Ultrasonic Lysis of Drosophila melanogaster Samples
Drosophila melanogaster is widely used in laboratories as model organism. Therefore, pre-analytical preparation steps such as lysis, cell disruption, protein extraction and DNA shearing of Drosophila melanogaster samples must be frequently carried out. Ultrasonic dismembrators are reliable and efficient and can used to perform various tasks such as lysis, protein extraction or DNA fragmentation at ease by only adjusting ultrasonic process parameters. Ultrasonic homogenizers are thereby flexible tools with a broad range of applications.
Ultrasonic Lysis and Protein Extraction
Lysis, cell solubilization, tissue homogenization and protein extraction are typical tasks for ultrasonic dismembrators in biological laboratories. Ultrasonic dismembrators and cell disruptors are well-suited to homogenize animal tissues, insects (e.g., Drosophila melanogaster, C. elegans) or plant specimens. Subsequent applications of ultrasonication are the lysis of cell suspensions and pellets as well as the extraction of intracellular proteins.
Ultrasonic lysis and protein extraction are highly reliable and reproducible processes, which can performed on basis of established protocols. Since ultrasonic process intensity can be exactly adjusted via sonication parameters such as amplitude, cycle / pulse mode, temperature and sample volume, once proven protocols can be repeated with the same outcome over and over.
- Highly efficient
- Adjustable to specific sample material
- Suitable for any volume
- Non-thermal treatment
- Reproducible results
- Simple and safe
Ultrasonic DNA and RNA Fragmentation
After cell lysis and protein extraction, a common required step in sample preparation is the shearing and fragmentation of DNA, RNA, and chromatin, e.g. before chromatin immunoprecipitation (ChIP). DNA and RNA fragmentation can be reliably achieved by breaking the covalent bonds that hold the DNA together by physical forces. Using physical shearing such as sonication, at first the DNA strands are broken, then the DNA is fragmented into smaller pieces.
Ultrasonic DNA fragmentation is reliable and efficient in shearing DNA to a targeted length, e.g. 500bp (base pairs). The major advantages of ultrasonic DNA fragmentation include the precise control of the ultrasonic process parameters and intensity. The ultrasonic process parameters can be adjusted by tuning sonication intensity, cycles and time precisely. This makes it possible to create desired DNA sizes and the targeted DNA length can be reliably produced as well as reproduced. Ultrasonic DNA shearing is also ideal to create high molecular weight DNA fragments.
Protocols for Ultrasonic Lysis of Drosophila melanogaster
Below you can find various protocols for the ultrasonically-assisted lysis, protein extraction, and DNA or chromatin fragmentation of Drosophila samples.
Ultrasonic Lysis for Cross-linking Immunoprecipitation (CLIP) Assay
CLIP assay was performed as previously reported with some modifications. Approximately 20 mg ovaries from 0- to 1-day-old wild-type females were UV crosslinked (3 × 2000 μJ/cm2), homogenized on ice in 1 mL RCB buffer (50 mM HEPES pH 7.4, 200 mM NaCl, 2.5 mM MgCl2, 0.1% Triton X-100, 250 mM sucrose, 1 mM DTT, 1× EDTA-free Complete Protease Inhibitors, 1 mM PMSF) supplemented with 300 U RNAseOUT and placed on ice for 30 min. The homogenate was sonicated on ice, at 80% power, five times in 20 s bursts with a 60 s rest in between using the Hielscher Ultrasonic Processor UP100H (100 W, 30 kHz) and centrifuged (16000 × g for 5 min at 4°C). Soluble extract was precleared with 20 μl Protein-G dynabeads for 20 min at 4°C. After removal of samples for immunoblotting and quantitation of RNA input (1%), HP1 was immunoprecipitated with anti-HP1 9A9 antibody from 450 μl precleared extract by incubation for 4 h with 50 μl Protein-G dynabeads. Immunoprecipitates were washed 4 times with RCB. To elute the immunoprecipitated RNAs, the pelleted beads were boiled in 100 μL of UltraPure DEPC-treated Water for 5 min. 900 μL Qiazol Reagent was added to the supernatant recovered for RNA preparation. The RNA purified was used as a template to synthesize cDNA using oligo dT, random hexamers and SuperScript reverse transcriptase III according to the manufacturer’s protocol.
(Cassale et al. 2019)
Ultrasonic Lysis for Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation was performed according to the method described by Menet with minor modifications. Approximately 20 mg ovaries from 0- to 1-day-old wild-type females were homogenized in 1 mL of NEB buffer (10 mM HEPES-Na at pH 8.0, 10 mM NaCl, 0.1 mM EGTA-Na at pH 8, 0.5 mM EDTA-Na at pH 8, 1 mM DTT, 0.5% NP-40, 0.5 mM Spermidine, 0.15 mM Spermine, 1× EDTA- free Complete Protease Inhibitors) with a homogenizer / immersion disperser 1 min (at 3000 rpm). The homogenate was transferred to a pre-chilled glass dounce and 15 full strokes were applied with a tight pestle. Free nuclei were then centrifuged at 6000xg for 10 min at 4°C. The nuclei-containing pellets were resuspended in 1 mL of NEB and centrifuged at 20000 × g for 20 min on sucrose gradient (0.65 mL of 1.6 M sucrose in NEB, 0.35 mL of 0.8 M sucrose in NEB). The pellet was resuspended in 1 mL of NEB and formaldehyde to a final concentration of 1%. Nuclei were cross-linked for 10 min at room temperature and quenched by adding 1/10 vol of 1.375 M glycine. The nuclei were collected by centrifugation at 6000 × g for 5 min. Nuclei were washed twice in 1 mL of NEB and resuspended in 1 mL of Lysis Buffer (15 mM HEPES-Na at pH 7.6, 140 mM NaCl, 0.5 mM EGTA, 1 mM EDTA at pH 8, 1% Triton X-100, 0.5 mM DTT, 0.1% Na Deoxycholate, 0.1% SDS, 0.5% N-lauroylsarcosine and 1× EDTA-free Complete Protease Inhibitors). Nuclei were sonicated using a Hielscher Ultrasonic Processor UP100H (100 W, 30 kHz) six times for 20 s on and 1 min on ice. Sonicated nuclei were centrifuged at 13000 × g for 4 min at 4°C. The majority of sonicated chromatin was 500 to 1000 base pairs (bp) in length. For each immunoprecipitation, 15 μg of chromatin was incubated in the presence of 10 μg of HP1 9A9 monoclonal antibody (3 h at 4°C in a rotating wheel). Then, 50 μl of dynabeads protein G was added and incubation was continued overnight at 4°C. The supernatants were discarded and samples were washed twice in Lysis Buffer (each wash 15 min at 4 °C) and twice in TE Buffer (1 mM EDTA, 10 mM TrisHCl at pH 8). Chromatin was eluted from beads in two steps; first in 100 μl of Eluition Buffer 1 (10 mM EDTA, 1% SDS, 50 mM TrisHCl at pH 8) at 65°C for 15 min, followed by centrifugation and recovery of the supernatant. Beads material was re-extracted in 100 μl of TE + 0.67% SDS. The combined eluate (200 μl) was incubated overnight at 65°C to reverse cross-links and treated by 50 μg/ml RNaseA for 15 min at 65°C and by 500 μg/ml Proteinase K for 3 h at 65°C. Samples were phenol–chloroform extracted and ethanol precipitated. DNA was resuspended in 25 μl of water. For maximising the molecular analyses with DNA immunoprecipitated, candidate genes were amplified in pairs through an optimized duplex-PCR protocol by using two different sets of primers having similar melting temperatures in a single reaction.
(Casale et al. 2019)
High-Performance Ultrasonic Cell Disruptors for Biological Samples
Hielscher Ultrasonics is your long-experienced partner when it comes to high-performance ultrasonicators for cell disruption, lysis, protein extraction, DNA, RNA, and chromatin fragmentation as well as other pre-analytical sample preparation steps. Offering a comprehensive portfolio of ultrasonic lab homogenizers and sample preparation units, Hielscher has the ideal ultrasonic device for your biological application and requirements.
Classic probe-type ultrasonicator with micro-tip such as the UP200St (200W; see picture left) or one of the ultrasonic sample preparation units VialTweeter or UP200ST_TD_CupHorn with VialHolder are favourite models in research and analytical laboratories. The classic ultrasonic probe is ideal, when prepare fewer samples must be lysed, extracted or fragmented. The sample preparation units VialTweeter and UP200St_TD_CupHorn allow for the simultaneous sonication of up to 10 or 5 vials, respectively.
If high sample numbers (e.g. 96-well plates, microtiter plates etc.) must be processed, the UIP400MTP is the ideal sonication setup. The UIP400MTP functions like a larger cuphorn, which is filled with water and has enough space to hold micro-well plates. Powered by a 400 watts powerful ultrasonic processor, the UIP400MTP delivers a very uniform and intense sonication of multi-well plates in order to disrupt cells, lyse samples, solubilize pellets, extract proteins or shear DNA.
Precise Control via Smart Software
All Hielscher sonication solutions from 200 watts upwards are equipped with a digital colour touch-screen and an intelligent software. Via the smart data protocolling all ultrasonic process parameters are automatically saved as CSV file on a built-in SD-card as soon as the ultrasonicator is started. This makes research and protocolling so much more convenient. After the sonication trials or sample preparation, you can simply review the sonication parameters of each sonication run and compare them.
Via the intuitive menu, numerous parameter can be preset before sonication: For instance, to control the temperature in the sample and to prevent its thermal degradation, an upper limit of sample temperature can be set. A pluggable temperature sensor, which comes with the ultrasonic unit, gives the ultrasonic processor feedback on the actual sonication temperature. When the upper temperature limit is reached, the ultrasonic device pauses until the lower limit of the set ∆T is reached and starts then automatically sonicating again.
If sonication with a specific energy input is required, you can preset the final ultrasonic energy of the sonication run. Of course, ultrasonic pulsation and cycle mode can be individually set, too.
To re-use your most successful sonication parameters, you can save various sonication modes (e.g. sonication time, intensity, cycle mode etc.) as pre-set modes, so that they can be easy and rapidly started again.
For more operational convenience, all digital ultrasonic units can be operated via browser remote control in any common browser (e.g., InternetExplorer, Safari, Chrome etc.). The LAN connection is a simple plug-n-play setup and requires no additional software installation.
We at Hielscher know that the successful sonication of biological samples requires precision and repeatability. Therefore, we designed our ultrasonicators as smart devices with all features that enable an efficient, reliable, reproducible and convenient sample preparation.
The table below gives you an indication of the approximate processing capacity of our ultrasonic systems from ultrasonic micro-tips and classic ultrasonic homogenizers to MultiSample ultrasonicators for the convenient, reliable preparation of numerous samples:
|Batch Volume||Flow Rate||Recommended Devices|
|96-well / microtiter plates||n.a.||UIP400MTP|
|10 vials à 0.5 to 1.5mL||n.a.||VialTweeter at UP200St|
|CupHorn for indirect sonication, e.g. up to 5 vials||n.a.||UP200St_TD_CupHorn|
|0.01 to 250mL||5 to 100mL/min||UP50H|
|0.01 to 500mL||10 to 200mL/min||UP100H|
|0.02 to 1L||20 to 400mL/min||UP200Ht / UP200St|
|10 to 2000mL||20 to 400mL/min||UP200Ht, UP400St|
|0.25 to 5L||0.05 to 1L/min||UIP500hdT|
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Literature / References
- Casale, A.M.; Cappucci, U.; Fanti, L.; Piacentini, L. (2019): Heterochromatin protein 1 (HP1) is intrinsically required for post-transcriptional regulation of Drosophila Germline Stem Cell (GSC) maintenance. Sci Rep 9, 4372 (2019).
- Ristola, M.; Arpiainen, S., Saleem, M.A.; Mathieson, P.W.; Welsh, G.I.; Lehtonen, S.; Holthöfer, H.(2009): Regulation of Neph3 gene in podocytes – key roles of transcription factors NF-κB and Sp1. BMC Molecular Biol 10, 83 (2009).
- Kharchenko, P.V: et al. (2011): Comprehensive analysis of the chromatin landscape in Drosophila. Nature. 2011 Mar 24; 471(7339): 480–485.
Facts Worth Knowing
Metabolomics is the study of small molecules, known as metabolites, present within cells, biofluids, tissues or organisms. These small molecules and their interactions within a biological system are summarized under the umbrella term “metabolome” and the research field is called metabolomics. The metabolome research is closely connected with the rapidly emerging field of precision medicine. The understanding of the metabolome and its relation to various diseases helps to develop disease prevention and clinical care strategies whilst individual variability in environment, lifestyle, genetics, and molecular phenotype. In order to release metabolite molecules from cells, ultrasonication is frequently used in biological laboratories for pre-analytical sample preparation such as cell disruption, lysis and the extraction of proteins, lipids and other molecules.