The Use Of Steel Homogenizer Probe Results In Sample Carryover Contamination
by Brian E Mace and Patrick M Sullivan
Duke University Medical Center, Durham, NC
IntroductionIn any experiment involving different treatment groups or varying starting material it is important to insure no crossover or contamination of sample material occurs. We routinely work with mouse models of varying genotype and then study the effects of different treatments in these mice. More specifically, we measure the levels of apolipoprotein E protein and mRNA in brain tissue using a very sensitive and quantitative assay system. Therefore, we tested two types of probes (Omni International) for potential contamination between tissue samples during the homogenization process. The probes we tested were the plastic tip disposable generator probe (Omni cat # 34725) and the steel tip probe (Omni cat #G7-95st)
ProcedureAfter removal from the skull the mouse brain is cut in half and then each hemisphere is cut into 5-8 mm pieces before being placed into a cryotube and then immediately frozen in liquid nitrogen. All samples were treated the same with the probe type being the only variable. One half of the brain was homogenized with the plastic probe and the other half was homogenized with the steel probe. The tissue is placed into 1 ml of guanidine buffer (5.0 M guanidine-HCL, 50 mM Tris pH 8.0) and allowed to thaw for 30 seconds before homogenizing on ice using a GLH homogenizer (Omni). Homogenization was done on low speed (setting 1) for 45 seconds, medium speed (setting 3) for 45 seconds, and then on high-speed (setting 6) for thirty seconds in a 2 ml cryogenic vial (cat # 430488 Corning Incorporated, Corning, NY). The probes were then cleaned in water on high speed for two minutes and wiped down with a kimwipe (Kimberly- Clark, Roswell, GA). The probe is then put in to a cryogenic vial containing only 1 ml of guanidine buffer and then turned on for 1 min to "clean" the probe. This is repeated one more time. The primary sample is rocked for 4 hours at room temperature to insure complete solubilization. Homogenates are diluted 1:10 in ice-cold casein buffer (0.25% casein/phosphate buffered saline pH 7.4, 0.05% sodium azide, 1X protease inhibitor cocktail, 0.5 M EDTA, pH 8.0,) and kept on ice. Diluted homogenates are spun in an Eppendorf microfuge at 14,000 rpm for 20 minutes at 4 C and the supernatant assayed for protein content.
The Micro BCA Protein Assay (Pierce) was used according to the manufacture's protocol. In brief, 150µl of sample is pipeted into duplicate wells of a 96 well plate (Costar cat # 3596, Corning Incorporated, Corning, NY). 150µl of working solution was added to each well and the plate was mixed for 30 seconds on a shaker. The plate was covered and incubated at 37 C for 2 hours and the absorbance was measured at 562 nm on a Theromomax microplate reader (Molecular Devices, Sunnyvale CA). The samples were averaged and background subtracted. Protein concentration was determined by using a known concentration of BSA as the standard. To correct for different weights of tissue the following formula was used:
Average protein concentration of wash/Average protein concentration of 1/2 brain *100%These numbers were then used to compare the amount of protein carryover between the plastic and steel probe.
ResultsBoth the Omni tip disposable generator probe # 34725 and the steel Omni tip probe #G7-95ST homogenized the brain tissue very efficiently. For comparison sake we corrected for different starting amounts of protein by looking at the percent of wash homogenate protein compared to the total protein detected in the 1/2 brain homogenate. The plastic Omni tip disposable generator probe # 34725 had a carry-over of 0.6% (0.12 µug/ml) of total protein from brain homogenate. The steel Omni tip probe #G7-95ST had a carry-over of 2.4% (0.34 µug/ml) of total protein from brain homogenate (fig.1).
It is worth noting that these numbers were obtained after processing of a single sample. Since the steel probe would be used for all subsequent samples we do not know if this carryover of protein contamination would accumulate over time. Extensive cleaning of the steel probe between each sample may minimize this risk, however the time required for autoclaving, cleaning and technician time does not warrant use of the steel probe in our opinion. Also, SDS-solubilized tissue may show even more carryover, since a much larger fraction of the tissue is not soluble in SDS. Obviously this is not an issue when using the disposable probes. For measuring small differences in mRNA between samples using a RT-PCR method, the plastic disposable probes would clearly be preferred.
About the authorsBrian E. Mace and Patrick M. Sullivan work at Duke University Medical Center, in Durham, North Carolina.