Use of Novel Abrasives for the Extraction of RNA from Small Amounts of Difficult Tissue
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Use of Novel Abrasives for the Extraction of RNA from Small Amounts of Difficult Tissue Scott Tighe 1 , Matt Cunningham 2 , and Tim Hunter 1 University of Vermont, Vermont Cancer Center, Burlington VT 1 and Johnson State College, Dept of Biological Sciences, Johnson, Vermont 2 Abstract Current techniques for isolating intact RNA from various tissues are a challenge with small sample sizes. Most methods rely on a homogenization technique that employs the use of equipment and supplies that are not guaranteed RNase-free. This often results in low recoveries and poor RNA quality. In this study, we describe and test the utility for RNase-free homogenization of small tissue sizes using novel abrasive particles including diamond, cubic boron nitride, silicon carbide, boron carbide, AlO 3 , and Duralum. The abrasives are used in conjunction with disposable RNase-free pestles, standard 1.5 ml microcentrifuge tubes, 1-5 mg of tissue, and 100µl of a RNA extraction reagent such as Trizol TM or guanidinium isothiocyanate. The homogenization process is a rapid technique using a motorized attachment on the pestle followed by filtration through a Qiashredder TM column and extracted using previously described methods specific to the RNA extraction reagent. Preliminary results indicate that this modified technique is effective at recovering intact RNA from small amounts of tissue. Although all abrasives performed well, several characteristics were noted during the evaluation. Diamond and cubic boron nitride appear to be the easiest to use based on tissue visualization during homogenization and filterability. AlO 3 and Duralum are two abrasives that possess a razor sharp morphology and are more suitable for tissues that require aggressive homogenization. However, tissue visualization is difficult due to their white colored appearance. Silicon carbide and boron carbide performed equally as well, allowed good tissue visibility, but formed black residue during the homogenization process because of fragmentation of the abrasive. Recovery data in pure RNA dosing and soft tissue experiments approaches 70 to 80% indicating sample retention in the abrasive matrix or Qiashredder TM column. Conversely, recoveries from hard tissues exceeded traditional homogenization techniques by two fold or greater without the sacrifice of RNA integrity. Acknowledgements The VCC Core Labs gratefully acknowledges the donation of cubic boron nitride and diamond by Warren-Amplex Superabrasives Corp. in Olyphant, PA., DSW and boron carbide by Washington Mills ElectroMinerals Corp in Niagara Falls NY, and AlO3 and silicon carbide by United Unibrite Corp. in Glen Cove NY. This work was supported in part by NCI grant P30ca22435 and Lake Champlain Cancer Research Organization (LCCRO). Materials The purpose of this study was to invent a efficient method that would recover RNA from tissue using all RNase-free disposable materials and microscopic abrasives. The need for an abrasive is justified because RNase-free homogenization is often a challenge for rigid tissues. In this study we evaluated six abrasives on four tissues types using two modified protocols using Trizol reagent and the Qiagen RNeasy kit. Qiagen RNeasy kit Trizol RNA extraction reagent MoBio 1.8 ml centrifuge tubes 1.5 ml pestle with motor unit Qiashredder columns Pellet Paint Chloroform Ethanol Isopropanol P2-P1000 pipets RNase Zap Analytical balance Biosafety hood Microcentrifu ge Abrasives Used in this Study Diamond Cubic Boron Nitride Silicon Carbide Aluminium Oxide Duralum Boron Carbide Motorized Pestle Goals Methods Abrasives were prepared RNase-free by soaking in ETOH for 30 minutes, RNase Zap for 10 minutes, washing in sterile water, autoclaving for 1 hour, and baking for 1 hour at 250F. Tissues Brain: Fresh Rat Melanoma: Snap Frozen Human Intestine: Frozen 1mM Sodium Citrate and RNase inhibitor. Modified Qiagen RNeasy Homogenization Procedure Add a fresh 100uL aliquot of RLT buffer to the original tube and vortex. Using the same pipet, transfer to the Qiashredder tube. Repeat with one additional aliquot of RLT buffer. Spin the Qiashredder at full speed for 2 minutes. Quantitatively measure the pass- through using a micropipet and transfer to a new microcentrifuge tube. Add 1 volume of 70% ethanol to the new tube containing the extract. Vortex for 3 seconds and centrifuge at full speed for 5 seconds. Pipet up and down several times and transfer to a Qiagen RNeasy mini silica column. Centrifuge the column for 1 minute at full speed. Remove the pass-through from the capture tube and reapply it to the same column. Spin again. DNase treat the column/sample according to Qiagen procedure. Centrifuge the column for 15 seconds. Discard pass-through and capture tube. Place Qiagen RNeasy mini silica basket into a new capture tube and apply 0.5 mL RW1 buffer and centrifuge at full speed for 15 seconds. Repeat this procedure with two aliquots of RPE buffer replacing capture tube each time. Place the Qiagen RNeasy mini silica basket into a new capture tube and centrifuge at full speed for 2 minutes to “dry” the silica column. Place the Qiagen RNeasy mini silica basket in a 1.5 mL centrifuge tube and apply 30 uL of DEPC water directly onto the silica membrane. Centrifuge the column under the same conditions as “drying” step. The pass through is the RNA sample. Remove the pass through and apply it again to the center of the column. Centrifuge again. This is the recovered RNA. Modified Trizol TM Homogenization Procedure Using a different pipet, add a fresh 100uL aliquot of Trizol TM to the original tube and vortex. Using the previous pipet, transfer to the Qiashredder tube. Repeat with one additional aliquot of Trizol TM . This is to insure that all homogenate is transferred to the shredder column. Centrifuge the shredder column for 2 minutes at full speed and transfer the pass-through to a Axygen TM ultra-clear 1.7 mL microcentrifuge tube. The resulting volume should be 280 uL. Add 60 ul of 100% chloroform and vortex. Allow sample to sit for 2 minutes followed by centrifuging at full speed 13,000 G for 15 minutes. Carefully remove supernatant and transfer to another Axygen TM ultra-clear 1.7 mL tube. Add 180ul of ice-cold 100% isopropanol and 1 uL of pellet paint. Centrifuge at 4C for 30 minutes. The precipitated RNA will appear as a tiny pink spot at the bottom of the centrifuge tube. Remove supernatant and add 1 mL of 80% ethanol. Invert tube 10-15 times and spin again at 4C for 15 minutes. Remove supernatant. Repeat this washing step one additional time. After removing the last aliquot of ethanol, perform a quick spin on the tube and use a P20 to remove as much of the ethanol as possible. Allow the tube to stand open for air drying for 30 minutes in hood. DO NOT allow tube to be in direct flow of air as this may cause the pellet to become aerosolized. Aseptically combine 1-10 mg of tissue, 20 mg of abrasive, and 100 uL of either RLT buffer containing b-mercaptoethanol for the modified Qiagen RNeasy procedure or 100 uL of Trizol TM for the Modified Trizol TM Homogenization and in a 1.8 mL MoBio tube. Using the motorized attachment for the 1.5 mL tube pestle, homogenized the sample for 1-5 minutes or until tissue is fully dissociated. Vortex and centrifuge briefly. Using a P1000 micropipet, pipet up and down to loosen the homogenate and than transfer the homogenate including the abrasive to a Qiashredder tube. Keep micropipet and tip for next transfer. Results 0 1000 2000 3000 4000 5000 6000 7000 SiC -1 SiC -2 BC -1 BC -2 Diam ond-1 Diam ond-2 DSW -1 DSW -2 AlO 3-1 AlO 3-2 C BN -1 C BN -2 P estle-1 P estle-2 Shredder A brasive ng R N A /m g M elanom a Qiagen Trizol Homogenization was performed in the presence of each abrasive using pure yeast RNA to determine if non-specific binding of RNA occurs. These data indicate that minimal RNA is lost (bound) Data not shown for silicon carbide. Binding Experim ent 81 92 75.9 100 42 0 20 40 60 80 100 120 C -Boron N itride DSW Al03 Diam ond Boron Carbide % Recovered Homogenization was performed on melanoma tissue ranging in size 4.2-5.7 mg. Abrasives were used in the range of 5- 20 mg. AlO 3 Performed best Melanoma Tissue: Moderately Tough Tissue Pure RNA Recovery Experiment Extraction of RNA from Rat Brain (Soft Tissue) Fresh rat brain tissue was homogenized using both techniques in replicate(s). Results of these soft tissue extractions indicate that the pestle sample had the highest recovery which is expected with soft tissue. Tough tissue however often requires a more aggressive approach. Rat Intestinal Lumen: A Tough Tissue! Fluorescence Time (seconds) 0 1 2 3 4 5 6 7 8 9 10 11 19 24 29 34 39 44 49 54 59 64 69 18 S 28 S Fluorescence Time (seconds) 0 5 10 15 20 25 30 35 19 24 29 34 39 44 49 54 59 64 69 Example of Rat Brain RNA Quality From Cubic Boron Nitride Modified Trizol TM Technique Downstream Analysis of Extracted RNA Using RT-QPCR Relative standard curve generated from cDNA that was reverse transcribed from the pestle lumen RNA sample. The resulting slope of >0.99 indicates exceptional PCR efficiency for 18S. Amplification plot of duplicates samples from the six rat lumen abrasive RNAs. These data indicate excellent reproducibility. Conclusio n All extracted RNA was analyzed using real time Q-PCR to determine if any inhibitory substances were present from the abrasive compounds. 2 ug of total RNA was reverse transcribed and amplified using rat 18s primers and probe. Homogenization was performed on 4-6 mg of rat intestine. Abrasives were used in the range of 5-20 mg. Homogenization was performed using both motorized and non- motorized rotation until visual dissociation for each tissue was observed. Dissociation was not observed for the pestle sample. 0 300 600 900 A brasive Q iagen ng RNA /m g Tissue Trizol ng RNA /m g Tissue Modified Qiagen Technique These data indicate that the use of abrasives on soft tissue such as brain is unnecessary as RNA recovery is adequate using just the pestle technique. However, increased recovery of RNA from tough tissues has been observed Pure RNA recovery experiments indicate that non-specific binding of RNA to the abrasive is insignificant. All Abrasives performed adequately but silicon carbide and AlO 3 seemed to facilitate better recoveries of RNA using the modified Qiagen and Trizol technique, respectively. AlO 3 and DSW have superior homogenization ability due to their sharp morphology. Additional studies on other tough tissues should be investigated. Secondary analysis including cDNA synthesis, RT-QPCR, spectrophotometry, and RNA assesment using the Agilent bioanalyzer indicate that the resulting RNA is of outstanding quality with no inhibitory substances. Laboratory handling of cubic boron nitride was most acceptable. It was easy to visualize the tissue and appeared to homogenize the sample effectively. Silicon carbide and boron carbide contributed a black residue to the homogenate and visualization was difficult. The diamonds used in this study seemed too small and tended to “float” during homogenization. Larger diamonds(150-200um) should be tested in future work. Although AlO 3 and DSW homogenize tissue very well, pipeting and visualization was difficult due to theconsistency and white color. Future consideration should be give to using two abrasives in combination such as silicon carbide with AlO 3. This method is a rapid and effective technique to extract RNA from small tissue sizes (1-15mg). It employs the use of all disposable RNase-free materials and is a small format that can be carried out in a biosafety Abrasive Designation Supplier Size Cubic Boron Nitride CBN Warren Diamond 140-170 m Diamond Dia Warren Diamond 74m Aluminum Oxide(AlO 3 ) AlO3 United Unibrite 130m Boron Carbide BC Washington Mills 141m Duralum Special White DSW Washington Mills 141m Silicon Carbide SiC United Unibrite 200 um Rat Intestinal Lumen: A Tough Tissue! Homogenization was performed on 4-6 mg of rat intestine. Abrasives were used in the range of 5-20 mg. Homogenization was performed using both motorized and non- motorized rotation until visual dissociation for each tissue was observed. Dissociation was not observed for the pestle sample. M odified Q iagen Hom ogenization 0 500 1000 1500 2000 ng RNA /m g Lum en M odified TrizolH om ogenization 0 3000 6000 9000 ng R N A /m g Lum en
description
Abrasive. Designation. Supplier. Size. Cubic Boron Nitride. CBN. Warren Diamond. 140-170 m. Diamond. Dia. Warren Diamond. 74 m. Aluminum Oxide(AlO 3 ). AlO3. United Unibrite. 130 m. Boron Carbide. BC. Washington Mills. 141 m. Duralum Special White. DSW. - PowerPoint PPT Presentation
Transcript of Use of Novel Abrasives for the Extraction of RNA from Small Amounts of Difficult Tissue
Use of Novel Abrasives for the Extraction of RNA from Small Amounts of Difficult TissueScott Tighe1, Matt Cunningham2, and Tim Hunter1
University of Vermont, Vermont Cancer Center, Burlington VT1 and Johnson State College, Dept of Biological Sciences, Johnson, Vermont 2
AbstractCurrent techniques for isolating intact RNA from various tissues are a challenge with small sample sizes. Most methods rely on a homogenization technique that employs the use of equipment and supplies that are not guaranteed RNase-free. This often results in low recoveries and poor RNA quality.
In this study, we describe and test the utility for RNase-free homogenization of small tissue sizes using novel abrasive particles including diamond, cubic boron nitride, silicon carbide, boron carbide, AlO3, and Duralum. The abrasives are used in conjunction with disposable
RNase-free pestles, standard 1.5 ml microcentrifuge tubes, 1-5 mg of tissue, and 100µl of a RNA extraction reagent such as TrizolTM or guanidinium isothiocyanate. The homogenization process is a rapid technique using a motorized attachment on the pestle followed by filtration through a QiashredderTM column and extracted using previously described methods specific to the RNA extraction reagent.
Preliminary results indicate that this modified technique is effective at recovering intact RNA from small amounts of tissue. Although all abrasives performed well, several characteristics were noted during the evaluation. Diamond and cubic boron nitride appear to be the easiest to use based on tissue visualization during homogenization and filterability. AlO3 and Duralum
are two abrasives that possess a razor sharp morphology and are more suitable for tissues that require aggressive homogenization. However, tissue visualization is difficult due to their white colored appearance. Silicon carbide and boron carbide performed equally as well, allowed good tissue visibility, but formed black residue during the homogenization process because of fragmentation of the abrasive. Recovery data in pure RNA dosing and soft tissue experiments approaches 70 to 80% indicating sample retention in the abrasive matrix or QiashredderTM column. Conversely, recoveries from hard tissues exceeded traditional homogenization techniques by two fold or greater without the sacrifice of RNA integrity.
Acknowledgements
The VCC Core Labs gratefully acknowledges the donation of cubic boron nitride and diamond by Warren-Amplex Superabrasives Corp. in Olyphant, PA., DSW and boron carbide by Washington Mills ElectroMinerals Corp in Niagara Falls NY, and AlO3 and silicon carbide by United Unibrite Corp. in Glen Cove NY. This work was supported in part by NCI grant P30ca22435 and Lake Champlain Cancer Research Organization (LCCRO).
Materials
The purpose of this study was to invent a efficient method that would recover RNA from tissue using all RNase-free disposable materials and microscopic abrasives. The need for an abrasive is justified because RNase-free homogenization is often a challenge for rigid tissues. In this study we evaluated six abrasives on four tissues types using two modified protocols using Trizol reagent and the Qiagen RNeasy kit.
Qiagen RNeasy kitTrizol RNA extraction reagentMoBio 1.8 ml centrifuge tubes1.5 ml pestle with motor unitQiashredder columnsPellet Paint Chloroform
EthanolIsopropanolP2-P1000 pipetsRNase ZapAnalytical balanceBiosafety hoodMicrocentrifuge
Abrasives Used in this Study
Diamond
Cubic Boron Nitride
Silicon Carbide
Aluminium Oxide
Duralum
Boron Carbide
Motorized Pestle
Goals
Methods
Abrasives were prepared RNase-free by soaking in ETOH for 30 minutes, RNase Zap for 10 minutes, washing in sterile water, autoclaving for 1 hour, and baking for 1 hour at 250F.
TissuesBrain: Fresh RatMelanoma: Snap Frozen HumanIntestine: Frozen 1mM Sodium Citrate and RNase inhibitor.
Modified Qiagen RNeasy Homogenization Procedure
Add a fresh 100uL aliquot of RLT buffer to the original tube and vortex. Using the same pipet, transfer to the Qiashredder tube.
Repeat with one additional aliquot of RLT buffer.
Spin the Qiashredder at full speed for 2 minutes. Quantitatively measure the pass-through using a micropipet and transfer to a new microcentrifuge tube.
Add 1 volume of 70% ethanol to the new tube containing the extract. Vortex for 3 seconds and centrifuge at full speed for 5 seconds. Pipet up and down several times and transfer to a Qiagen RNeasy mini silica column.
Centrifuge the column for 1 minute at full speed. Remove the pass-through from the capture tube and reapply it to the same column. Spin again.
DNase treat the column/sample according to Qiagen procedure. Centrifuge the column for 15 seconds. Discard pass-through and capture tube.
Place Qiagen RNeasy mini silica basket into a new capture tube and apply 0.5 mL RW1 buffer and centrifuge at full speed for 15 seconds. Repeat this procedure with two aliquots of RPE buffer replacing capture tube each time.
Place the Qiagen RNeasy mini silica basket into a new capture tube and centrifuge at full speed for 2 minutes to “dry” the silica column.
Place the Qiagen RNeasy mini silica basket in a 1.5 mL centrifuge tube and apply 30 uL of DEPC water directly onto the silica membrane.
Centrifuge the column under the same conditions as “drying” step. The pass through is the RNA sample. Remove the pass through and apply it again to the center of the column. Centrifuge again. This is the recovered RNA.
Modified TrizolTM Homogenization Procedure
Using a different pipet, add a fresh 100uL aliquot of TrizolTM to the original tube and vortex. Using the previous pipet, transfer to the Qiashredder tube.
Repeat with one additional aliquot of TrizolTM. This is to insure that all homogenate is transferred to the shredder column.
Centrifuge the shredder column for 2 minutes at full speed and transfer the pass-through to a AxygenTM ultra-clear 1.7 mL microcentrifuge tube. The resulting volume should be 280 uL.
Add 60 ul of 100% chloroform and vortex. Allow sample to sit for 2 minutes followed by centrifuging at full speed 13,000 G for 15 minutes.
Carefully remove supernatant and transfer to another AxygenTM ultra-clear 1.7 mL tube. Add 180ul of ice-cold 100% isopropanol and 1 uL of pellet paint. Centrifuge at 4C for 30 minutes.
The precipitated RNA will appear as a tiny pink spot at the bottom of the centrifuge tube.
Remove supernatant and add 1 mL of 80% ethanol. Invert tube 10-15 times and spin again at 4C for 15 minutes. Remove supernatant. Repeat this washing step one additional time.
After removing the last aliquot of ethanol, perform a quick spin on the tube and use a P20 to remove as much of the ethanol as possible. Allow the tube to stand open for air drying for 30 minutes in hood. DO NOT allow tube to be in direct flow of air as this may cause the pellet to become aerosolized.
Aseptically combine 1-10 mg of tissue, 20 mg of abrasive, and 100 uL of either RLT buffer containing b-mercaptoethanol for the modified Qiagen RNeasy procedure or 100 uL of TrizolTM for the Modified TrizolTM Homogenization and in a 1.8 mL MoBio tube.
Using the motorized attachment for the 1.5 mL tube pestle, homogenized the sample for 1-5 minutes or until tissue is fully dissociated. Vortex and centrifuge briefly.
Using a P1000 micropipet, pipet up and down to loosen the homogenate and than transfer the homogenate including the abrasive to a Qiashredder tube. Keep micropipet and tip for next transfer.
Results
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1000
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SiC
-1
SiC
-2
BC
-1
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-2
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Dia
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-2
DS
W-1
DS
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Qiagen
Trizol Homogenization was performed in the presence of each abrasive using pure yeast RNA to determine if non-specific binding of RNA occurs. These data indicate that minimal RNA is lost (bound) Data not shown for silicon carbide.
Binding Experiment
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75.9
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C-BoronNitride
DSW Al03 Diamond BoronCarbide
% R
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Homogenization was performed on melanoma tissue ranging in size 4.2-5.7 mg. Abrasives were used in the range of 5-20 mg. AlO3 Performed best
Melanoma Tissue: Moderately Tough TissuePure RNA Recovery Experiment
Extraction of RNA from Rat Brain (Soft Tissue)
Fresh rat brain tissue was homogenized using both techniques in replicate(s). Results of these soft tissue extractions indicate that the pestle sample had the highest recovery which is expected with soft tissue. Tough tissue however often requires a more aggressive approach.
Rat Intestinal Lumen: A Tough Tissue!
Flu
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19 24 29 34 39 44 49 54 59 64 69
Example of Rat Brain RNA Quality From Cubic Boron Nitride
Modified TrizolTM Technique
Downstream Analysis of Extracted RNA Using RT-QPCR
Relative standard curve generated from cDNA that was reverse transcribed from the pestle lumen RNA sample. The resulting slope of >0.99 indicates exceptional PCR efficiency for 18S.
Amplification plot of duplicates samples from the six rat lumen abrasive RNAs. These data indicate excellent reproducibility.
Conclusion
All extracted RNA was analyzed using real time Q-PCR to determine if any inhibitory substances were present from the abrasive compounds. 2 ug of total RNA was reverse transcribed and amplified using rat 18s primers and probe.
Homogenization was performed on 4-6 mg of rat intestine. Abrasives were used in the range of 5-20 mg. Homogenization was performed using both motorized and non-motorized rotation until visual dissociation for each tissue was observed. Dissociation was not observed for the pestle sample.
Modified Qiagen Homogenization
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Modified Trizol Homogenization
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Abrasive
Qiagen ng RNA / mg Tissue
Trizol ng RNA / mg Tissue
Modified Qiagen Technique
These data indicate that the use of abrasives on soft tissue such as brain is unnecessary as RNA recovery is adequate using just the pestle technique. However, increased recovery of RNA from tough tissues has been observed
Pure RNA recovery experiments indicate that non-specific binding of RNA to the abrasive is insignificant.
All Abrasives performed adequately but silicon carbide and AlO3 seemed to facilitate better recoveries of RNA using the modified Qiagen and Trizol technique, respectively.
AlO3 and DSW have superior homogenization ability due to their sharp morphology. Additional studies on other tough tissues should be investigated.
Secondary analysis including cDNA synthesis, RT-QPCR, spectrophotometry, and RNA assesment using the Agilent bioanalyzer indicate that the resulting RNA is of outstanding quality with no inhibitory substances.
Laboratory handling of cubic boron nitride was most acceptable. It was easy to visualize the tissue and appeared to homogenize the sample effectively. Silicon carbide and boron carbide contributed a black residue to the homogenate and visualization was difficult. The diamonds used in this study seemed too small and tended to “float” during homogenization. Larger diamonds(150-200um) should be tested in future work. Although AlO3 and DSW homogenize tissue very well, pipeting and visualization was difficult due to theconsistency and white color.
Future consideration should be give to using two abrasives in combination such as silicon carbide with AlO3.
This method is a rapid and effective technique to extract RNA from small tissue sizes (1-15mg). It employs the use of all disposable RNase-free materials and is a small format that can be carried out in a biosafety cabinet.
This technique may be suitable for other biomolecules such as DNA and protein.
Other studies being carried out in the Lounsbury lab using this technique with silicon carbide have successfully isolated RNA from 1mg samples of rat brain blood vessels and used it in microarray studies with excellent results. They are currently using it for isolating proteins with reasonable success.
Abrasive Designation Supplier Size
Cubic Boron Nitride CBN Warren Diamond 140-170 m
Diamond Dia Warren Diamond 74m
Aluminum Oxide(AlO3) AlO3 United Unibrite 130m
Boron Carbide BC Washington Mills 141m
Duralum Special White DSW Washington Mills 141m
Silicon Carbide SiC United Unibrite 200 um
Rat Intestinal Lumen: A Tough Tissue!
Homogenization was performed on 4-6 mg of rat intestine. Abrasives were used in the range of 5-20 mg. Homogenization was performed using both motorized and non-motorized rotation until visual dissociation for each tissue was observed. Dissociation was not observed for the pestle sample.
Modified Qiagen Homogenization
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Modified Trizol Homogenization
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