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The Key Considerations
When selecting the appropriate tissue disruption technique consider the following characteristics of your experiment:
- Sample Type: What are you lysing? Samples can vary in resilience. For instance soft tissue, such as brain and spleen, is easier to homogenize compared to hard tissue, such as bone. A more aggressive method, such as bead-beating with stainless steel, may be necessary for harder samples.
- Target Molecules: What are you trying to isolate and where is it located? Different methods and/or reagents are needed for nucleic acid isolation versus protein isolation. Similarly, methods for mitochondrial protein isolation versus total protein isolation will vary.
- Required Yield: How much DNA, RNA, or protein do you need to perform the next step? Different methods will result in higher yield, but may be more time-consuming or expensive.
- Intended Downstream Application: What are you doing with the isolated material? Certain reagents commonly used in tissue lysis are incompatible with specific experiments. For instance, if you intend on performing enzymatic assays with your isolated protein of interest, you should choose a method that will retain or restore protein function (i.e. the protein will not be denatured).
- Sample Quantity: Do you need a high-throughput method and/or will you be performing the method frequently? If so, consider investing in specialized equipment that is reliable and efficient, such as the FastPrepTM Instruments that are optimized for sample preparation for a variety of cells and tissues.
- Budget: What equipment do you already have and how much can you spend? Tissue lysis methods can vary significantly in cost, with do-it-yourself methods being cheaper and high-throughput methods being more expensive. While affordable "home-brew" methods can be sufficient for certain applications and labs, it may require more time spent troubleshooting and optimizing. Consider using ready-to-use buffers and best-in-class bead beating technology to avoid research delays.
FastDNA-96 Tissue & Insect DNA Kit, 2 x 96 Preps
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Using Physical Disruption to Lyse Tissues
Physical disruption uses equipment, such as beads or mortar and pestle, and high-speed shaking (or manual grinding) to homogenize tissue and extract nucleic acids or proteins. Bead beating methods can range from low to high impaction, breaking down any sample type whether the tissue is hard (e.g. bone) or soft (e.g. brain). It is important to note that physical disruption methods can produce localized heat, particularly when dealing with resilient sample types, so maintaining cold conditions (i.e. cryogenic grinding) may be required to keep target molecules intact.
Physical disruption can range in cost and throughput. Manual grinding with a mortar and pestle is affordable, but time-consuming and often inconsistent across samples. Bead-beating methods range from affordable to expensive, but are consistently effective and can easily be converted to automated systems for high-throughput needs.
Bead beating lysing matrices commonly used for tissue samples to extract nucleic acids and/or proteins:
- Lysing Matrix A: A combination of garnet matrix and large ceramic spheres
- Lysing Matrix D: Ceramic spheres
- Lysing Matrix S: Stainless steel
- Lysing Matrix Z: Yttria-stabilized zirconium oxide beads
Additionally, you can streamline tissue sample preparation using commercially available kits, such as the FastDNA-96 Tissue & Insect DNA Kit.
Get reproducible homogenization with MP Bio’s bead beating tubes and ready-to-use lysing matrices for any sample type.View Products
Liquid-Based Tissue Lysis
Liquid-based cell lysis involves solutions containing detergents or buffers that can weaken or disrupt cell or tissue membranes. Use nonionic (e.g., Triton-X, NP-40) and zwitterionic (e.g., CHAPS) detergents as a mild approach to solubilize membrane proteins while maintaining protein function. Ionic detergents (e.g., sodium deoxycholate, Sodium Dodecyl Sulfate) are strong solubilizing agents and denature proteins, destroying protein activity.
RIPA buffer is a common buffer used to disrupt soft tissue for protein extraction. It is compatible with SDS-PAGE, but not ideal for immunoprecipitation, so dialysis may be required depending on the downstream application to avoid interference with protein interactions. RIPA is a Tris-based solution that contains three detergents: NP40, sodium deoxycholate, and sodium dodecyl sulfate (SDS).
- RIPA buffer recipe: 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS).
A common alternative to RIPA buffer is using a hypotonic lysis buffer containing a protease and phosphatase inhibitor cocktail to swell the cells, weakening the membrane, to facilitate lysis while retaining protein activity.
Sonication disrupts cells using high frequency sound waves using an ultrasonic bath or probe. It utilizes more power than other methods to lyse tissues, so it generally takes less time, but the equipment is expensive and can generate intense heat within samples. This approach often disrupts DNA integrity, so it may not be appropriate for downstream applications analyzing nucleic acids, but it does make nuclear protein extraction easy.
Deciding between bath sonication and sonication with a probe comes down to sample volume and quantity.
Sonication with a probe is useful for small volumes and can be adapted with a microprobe for especially low volumes, but only one sample can be sonicated at a time, and the probe needs to be thoroughly cleaned between uses to avoid contamination.
For many applications, a hybrid approach is ideal. For instance, using MP Bio’s lysing matrices, a bead beading approach, in combination with guanidine thiocyanate lysis buffer has proven effective and efficient for homogenizing skin tissue—infamous for its resistance to mechanical shearing, RNases, and proteases.
If your starting sample is particularly large, utilizing all three methods may be beneficial. Mincing and grinding the sample followed by homogenization with lysis buffer and vortexing can produce a semi-clear lysate, which can then be fully lysed with a sonicator.
Quick Tips on How to Optimize Your Lysing Method
Tissue lysis involves numerous steps, materials, and settings. Each of these variables affects the end-result, which is why you should prioritize optimization.
Regardless of your approach, you should first identify all components within the sample preparation method(s) because each of those variables can influence the downstream application.
After you’ve determined the ideal lysing approach (e.g., bead beating using lysing matrix A), you’ll want to consider the following:
- Sample size and abundance (weight/volume)
- Lysing duration and intervals (range between 10 sec to 3 mins, in 10-20 secs sequential replicates, so 10 sec, 20 sec, 30 seconds, 50 seconds)
- Lysing speed range
Finally, you’ll want to assess the post-processing quality/endpoint. Here’s how:
- Visual Check for Homogenous Lysis: The sample needs to look like it’s a homogenate, meaning it lacks chunks and pieces of tissue in the liquid.
- Visual Check Post-Centrifugation: Centrifuge samples at the maximum RPM for 10 minutes or less (duration should be optimized) and evaluate the tube for target distribution. For instance fat tissue homogenization should yield 3 layers, with the fat layer on top.
- Downstream Analysis: Use a biological target or metric, such as DNA, RNA, or protein concentration, to determine which upstream protocol is suitable for your downstream application.
Selecting the appropriate tissue disruption technique is an important step in setting up your experiment for success. There are several factors to consider when choosing between physical disruption, liquid homogenization, and sonication, but oftentimes a hybrid approach is needed.
MP Bio offers a wide range of solutions and resources to meet your tissue homogenization needs.