Effect of Homogenization Method on DNA Yield and Fragment Size
By Myan Colatat, OPS Diagnostics, LLC
A comparison of four methods used to homogenize mouse muscle for DNA isolation was performed. Liquid nitrogen chilled mortar and pestle, liquid nitrogen chilled CryoGrinder, rotor-stator, and high throughput bead beater were compared using the parameters of DNA yield, purity, and fragment size. Rotor-stator homogenizer generated the smallest fragments while the CryoGrinder produced the largest. The bead beater generated the highest yield while the traditional liquid nitrogen chilled mortar and pestle produced the least. The highest purity sample was produced with the bead beater while the rotor-stator had the lowest. No one method produced the greatest yield, purity, and fragment size. Depending upon the application, different homogenization methods can be matched to selected assays to gain the best results.
Sample disruption is a necessary early step in the isolation of RNA, DNA, and proteins. Both chemical and physical methods are used to disrupt samples. Chemical methods typically rely on detergents and chaotropes while tools for physically disrupting samples include mixer mills (bead beaters), sonicators, mortar and pestles, and hand-held homogenizers (rotor-stators).
Though chemical lysis is often the most efficient method, it is not always completely reliable for disrupting solid tissues. An alternative approach involves mechanically disrupting the sample. Two common methods of mechanical disruption are shearing through the use of a rotor-stator, which involves slicing, dicing, and/or cutting, and pulverizing/smashing through the use of a mixer mill or a mortar and pestle.
Rotor-stators are used to quickly shear samples. They have a blade inside of a stationary, perforated tube which rotates at high speeds. It is essentially a circular scissor, very similar to a handheld blender. Rotor-stators are a classical and widely used tool for homogenizing samples. Mixer mills, or bead beaters, are used for individual samples and high throughput applications. Paint shakers and dental amalgamators were the original mixer mills, but have been replaced with machines designed for scientific use. The paint shakers' figure "8" motion has been superseded by the mixer mill's reciprocating linear motion. The mortar and pestle is a historic tool used for grinding samples. It is still popular, and when partnered with liquid nitrogen, it is effective for disrupting samples for DNA and RNA isolation. A miniaturized version of the mortar and pestle, the CryoGrinder™ is now available for grinding very small samples (up to 100 mg).
Much effort has been expended to optimize the conditions for isolating DNA from various tissues, however little has been reported as to the best homogenization method. In an effort to clarify the effect homogenization has on DNA isolation, a comparison was made between four tissue homogenization methods used to disrupt solid tissue in preparation for processing using a commercially available kit.
Materials and Methods
Tissue Samples: Female CD1 mice, 42 days (Charles River Laboratories) were used and leg muscles were extracted, frozen, and stored at -140°C until use. For homogenization, tissue was thawed and processed as described below.
DNA Purification from Tissue: Mouse muscle (25 mg) was homogenized as described below and DNA was purified using a modified QIAamp DNA Mini Kit protocol to isolate RNA-free DNA. As an alternative to the QIAamp DNA Mini Kit ATL lysis buffer which contains SDS and foams extensively during homogenization, 140μl TEN9 lysis buffer (50mM Tris pH 9, 100mM EDTA, 200mM NaCl) was used during homogenization or added to pulverized tissue following grinding. To this mixture, 20 μl SDS and 20 μl Proteinase K (10 mg/ml in water) were added. This TEN9 buffer with SDS and Proteinase K is essentially the same as ATL lysis buffer.
Muscle homogenized cryogenically with the CryoGrinder or mortar and pestle were transferred to a container containing TEN9. Sample processing with rotor-stator and HT Homogenizer (bead beater) were homogenized in TEN9 buffer. In both instances, SDS and Proteinase K were added subsequently.
Purified DNA was analyzed spectrophotometrically at 260 and 280 nm to measure concentration and purity. Agarose gel electrophoresis (1% agarose in 1X TAE buffer) was used to assess the fragment sizes of isolated DNA. For visualization, DNA samples were concentrated 20X by alcohol precipitation before electrophoresis.
Homogenization: Homogenization was performed with liquid nitrogen chilled mortar and pestle (Coors), liquid nitrogen chilled CryoGrinder (OPS Diagnostics), rotor-stator homogenizer (Virtis), and HT Homogenizer mixer mill (OPS Diagnostics). A summary of each method is found in Table 1.
Table 1. Methods used to homogenize mouse muscle.
Mortar and Pestle
The sample (25 mg) was frozen with liquid nitrogen, placed in a cryogenically chilled 150 mm mortar, and then pulverized with a chilled pestle. Pulverized muscle was transferred to a chilled 2ml microfuge tube and suspended in 140 μl TEN9 lysis solution followed by 20 μl SDS and 20 μl Proteinase K.
CryoGrinder™ (miniature mortar and motorized pestle)
The sample (25 mg) was frozen in a CryoCooler with liquid nitrogen, placed into a pre-chilled CryoGrinder mortar, and then pulverized first with the small and then large motor driven pestles. Ground sample was transferred to a chilled 2ml microfuge tube and suspended in 140 μl TEN9 lysis solution followed by 20 μl SDS and 20 μl Proteinase K.
The sample (25 mg) was placed in a 96-well plate with one 5/32" stainless steel grinding ball (OPS Diagnostics, GBSS 156-5000-01), 140μl TEN9 (without 20 μl SDS and 20 μl Proteinase K), and then capped with a press on mat. The plate with sample was placed into the mixer mill (HT Homogenizer, OPS Diagnostics) and homogenized at 1200 rpm for 2 minutes. The lysate was then transferred to a 2ml microfuge tube, followed by the addition of 20 μl SDS and 20 μl Proteinase K.
The sample (25 mg) was placed into a 4 ml plastic tube containing 140 μl TEN9 (without SDS and Proteinase K) and mechanically homogenized with a 6mm tip (generator) at 10 K rpm for 2 min with a Tempest rotor-stator homogenizer (Virtis, Gardner, NY). The lysate was transferred to a 2 ml microfuge tube and 20 μl SDS and 20 μl Proteinase K were added.
DNA yield, purity and fragmentation are affected by the method used to homogenize the sample. The greatest DNA yield was obtained by bead beating samples with the HT Homogenizer followed by the rotor-stator and CryoGrinder while the mortar and pestle had the lowest yield (Table 2). Likewise, the HT Homogenizer generated the DNA with the highest purity as measure by 260/280 ratio (Table 2). The CryoGrinder also had good purity followed by the mortar and pestle. The rotor-stator was had the poorest purity.
Fragment size was significantly impacted by the homogenization method as the shearing method (rotor-stator) generated a continuum of fragments starting at a high of around 15 kb (Fig. 1). The HT Homogenizer generated a narrower range of larger fragments than the rotor-stator, as did the mortar and pestle. The CryoGrinder produced a much broader range of large fragments than the other methods. In addition to the fragments, significant fluorescence (larger molecular weight molecules) was present in the wells corresponding to the samples of the HT Homogenizer, rotor-stator, and CryoGrinder.
|Figure 1. Purified DNA analyzed on 1% agarose gel (1X TAE) with Lambda-HinDIII size markers.
Lane 1 - Lambda HinDIII digest
Lane 2 - HT Homogenizer
Lane 3 - Rotor-stator
Lane 4 - Mortar and pestle
Lane 5 - CryoGrinder
Lane 6-Lambda HinDIII digest
Table 2. Optical Density, Ratio, and Yield of Purified DNA.
|260 nm||280 nm||Ratio||DNA Yield (μg)|
|Mortar and Pestle||0.20||0.14||1.41||4.0|
Methods used to homogenize tissue for DNA isolation impact yield, fragment size, and purity. Bead beating with the HT Homogenizer produced the highest yield and best purity. Cryogenic grinding with the CryoGrinder produced large DNA fragments, but lower yield and purity. The rotor-stator had a good yield, but the lowest purity and smallest fragments. The mortar and pestle had good size fragments, but relatively low purity and very poor yield. In short, no one method produced the highest yield, the highest purity, and largest fragments.
A significant effect as a direct consequence of homogenization is DNA fragment size. Rotor-stators, which rely on a rotating knife for shearing tissues produce relatively small fragments as compared to bead beating and grinding. This result is not particularly surprising as large molecular weight DNA can be easily sheared by the simple act of pipetting. While the other three methods evaluated also generated a range of fragment sizes, none were nearly as heterogeneous as those produced by the rotor-stator. The bead mill generated larger fragments which may be a result of the high impact of the grinding ball a limited number of times (approximately 2500) compared with 20,000 rotations of the rotor-stator "knife." The mortar and pestle, including the CryoGrinder, disrupt more by crushing and grinding, and as such generated relatively large fragments. This was very apparent with the CryoGrinder. Grinding and crushing apparently have less of a deleterious effect on DNA than shearing.
All four homogenization methods started with the same mass of mouse tissue, yet the yields were significantly different. Bead beating the muscle has previously been demonstrated to very effectively disrupt muscle fibers for protein release (see link). In comparison, disrupting muscle with a rotor-stator produced homogenates that retain muscle fibers and filaments. These small pieces of tissue are not thoroughly disaggregated by further treatment with SDS and Proteinase K, thus significant DNA is lost when the lysate is cleared by centrifugation prior to purification. The CryoGrinder was less effective than both the rotor-stator and the HT Homogenizer. With the CryoGrinder, some sample is not recovered from the mortar while grinding efficiency is similar to the rotor-stator. This leads to overall lower yields. The mortar and pestle had the lowest yield. However, this poor showing is less a factor of grinding efficiency as it is recovery. With small sample mass, grinding in a relatively large mortar makes collecting the pulverized sample difficult. The result is very low DNA yields. This yield issue with the mortar and pestle was the rationale for developing the CryoGrinder. The CryoGrinder, which works on the sample principle as the mortar and pestle, has a miniature mortar (approximately 1/2 inch in diameter) that limits the spread of the pulverized sample and allows for efficient collection following grinding.
The different degrees of sample purity resulting from the processing are difficult to explain (Table 2). Once homogenized, all samples had SDS and Proteinase K added and then were purified using the QIAamp DNA Mini Kit. This kit is designed to remove impurities yet results varied based on the 260/280 ratio from 1.29 to 1.77 OD260/OD280. It is possible that contaminants other than those from the sample were introduced from the rotor-stator and mortar and pestle, both of which have been routinely used. The HT Homogenizer relies on consumable plates and balls which would limit contamination. In this test, the CryoGrinder was relatively new. Regardless of the source, it is important to note that intrinsic or extrinsic factors can affect the final purity of the DNA.
Depending upon the application, each of these disruption methods has value. For large samples, grinding with liquid nitrogen in a mortar and pestle is still a viable option for DNA isolation. Fragment size is relatively large, though potential purity problems must be considered. For smaller samples, the CryoGrinder yields large fragments and decent yield. Often small fragments are desirable, in which case the rotor-stator can be used. Bead beating with the HT Homogenizer was effective at generating the most DNA with relatively large fragment size. It has the further advantage of being capable of processing hundreds of samples per hour. Thus, depending upon the objective, each of these tools is valuable for isolating DNA.