RNA extraction
Cell lysis technique and homogenization for RNA extraction
Effective sample lysis and homogenization are critical for RNA isolation. These steps release and set RNA free by breaking down tissue, cells, and other biological materials, ensuring high-quality and robust yield for downstream RNA applications. However, extracting RNA from biological sources comes with several challenges, and no single method of lysis or homogenization works well for all sample types.
The selection of sample lysis and sample homogenization methods requires careful consideration of various factors that influence its success. These include the type of RNA (e.g., mRNA, miRNA, cell-free RNA, rRNA, tRNA, and non-coding RNAs), the subcellular localization of RNA (cytoplasmic, nuclear, or mitochondrial), and the inherent complexity of the biological material (e.g., tissue, cell culture, plants, blood, stool). Cellular structures, such as cell walls, provide significant barriers, while the presence of endogenous RNases and RNA-binding proteins poses additional challenges to RNA integrity and yield.
Furthermore, different sample types, ranging from microbial cells to animal tissues, require specialized disruption and homogenization techniques. The chosen methods must also align with the requirements of downstream applications, required threshold, and research objectives, which demand high-quality, intact RNA and workable concentration. Addressing these challenges necessitates a systematic approach to ensure efficient lysis, homogenization, and RNA isolation.
Ian: Dr. Ray, I’m getting consistently low RNA yields, even when following the protocol. What could be going wrong?
Dr. Ray: Low yield is often due to insufficient lysis or incomplete RNA recovery, Ian. Ensure your sample preparation is thorough, your homogenization is consistent and optimize the cell lysis step. RNA extraction can be tricky, but I can simplify the process for you. Are you ready to boost your RNA yield?
Cell lysis methods and sample homogenization types
Efficient lysis and homogenization of the starting material is an absolute requirement for all RNA isolation procedures. Disruption and homogenization are two distinct steps.
- Lysis – also called cell disruption, involves breaking down the physical barriers of the sample such as tissues, cell walls, and plasma membranes to release the RNA and other intracellular contents. This step ensures that all RNA is available for subsequent processing. Incomplete disruption can trap RNA within cells or organelles (e.g., nucleus), leading to reduced yields.
- Homogenization – refers to creating a uniform lysate by reducing the viscosity caused by the massive release of biomolecules such as genomic DNA, proteins, and other cellular components. Homogenization ensures that RNA is completely dissolved or suspended in the solution, binds effectively during column-based isolation, and prevents clogging of purification systems.
Lysis and homogenization methods are broadly categorized as:
- Mechanical: Involves the use of mechanical forces to break open a cell. This includes rotor–stator homogenizers, bead mills, syringes and needles, vortexing, mortar and pestle, and sonication.
- Chemical: Uses chemical reagents to break down the cell membrane or cell wall. This includes the use of detergents, alkali, and specialized lysis buffers.
- Biological: Involves the use of biological agents or enzymes to release cellular components. This includes the use of lysozyme or zymolase for breaking down the cell wall of bacteria and yeast cells, respectively.
- Physical: Uses different physical conditions to alter the physical state of different cellular components. This includes the use of heat, osmotic shock or freeze-thawing, which are often combined with other methods for enhanced efficiency.
Each method is suited to specific types of samples, and choosing the right approach can significantly impact the success of RNA isolation.
Sample lysis techniques for various biological materials
Effective sample processing depends on matching the method to the sample type. The table below presents the recommended cell lysis technique and sample homogenization methods based on the source biological material. However, when deciding between various lysis and homogenization methods, please consider compatibility with downstream applications and resource availability
Considerations for using bead mills in sample preparation
Bead mills are powerful tools for simultaneous disruption and homogenization, offering versatility for a range of sample types. Key considerations include:
- Bead size and composition
- Use 0.1 mm glass beads for bacteria, 0.5 mm for yeast, and 3–7 mm stainless steel beads for animal and plant tissues.
- Stainless steel beads are ideal for tough tissues due to their durability and efficiency.
- Buffer-to-bead ratio: Proper proportions ensure effective shearing while preventing overheating and sample loss. Optimize this ratio based on the sample type.
- Cryogenic grinding: For temperature-sensitive samples, pre-cool beads, vessels, and plant material in liquid nitrogen. This method prevents RNA degradation while enhancing tissue disruption.
- Pre-treatment of beads: Clean glass beads with concentrated nitric acid or purchase pre-treated acid-washed beads to avoid contamination.
Effective use of rotor-stator homogenizers for cell and tissue processing
Rotor-stator homogenizers are a go-to solution for processing animal tissues and lysates. They work by combining high-speed mechanical shear forces with turbulence to ensure thorough disruption and homogenization. Key features include:
- Speed and efficiency: Process tissues in 5–90 seconds, depending on the toughness of the sample.
- Probes and volumes:
- Probes with diameters of 5–7 mm are suited for small volumes (<300 μL).
- Larger probes (10+ mm) are needed for higher volumes.
- Foam prevention: Avoid foaming by ensuring proper immersion of the probe in a correctly sized vessel. Hold the probe to one side of the tube to minimize turbulence.
Notes on mortar and pestle methods for sample homogenization
A mortar and pestle remains one of the simplest yet effective tools for disrupting tough biological samples like plants and fungi. Cell disruption using mortar and pestle involves the following steps:
- Freeze the sample in liquid nitrogen immediately upon collection to prevent RNA degradation.
- Grind the sample to a fine powder under liquid nitrogen to ensure thorough disruption.
- Transfer the powdered sample into a pre-cooled tube. Allow the liquid nitrogen to evaporate without thawing the sample.
- Add lysis buffer quickly to maintain RNA integrity and proceed with homogenization.
While this method effectively disrupts the cells, it does not homogenize the sample. Additional homogenization using a bead mill or rotor-stator homogenizer is required to ensure uniformity.
Using syringe and needle for cell lysis and sample homogenization
Homogenization using a syringe and needle is a straightforward and cost-effective method for processing viscous lysates. This method is highly effective for shearing high molecular weight DNA and reduces viscosity, ensuring high RNA binding efficiency in downstream applications. Key steps in performing the homogenization include:
- Load the lysate into a 20-gauge needle attached to a sterile syringe.
- Pass the lysate through the needle at least 5–10 times until homogenization is achieved.
- Adjust the volume of the lysis buffer to improve flow and minimize sample loss.
This method is particularly useful for bacterial lysates and small-scale animal tissue samples where mechanical homogenizers are unavailable.