One of the most efficient ways to extract nucleic acids from a sample is by smashing it against a hard surface repeatedly under high speed until cell walls and membranes crush from the pressure and release their internal contents. In other words: bead beating.

Bead beating is a great way to do what enzymes take hours to accomplish and sometimes never fully succeed in, which is cell lysis to release DNA or RNA for isolation. While enzymes can be successful for DNA isolation from a limited number of sample types, results are achieved a lot faster if you break down the matrix first. And RNA cannot be isolated in a timely fashion without the use of some kind of mechanical maceration.

The questions inevitably arise though, how hard do I need to beat to lyse my sample and how do I know what bead type to use?  The answers depend on a great number of variables, so to avoid beating your head against the wall to sort through them all, I have written this two-part blog series offering advice on the methods that we have used at MO BIO and found to work best for us.

This first article will focus on guidelines for RNA from tissues and plants.  There is so much to discuss about soil and microbes, so we’ll keep that for the second blog article, which will include data from our own research.

The Old Way to Isolate RNA- Liquid Nitrogen

RNA from tissues always requires serious pulverization. In the past, the most common method was using liquid nitrogen to freeze the sample and a mortar and pestle to grind the tissue to a powder. Although this approach works well, it is not complete. Once the sample is powdered and resuspended in a chaotropic lysis buffer, the genomic DNA is still high molecular weight and will add viscosity to the sample that can clog spin filters. To overcome this, the next step is to shear the genomic DNA with a needle and syringe which improves the efficiency of removing the genomic DNA from the column.

Drawbacks of Liquid Nitrogen Processing and Rotor Stator Homogenizers

Now, however, it’s the year 2010 and liquid nitrogen/ mortar and pestles are not the preferred method. Using these outdated methods, you need to either clean tools between samples, or you need to have a lot of them on hand and ready for use. The same is true for hand held rotor-stator homogenizers. This method is excellent for breaking a tissue down quickly and thoroughly so that the RNA is isolated with minimal degradation. However, the probe also needs to be cleaned between samples and there is always risk of cross-contamination. If you can use disposable probes on your rotor-stator, it is a better way to go. The drawback, however, is that you still can only process one sample at a time.

High Velocity Bead Beating- More Samples, No Cross-Contamination

This is where the high powered bead beaters come in and supersede the abilities of one-at-a-time methods. At MO BIO, we’ve developed a new instrument called a PowerLyzer™ bench top homogenizer. After working with and testing many models, we custom designed a machine with features that had everything we wanted for our own use, knowing that everyone else using bead beaters would desire the same changes. Basically, it’s a lot quieter and doesn’t vibrate your entire bench top.  And, the homogenization time needed for best results is also shortened (resulting in less heat and damage to the nucleic acids) because the horizontal positioning of the bead tube, (similar to the vortex adapter), is more efficient at grinding.   Less time means better RNA integrity.

What Bead Tubes do I Use?

For RNA from plant and animal tissues, we use the 2.8 mm ceramic bead tubes for two reasons. First, the 2.38 mm bead size is perfect for a 10-20 mg piece of animal tissue or 50 mg of plant tissue. We tested the 1.4 mm beads for tissues and the small size does not give as good a result with short homogenization times.  The longer time needed to liquify the sample increase degradation of RNA.

Using a single bead type (vs. a bead mix) allows for better consistency and homogeneity from prep to prep, since the larger more effective beads have complete access to the tissue and are not blocked by smaller less effective beads. The second reason for using the 2.8 mm ceramic bead tubes is because they are pre-loaded in a “Tough Tube” which is a specially made plastic bead tube that can withstand high force without breaking.

What is the Best Speed and Number of Cycles for Homogenization

This will vary based on the instrument used.  For animal tissues, we tested a wide range of speeds from 3500 to 5000 RPM, and while they all worked, the optimal RNA recovery was observed using 3500 RPM for 2 x 45 seconds using a 30 second rest between cycles.  This speed is about equivalent to a setting of 5.5 on the FastPrep. However, unlike the FastPrep, the PowerLyzer allows you to program any number of cycles and rest  time in between cycles, so you can save the protocol and program others and keep them handy for next time.

For Plant RNA, we found the optimal setting to be 4200 RPM for 2 x 45 second cycles with a 30 second rest in between cycles.  When 3500, 4200, and 5000 RPM were compared, 4200 gave the highest yield RNA from a variety of samples that included leaf, stem, roots, and seeds.

Summary

High powered bead beaters have many advantages over methods that process only one sample at a time. But for many people, this means re-optimizing current protocols. Fortunately, we’ve done a lot of the optimization already so you can get up and running right at the start. And RNA yields and integrity are going to be better when you can homogenize everything quickly and at once, and not have long lag times with your samples on ice while you move through all your preps.

If you are interested in ready-to-use kits for the PowerLyzer™ or other high powered bead beaters that are complete with the validated and optimal bead tube, you can find more information at these links.

For RNA from tissues go to the PowerLyzer UltraClean Tissue & Cells RNA Isolation Kit page and for RNA from plants, go to the PowerLyzer UltraClean Plant RNA Isolation Kit page. 

The PowerPlant DNA Isolation Kit is ready-to-use on high powered bead beaters, with tough tubes containing 2.38 mm stainless steel beads.

For DNA from soil and microbes, we will discuss this in an upcoming article and will present to you some very interesting data!

~Suzanne

This entry was posted on Friday, June 18th, 2010 at 4:02 pm and is filed under Tech Tips. You can follow any responses to this entry through the RSS 2.0 feed. Why not leave a response, and share your opinion?