The significance of modifying cereal crops by breeding and genetic modification has never been greater as the impact of climate change and increasing disease affects yields. In generating modified and new varieties of cereal crops, the isolation of DNA becomes a routine activity for either tracking or identifying key markers. The DNA isolation process can also be a major bottleneck in the research effort as the disruption of seeds and leaf tissue is often performed on individual samples, whether it is a single leaf punch or pool of seeds from a field trial. The use of mortar and pestle, handheld homogenizer, coffee grinder, and blender on hundreds to thousands of samples is slow and painfully monotonous.
OPS Diagnostics, in collaboration with multiple companies in both the agricultural biotechnology and laboratory equipment sectors, has developed tools aimed at reducing the bottleneck of sample preparation for DNA, RNA, and protein isolation. These tools are summarized below.
High Throughput Homogenizing - No single tool has lead to a decrease in sample processing time as the linear motion mixer mill, a bead beater that accommodates multiple samples per processing run. There are many different commercially available mixer mills, but only two provide the linear motion (i.e., straight up and down, or back and forth) that is needed to standardize the processing of each individual sample. The GenoGrinder 2010 and HT Homogenizer can handle thousands of samples daily. The units are very similar except in their capacity.
Leaf Homogenization - Leaf tissue can be processed in high throughput homogenizers very effectively. Though some researchers freeze dry leaves prior to grinding, this is generally not necessary as samples from leaf punches to full leaves can be effectively disrupted in less than two minutes. The factor that limits sample throughput is sample size. Leaf punches, i.e., samples made with a paper punch, can be homogenized in a deep well plate with a 5/32" grinding ball. The microplate format permits up to 192 samples per run in a high throughput homogenizer. Homogenates are generally fluid and can readily be processed by a liquid handling workstation.
For larger leaf samples, such as multiple leaf punches or full leaves, grinding vials are required. Two standard containers are used for leaf homogenization, namely 4 ml and 15 ml polycarbonate vials. When it comes to grinding by bead beating, polycarbonate vials provide superior results as they are strong and rigid. Vials made of more popular polymers, such as polypropylene, tend to heat during grinding and become pliable. Soft plastics make poor grinding vials. In 4 ml vials, up to 500 mg of leaf tissue can be processed, while the 15 ml vials can handle 2-3 grams of leaf. These vials use one 3/8" grinding ball for the 4 ml vial and up to two 7/16" balls for the 15 ml vials.
Many times leaves are the target for RNA isolation for gene expression profile studies. As with all RNA isolation procedures, it is important that the RNA is not degraded during the homogenization process. One common method for preserving the RNA is to homogenize or grind the sample while it is cryogenically frozen, i.e., below -120°C. Ceramic mortar and pestles chilled with liquid nitrogen can be used effectively for grinding, but once again the throughput is poor. A miniaturized and motorized version of the mortar and pestle, the CryoGrinder™, has been developed for processing of multiple small samples. The mortar is a rectangular block, 1" square and 1.5" tall, which is matched up with a motor driven porcelain-zirconium pestle that effectively grinds leaf punches and other small samples. The small mortar size allows for efficient transfer of the homogenized sample to tubes for further processing.
Seed Grinding -Seeds can be one of the more difficult substances to grind in a high throughput format. This is especially true for dried kernels. Even rice grains are difficult to grind in comparison to leaf tissue. For instance, one grain of rice weighs less than a leaf punch however a 5/32" grinding ball has no effect on the grain. To homogenize the grain, a 3/8" grinding ball and 4 ml polycarbonate vial are needed. Thus, the major obstacle in homogenizing seeds are their inherent toughness which requires larger grinding balls. This immediately impacts throughput by as larger vials are needed. However even corn kernels can be homogenized in a 24 well format in 2-3 minutes. Pooled field samples can be ground in larger 15 ml polycarbonate vials. These 4 and 15 ml vials have been formatted into vial sets which simplify the processing. The 24 well vial sets can even have grinding balls removed using a high throughput 24 pin magnet.
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| HT Homogenizer | GenoGrinder 2010 | Vial Set | Soy homogenized in a polycarbonate vial |