Complete Disruption –A Critical Step

Cellular disruption is the first step in RNA isolation and one of the most critical steps affecting yield and quality of the isolated RNA. Typically, cell disruption needs to be fast and thorough. Slow disruption, for example placing cells or tissue in guanidinium isothiocyanate (GITC) lysis solution without any additional physical shearing, may result in RNA degradation by endogenous RNases released internally, yet still inaccessible to the protein denaturant, GITC. This is especially a concern when working with tissues high in endogenous RNase such as spleen and pancreas. Incomplete disruption may also result in decreased yield because some of the RNA in the sample remains trapped in intact cells and, therefore, is unavailable for subsequent purification. For most samples, thorough disruption can be monitored by close inspection of the lysate after disruption.

There should be no visible particulates, except when disrupting materials containing hard, non-cellular components, such as connective tissue or bone. Finding the most appropriate method of cell or tissue disruption for your specific starting material is important for maximizing the yield and quality of your RNA preparation. The following article describes various disruption methods, and suggests which method to use for specific tissues/cell types. All of the disruption methods described here are compatible with Ambion's RNA isolation kits.

samples of total RNA isolated using the Ambion ToTALLY RNA™ Isolation Kit.

Figure 1. Representative samples of total RNA isolated using the Ambion ToTALLY RNA™ Isolation Kit. Total RNA was isolated from each sample as per protocol. Approximately 2 μg of each was electrophoresed on a 1% denaturing agarose gel and stained with ethidium bromide. Note that smaller rRNA bands visible in the leaf sample are derived from plant organelles such as plastids and chloroplasts.

Mechanical Or Enzymatic Disruption ?

Mechanical

Cell and tissue disruption methods are usually mechanical or enzymatic. Mechanical methods for disrupting fresh tissue and cells include homogenization with a Dounce or with a mechanical homogenizer (such as the Brinkmann Polytron®), vortexing, sonication, French press, bead milling, and even grinding in a coffee grinder! Disrupting frozen tissue is more time consuming and cumbersome than processing fresh tissue, but freezing tissue samples is sometimes necessary. Samples are usually frozen when, 1) they are collected over a period of time and thus, cannot be processed simultaneously; 2) there are many samples, 3) samples are collected in the field, or 4) mechanical processing of fresh samples is insufficient for thorough disruption. A mortar and pestle or bag and hammer are typically used when the starting material is frozen. Ambion's novel RNAlater™ Tissue Storage/RNA Stabilization Solution provides an alternative to freezing samples by stabilizing the RNA within a tissue sample until disruption is performed. RNA will remain intact in tissues for a day at 37°C, a week at 25°C, a month at 4°C, and indefinitely at subzero temperatures.

Enzymatic

Lysozyme, zymolase and lysostaphin digestion are among the enzymatic methods frequently used with bacteria and yeast to dissolve a coat, capsule, capsid or other structure not easily sheared by mechanical methods alone. (Ambion supplies both lysozyme and lysostaphin in its GramCracker™ Reagents, a front-end module to treat Gram-positive and Gramnegative bacteria and yeast.) Enzymatic treatment is usually followed by sonication, homogenization or vigorous vortexing in a GITC lysis buffer. Enzymatic methods may also be used for specific eukaryotic tissues, i.e. collagenase to break down collagen prior to cell lysis. For a quick review of disruption options, please see the chart below.

Animal Tissues And Cells

Most animal tissues can be processed fresh (unfrozen). It is important to keep fresh tissue cold and to process it quickly (within 30 minutes) after dissection. Tissues stored in Ambion's RNAlater Buffer can be treated as though they are fresh and processed identically. When disrupting fresh tissue, the cells need to be sheared immediately at the time the GITC lysis solution is added. This can be done by dispensing the lysis solution in the Dounce or tube, adding the tissue and immediately sonicating or homogenizing. Samples should never be left sitting in lysis solution, undisrupted. Hard tissues such as bone, teeth and some hard tumors may require milling. SPEX CertiPrep, Inc. of Metuchen, NJ (732-549-7144) manufactures freezer mills that pulverize samples by shuttling an impactor back and forth magnetically at cryogenic temperatures. Some Ambion customers have reported successful sample disruption using such mills. Mills may also be useful for other hard materials - Ambion's Technical Services Department has even had requests for protocols to isolating RNA from rock!

Animal tissues that have been frozen after collection are disrupted by grinding in liquid nitrogen with a mortar and pestle. During this process, it is important that the equipment and tissue remain at cryogenic temperatures. Pre-chill the mortar on dry ice and add liquid nitrogen to the mortar as the tissue is ground. The tissue should be dry and powdery after grinding. Grinding should be followed by thorough homogenization with a Dounce or mechanical homogenizer in a GITC lysis buffer. Processing frozen tissue in this way is cumbersome and time consuming, but effective. Alternatively, some samples can be stored in Ambion's RNAlater Buffer and processed as fresh tissue. Cultured cells are normally easy to disrupt. Cells grown in suspension are collected by centrifugation, washed and resuspended in a GITC lysis solution. Lysis is made complete by immediate vortexing or vigorous pipetting of the solution. Attached cells can be lysed directly on the culture plate. GITC lysis solution is added directly to the plate or flask and cells are scraped into the solution. The lysate is then transferred to a tube and vortexed or pipetted to ensure complete cellular disruption.

Alternatively, cells can be detached, collected, rinsed with PBS to remove culture medium, and then lysed by vortexing or sonicating in the lysis solution. Placement of the flask or plate on ice while washing and lysing the cells will further protect the RNA from endogenous RNases released during the disruption process.

 

Mechanical

Enzymatic

Soft Animal Tissues(Spleen)

Hard Animal Tissues (Bone)

Plant Tissues

Yeast Fungi Bacteria

Soil, Rock

Grind in liquid
Nitrogen with
Mortar & Pestle

 

 

 

 

 

 

 

   
Homogenize in Dounce or
Polytron®

 

 

 

 

 

 

 

 

 
Bead Mill

       

 

Freezer Mill

   

       

Sonication

           

 
Lysozyme

 

         

 
Lyticase,
Zymolase,
Glucalase

 

 

     

 

     

Plant Tissues

Soft, fresh plant tissues from plants such as Nicotiana and Arabidopsis can often be disrupted by homogenization in lysis buffer alone. (RNA yields from Arabidopsis are typically low; please see Figure 2 for typical plant RNA profiles.) Other plant tissues, like pine needles, need to be ground dry, without liquid nitrogen. Some hard, woody plant materials may require freezing and grinding in liquid nitrogen or milling. Plant cell suspension cultures and calluses can be lysed by sonication in a lysis buffer for 0.5 – 2 minutes (3). The diversity of plants and plant tissue make it impossible to give a single recommendation for all. However, most plant tissues typically contain polysaccharides and polyphenols that can coprecipitate with RNA and inhibit downstream assays. Treating a plant tissue lysate with polyvinylpyrrolidone (PVP) will precipitate such problematic components from the lysate before the actual RNA isolation is carried out (4). Ambion’s Plant RNA Isolation Aid is a ready-to-use PVP solution with which plant tissue lysates can be treated.

Yeast And Fungi

Yeast can be extremely difficult to disrupt because their cell walls may form capsules or nearly indestructible spores. There are several ways to approach yeast cell disruption. One of the most common and probably the most straightforward methods is mechanical disruption using a bead mill. Bead mills vigorously agitate a tube containing the sample, lysis buffer and small glass beads (0.5 - 1 mm). Bead mills are available from Biospec Products, Inc. Bartlesville, OK (800-617-3363). In a few minutes, cells are completely disrupted. Alternatively, yeast cell walls can be digested with zymolase, glucalase and / or lyticase to produce spheroplasts that are readily lysed by vortexing in a guanidinium-based lysis buffer. (Ambion’s GramCracker™ Reagents may also be useful here.) Some specialized isolation methods for yeast exist which use such methods as boiling SDS or boiling phenol treatment (yikes!) to insure complete cell lysis. To disrupt filamentous fungi, scrape the mycelial mat into a cold mortar, add liquid nitrogen and grind to a fine powder with a pestle. The powder can then be thoroughly homogenized or sonicated in lysis buffer to solubilize completely. As fungi may also be rich in polysaccharides, treatment with PVP may be helpful here too.

Total RNA isolation from various plant tissues using RNAqueous™ and Plant RNA Isolation Aid

Figure 2. Total RNA isolation from various plant tissues using RNAqueous™ and Plant RNA Isolation Aid. The two large ribosomal RNA bands are clearly visible. Note that the two kits may also be used on a fungus.

Bacteria

Bacteria, like plants, are extremely diverse; therefore, it is difficult to make one recommendation for all bacteria. Bead milling will lyse most Gram positive and Gram negative bacteria, including mycobacteria. It can be performed by adding glass beads and lysis solution to a bacterial cell pellet and milling for a few minutes. It is possible to lyse some Gram negative bacteria by sonication in lysis solution alone. At Ambion, we have found this to be sufficient for small cultures (milliliters), but not large cultures (liters). Bacterial cell walls can be digested with lysozyme to form spheroplasts. Gram positive bacteria usually require more rigorous digestion (increased incubation time, increased incubation temperature, etc.) than Gram negative organisms. The spheroplasts are then easily lysed with vigorous vortexing or sonication in GITC lysis buffer. GramCracker™ Reagents may prove useful for such isolations.

Soil And Sediments

Disruption of cells found in soil and sediments is accomplished one of two ways. One technique isolates the bacterial cells from the material prior to the RNA isolation procedure. This is accomplished by homogenization of wet soil in a Waring blender followed by a slow speed centrifugation to remove fungal biomass and soil debris. The supernatant is centrifuged again at a higher speed to pellet the bacterial cells (5). From this point, cells can be lysed as described above for bacteria. Other techniques describe RNA isolation from the soil or sediment directly. For example, one method requires soil to be added to a bead mill along with diatomaceous earth and lysis buffer. The sample is then agitated for a few minutes and centrifuged to remove solid debris.

Getting the RNA Out

All of the disruption methods described here are compatible with Ambion's RNA isolation kits. Cellular disruption in a strong denaturant such as GITC, provided as a component of Ambion's RNA isolation kits, yields a cell lysate from which RNA will then be isolated. The cell lysate can be used immediately or frozen for future use. Contact Ambion (800-888-8804 option 2) for more information about its RNA isolation products.

References

  1. Rapley, Ralph, Manning, David L. RNA Isolation and Characterization Protocols. 1998. Humana Press, Inc. Totowa, New Jersey.
  2. Farrell, Robert E., Jr. RNA Methodologies. 1993. Academic Press, Inc. San Diego, California.
  3. Liao, Yu-Cai, Drossard, Jurgen, Nahring, Jorg M., Fischer, Rainer. Isolation of RNA from Plant Cell Suspension Cultures and Calli by Sonication. BioTechniques (1997) 6:996-1000.
  4. Wilkins, T.A. and Smart, L.B., Isolation of RNA from Plant Tissue, in A Laboratory Guide to RNA: Isolation, Analysis, and Synthesis, edit. P.A. Krieg. ISBN 0-471-12536-9; Wiley-Liss, Inc. 1996.
  5. Holben, William E., Jansson, Janet K., Chelm, Barry K., Tiedje, James M. DNA Probe Method for the Detection of Specific Microorganisms in the Soil Bacterial Community. Applied and Environmental Microbiology. (1988) 54:3. 703- 711.