 700× g and 3,000× g.
- Perform two additional washing steps with buffer C.
DNA Isolation:
- The pellet from the first centrifugation at 700× g was used to isolate nuclear DNA (nDNA).
- DNA was isolated using spin columns.
- Mitochondrial DNA (mtDNA) was extracted from isolated mitochondria.
- Nuclear DNA (nDNA) was extracted from crude nuclear extracts, both from mouse liver tissue.
DNA Fragmentation:
- DNA was fragmented on ice using a 30-kHz/50-W ultrasonic sonicator UP50H with a 0.5-mm micro-tip sonotrode at 14 μm for 2 × 30 seconds.
Fragmentation Visualization and Quantification:
- Fragmentation after ultrasonification was visualized on a 1% agarose gel with SYBR safe DNA dye.
- The relative abundance of mtDNA and nDNA was determined by qPCR.
(cf. Mariero et al., 2019)
For high-throughput sample preparation, the multi-well plate sonicator UIP400MTP facilitates the preparation of large sample numbers in standard 96-well, multi-well and microtiter plates.
Read more about advantages of high-throughput sample prep using the UIP400MTP!
Tips for Optimal Mitochondria Isolation Using Sonication:
- Temperature Control: Conduct all steps at a temperature range of 0°C to 4°C. This is critical to maintaining the integrity and functionality of the mitochondria.
- Efficiency and Speed: Work swiftly and purify the mitochondria only to the extent required for your specific application. Excessive manipulation can lead to significant losses of mitochondrial content.
- Dilution of Suspensions: Maintain low concentrations of cell and organelle suspensions throughout the isolation process. This helps to minimize the risks of trapping and agglutination, thereby improving the purity of the isolated mitochondria.
- Sample Volume: Opt for multiple small-scale preparations rather than a single large one. This approach typically results in better yields, as scaling up does not proportionally increase the amount of recoverable mitochondria. The multi-well plate sonicator UIP400MTP facilitates the rapid and reliable lysis of cell for mitochondria isolation as well as the protein extraction from mitochondria.
These guidelines will make the isolation process using ultrasonic lysis and centrifugation more efficient, yielding high-quality mitochondrial preparations suitable for downstream applications.
Hielscher Sonicators – Quality Made in Germany
Hielscher ultrasonicators are well-known for their highest quality and design standards. Robustness and easy operation allow the smooth integration of our ultrasonicators into industrial facilities. Rough conditions and demanding environments are easily handled by Hielscher ultrasonicators.
Hielscher Ultrasonics is an ISO certified company and put special emphasis on high-performance ultrasonicators featuring state-of-the-art technology and user-friendliness. Of course, Hielscher ultrasonicators are CE compliant and meet the requirements of UL, CSA and RoHs.
96-well plate sonicator UIP400MTP for the sonication of microtiter and multiwell plates
Literature / References
- FactSheet UIP400MTP Multi-well Plate Sonicator – Non-Contact Sonicator – Hielscher Ultrasonics
- UIP400MTP-Multi-well-Plate-Sonicator-Infographic
- De Oliveira A, Cataneli Pereira V, Pinheiro L, Moraes Riboli DF, Benini Martins K, Ribeiro de Souza da Cunha MDL (2016): Antimicrobial Resistance Profile of Planktonic and Biofilm Cells of Staphylococcus aureus and Coagulase-Negative Staphylococci. International Journal of Molecular Sciences 17(9):1423; 2016.
- Martins KB, Ferreira AM, Pereira VC, Pinheiro L, Oliveira A, Cunha MLRS (2019): In vitro Effects of Antimicrobial Agents on Planktonic and Biofilm Forms of Staphylococcus saprophyticus Isolated From Patients With Urinary Tract Infections. Frontiers in Microbiology 2019.
- Lauren E. Cruchley-Fuge, Martin R. Jones, Ossama Edbali, Gavin R. Lloyd, Ralf J. M. Weber, Andrew D. Southam, Mark R. Viant (2024): Automated extraction of adherent cell lines from 24-well and 96-well plates for multi-omics analysis using the Hielscher UIP400MTP sonicator and Beckman Coulter i7 liquid handling workstation. Metabomeeting 2024, University of Liverpool, 26-28th November 2024.
- Dreyer J., Ricci G., van den Berg J., Bhardwaj V., Funk J., Armstrong C., van Batenburg V., Sine C., VanInsberghe M.A., Marsman R., Mandemaker I.K., di Sanzo S., Costantini J., Manzo S.G., Biran A., Burny C., Völker-Albert M., Groth A., Spencer S.L., van Oudenaarden A., Mattiroli F. (2024): Acute multi-level response to defective de novo chromatin assembly in S-phase. Molecular Cell 2024.
Frequently Asked Questions about Mitochondria and Mitochondrial Diagnostic
What are Mitochondria?
Mitochondria are membrane-bound organelles found in the cells of most eukaryotic organisms. They are known as the powerhouses of the cell because they produce energy in the form of adenosine triphosphate (ATP) through the process of cellular respiration. Additionally, mitochondria have their own DNA and play key roles in other cellular processes, including the regulation of the cell cycle and cell death.
What differentiates mtDNA from Genomic DNA?
Mitochondrial DNA (mtDNA) differs from genomic DNA (gDNA) in several key ways. MtDNA is located in the mitochondria, is circular, and is maternally inherited, whereas gDNA is located in the cell nucleus, is linear, and is inherited from both parents. MtDNA is much smaller, encoding only 37 genes, while gDNA contains about 20,000-25,000 genes. MtDNA is present in multiple copies per cell, has a higher mutation rate, and primarily codes for proteins involved in mitochondrial function. In contrast, gDNA is typically diploid, has a lower mutation rate, and encodes a vast array of genes necessary for the organism’s development and function. Additionally, mtDNA transcription and translation occur within the mitochondria, whereas gDNA transcription occurs in the nucleus, and translation occurs in the cytoplasm. These differences reflect their distinct roles and evolutionary origins.
What is Cell-Free Extract?
A cell-free extract is a solution containing the contents of lysed cells, including proteins, nucleic acids, and other cellular components, but without intact cell membranes. This extract is used in biochemical and molecular biology research to study cellular processes in vitro, enabling researchers to analyze reactions and mechanisms outside of living cells.
What Role Play PCR Tests in Mitochondrial Diagnostics?
PCR tests play a critical role in mitochondrial diagnostics by enabling the detection of mutations, deletions, or copy number variations in mitochondrial DNA (mtDNA). They allow for the amplification of specific mtDNA regions to identify pathogenic variants associated with mitochondrial disorders. PCR-based techniques, such as quantitative PCR (qPCR) and long-range PCR, are also used to assess mtDNA integrity, heteroplasmy levels, and mtDNA depletion, providing essential insights into mitochondrial function and disease.
Learn how the UIP400MTP Microplate Sonicator facilitates PCR tests and assays!
What is Differential Centrifugation?
Differential centrifugation is a widely used technique for cell fractionation and mitochondrial isolation. This method separates cellular structures based on their sedimentation coefficient, which depends on both density and shape. The process involves applying varying levels of centrifugal force to samples in buffered salt solutions with specific densities. Structures with similar sedimentation coefficients will settle at the bottom of the collection tube at the same time, allowing for their recovery.
How is Differential Centrifugation used for Mitochondria Isolation?
Differential centrifugation allows researchers to effectively separate mitochondrial fractions from other cellular components. Isolation of mitochondria involves several centrifugation steps and the subsequent recovery of the isolate.
Initial Centrifugation: Apply a low centrifugal force to sediment large cellular debris and nuclei.
Subsequent Centrifugations: Increase the centrifugal force stepwise to pellet fractions enriched with mitochondria. Each centrifugation step removes structures with progressively higher sedimentation coefficients.
Fraction Recovery: After each centrifugation, the pellet is collected, and the supernatant is subjected to higher g-forces to isolate the next fraction. This is repeated until the desired purity of mitochondria is achieved.