Use of Novel Abrasives for the Extraction of RNA from Small Amounts of Difficult Tissue

1. Abrasive Designation Supplier Size Cubic Boron Nitride CBN Warren Diamond 140-170 m Diamond Dia Warren Diamond 74m Aluminum Oxide(AlO3) AlO3 United Unibrite 130m Boron Carbide BC Washington Mills 141m Duralum Special White DSW Washington Mills 141m Silicon Carbide SiC United Unibrite 200 um Use of Novel Abrasives for the Extraction of RNA from Small Amounts of Difficult Tissue Scott 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 Abstract Results Methods 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, 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. Melanoma Tissue: Moderately Tough Tissue Pure RNA Recovery Experiment 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. 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. 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 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. Rat Intestinal Lumen: A Tough Tissue! 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. 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. Goals Downstream Analysis of Extracted RNA Using RT-QPCR 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. 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. Extraction of RNA from Rat Brain (Soft Tissue) Materials Ethanol Isopropanol P2-P1000 pipets RNase Zap Analytical balance Biosafety hood Microcentrifuge 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 Tissues Brain: Fresh Rat Melanoma: Snap Frozen Human Intestine: Frozen 1mM Sodium Citrate and RNase inhibitor. 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 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. Motorized Pestle 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. Abrasives Used in this Study Cubic Boron Nitride Aluminium Oxide Example of Rat Brain RNA Quality From Cubic Boron Nitride Modified Qiagen Technique Modified TrizolTMTechnique Diamond Duralum 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). 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. Boron Carbide Silicon Carbide