One of the most efficient ways to extract nucleic acids from a sample is to use mechanical force to release them. This is done repeatedly under high speed, until cell walls and membranes are disrupted 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, i.e., cell lysis that facilitates subsequent DNA or RNA isolation. While enzymes can be successful for DNA isolation from a limited number of sample types, results are achieved a lot faster if the matrix is broken down first. Furthermore, RNA cannot be isolated in a timely fashion without the use of mechanical maceration.
Questions inevitably arise regarding how hard to beat to lyse a sample and how to know what bead type to use. The answers depend on many variables, so to avoid the frustration of sorting through them all, here is our best advice on the methods that we have used at QIAGEN and found to work best for us.
The old way to isolate RNA – liquid nitrogen
RNA isolation from tissues always requires extensive 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
Nowadays, liquid nitrogen/mortar and pestles are not the preferred methods. For those still using this method, tools need to be cleaned between samples, or many of them need to be available on hand for use. The same is true for handheld 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 a risk of cross-contamination. If disposable probes are used on the rotor-stator, it is a much better method. The drawback, however, is that only one sample can be processed at a time.
High velocity bead beating – more samples, no cross-contamination
High powered bead beaters are now the preferred homogenizing method, easily superseding the abilities of their one-at-a-time method predecessors. Therefore, we have recently added the PowerLyzer® 24 Homogenizer to our portfolio. The PowerLyzer 24 Homogenizer is the quietest homogenizer on the market, and unlike other models, it doesn’t cause a bench top to vibrate. Additionally, the homogenization time needed for best results is shortened due to the horizontal positioning of the bead tube. This results in less heat and damage to the nucleic acids as well as high grinding efficiency. Less time also means better RNA integrity.
Which bead tubes should be used?
For RNA from plant and animal tissues, we recommend the 2.8 mm ceramic bead tubes for two reasons. First, the 2.8 mm bead size is perfect for 10–20 mg of animal tissue or 50 mg of plant tissue. We tested the 1.4 mm beads for tissues but the small size does not give as good a result with short homogenization times. The longer time needed to liquefy samples increases RNA degradation.
In addition, a single bead type should be used rather than a bead mix. This 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 that 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 optimal 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. While they all worked, 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®-24. However, unlike the FastPrep-24, the PowerLyzer 24 Homogenizer can be programmed for any number of cycles as well as rest time in between cycles, and the protocol and program can be saved for future use.
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.
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 experiments can be up and running right from the start. Furthermore, RNA yields and integrity are going to be better when everything can be homogenized immediately without long lag times with samples on ice.
We offer ready-to-use kits for plant and tissue and cell samples for the PowerLyzer 24 Homogenizer or other high-powered bead beaters that are complete with the validated and optimal bead tubes. Additionally, 2.8 mm ceramic bead tubes can be purchased separately.
We will discuss best practices for soil microbial DNA extraction in an upcoming post so stay tuned for more tips and tricks for your microbial studies. Another way to learn how to optimize your microbial research workflow is to check our webinar page for upcoming live webinars and a library of recordings and slide decks of past webinars.
Trademarks: FastPrep® is a trademark of MP Biomedicals.
Authors: This post was originally published on mobio.com, and has been updated and modified by Heather Martinez and Miranda Hanson-Baseler.