Thermo Fisher Scientific TOTALLY RNA 1910, COIL Owner's Manual
Thermo Fisher Scientific TOTALLY RNA 1910, COIL is a kit designed for isolating total RNA from various samples, including animal and plant tissues, cultured cells, bacteria and yeast. This kit allows researchers to obtain high-quality RNA for further downstream applications such as gene expression analysis or microarrays.
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USER GUIDE TōTALLY RNA Kit Publication Part Number 1910M Rev. C Revision Date November 2011 For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject to change without notice. LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. NOTICE TO PURCHASER: LIMITED USE LABEL LICENSE: Research Use Only The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser's activities for a fee or other form of consideration. For information on obtaining additional rights, please contact [email protected] or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008. TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. LabChip is a trademark of Caliper Life Sciences Inc. Agilent and Bioanalzyer are trademarks of Agilent Technologies, Inc. © 2011 Life Technologies Corporation. All rights reserved. Part Number 1910M Rev. C November 2011 Contents About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 User attention words . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 ■ TōTALLY RNA Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Materials Provided with the Kit and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Sample Disruption and Homogenization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Equipment and Solution Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Animal or Plant Tissue Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Cultured Eukaryotic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 RNA Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Preparation of Lysate for RNA Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Phenol:Chloroform:IAA Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Acid Phenol Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Isopropanol Precipitation of the RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Long Term Storage of RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Assessing RNA Yield and Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Assessing RNA Yield and Purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Denaturing Agarose Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Low RNA Yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Problems During Denaturing Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 RNA Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Contaminants in RNA; RNA Inhibits Enzymatic Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Optional RNA Clean-up Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 (optional) LiCl Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Removal of Trace DNA Contamination with DNase I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 ■ APPENDIX A Materials Not Provided With the Kit . . . . . . . . . . . . . . . . . 25 Required Materials Not Provided With the Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Related Products Available from Life Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 TōTALLY RNA Kit User Guide 3 Contents ■ APPENDIX B Recipes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 10X TBE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 RNase-free water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Phosphate Buffered Saline (PBS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 ■ APPENDIX C Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 General Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Obtaining SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4 TōTALLY RNA Kit User Guide About This Guide WARNING! ABBREVIATED SAFETY ALERTS. Hazard symbols and hazard types specified in procedures may be abbreviated in this document. For the complete safety information, see the “Safety” appendix in this document. IMPORTANT! Before using this product, read and understand the information the “Safety” appendix in this document. Purpose The TōTALLY RNA Kit User Guide provides detailed procedures, reference information and troubleshooting for the kit. User attention words Five user attention words may appear in this document. Each word implies a particular level of observation or action as described below: Note: Provides information that may be of interest or help but is not critical to the use of the product. IMPORTANT! Provides information that is necessary for proper instrument operation or accurate chemistry kit use. CAUTION! Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. WARNING! Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury. DANGER! Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury. TōTALLY RNA Kit User Guide 5 About This Guide User attention words Except for IMPORTANTs, the safety alert words in user documentation appear with an open triangle figure that contains a hazard symbol. These hazard symbols are identical to the hazard symbols that are affixed to the instrument. See the “Safety” appendix for descriptions of the symbols. 6 TōTALLY RNA Kit User Guide TōTALLY RNA Kit Introduction Background Classical RNA isolation procedures are based on disruption of cells in guanidinium thiocyanate/cationic detergent solutions, followed by organic extraction and alcohol precipitation of the RNA. Guanidinium solutions are powerful reagents for the inactivation of endogenous RNases, but the tendency for DNA, heme, and other pigments and impurities to copurify with the RNA can be a problem. The Ambion ToTALLY RNA™ Total RNA Isolation Kit is designed to remove undesirable impurities from cellular RNA without sacrificing speed, convenience, or RNA yield. Samples are lysed in a guanidinium based lysis solution and are then extracted sequentially with Phenol:Chloroform:IAA and Acid-Phenol:Chloroform. The RNA is then precipitated with isopropanol, and reagents are provided for an optional lithium chloride (LiCl) precipitation as well. The kit includes all the reagents (except isopropanol) required for isolation of total RNA from up to 10 g of tissue or about 109 cultured cells, and also includes ancillary reagents for analysis and storage of the RNA. Note: The isopropanol is not provided due to environmental concerns associated with the packaging that would be required for co-shipment of alcohol and phenol solutions. Materials Provided with the Kit and Storage Amount Component Storage 1 mL Formaldehyde Load Dye –20°C 100 mL Phenol:Chloroform:IAA 4°C 100 mL Acid-Phenol:Chloroform 4°C 15 mL Sodium Acetate Solution 4°C 3.0 M sodium acetate pH 4.5 50 mL Lithium Chloride Precipitation Solution 4°C 7.5 M lithium chloride/50 mM EDTA 5 mL 5 M Potassium Acetate 4°C 100 mL Denaturation Solution room temp 50 mL Elution Solution (0.1 mM EDTA) any temp† † Store Elution Solution at –20°C, 4°C, or room temp. TōTALLY RNA Kit User Guide 7 TōTALLY RNA Kit Sample Disruption and Homogenization CAUTION! Phenol is a highly corrosive chemical capable of causing severe burns. Wear protective clothing, gloves, and safety glasses at all times when handling the Phenol:Chloroform:IAA and the AcidPhenol:Chloroform. We recommend using a chemical safety hood for phenol extractions. See the Material Data Safety Sheet. Sample Disruption and Homogenization The first and most important step in any RNA isolation procedure is the rapid disruption and homogenization of the sample. Sample disruption must be rapid enough to expose the intracellular contents to the Denaturation Solution before endogenous RNases have a chance to degrade the RNA. Once homogenized, lysates can be processed immediately or stored frozen at –80°C for several months. Equipment and Solution Preparation Make sure that the Denaturation Solution is completely dissolved It is common for a precipitate to form in the Denaturation Solution. Before using it, check the bottle carefully to see if a precipitate has formed during storage, and if so, heat the solution to 37°C, swirling the bottle occasionally, until the precipitate goes back into solution. Lab bench and pipettors Before working with RNA, clean the lab bench, and pipettors with an RNase decontamination solution such as Ambion RNaseZap® RNase Decontamination Solution. Gloves and RNase-free technique Wear laboratory gloves at all times during this procedure and change them frequently. They will protect you from the reagents, and they will protect the RNA from nucleases that are present on skin. Use RNase-free pipette tips to handle the kit reagents. Washing/sterilization of equipment The equipment used for tissue disruption/homogenization should be washed well with detergent and rinsed thoroughly. Baking is unnecessary, because the Denaturation Solution will inactivate any low level RNase contamination. IMPORTANT! If samples will be ground in a mortar and pestle, pre-chill the equipment in dry ice or liquid nitrogen. Be sure to use RNase-free labware for all manipulations downstream of the AcidPhenol:Chloroform extraction, because once the RNA is precipitated out of the Denaturation Solution, it will be highly susceptible to degradation by RNase. 8 TōTALLY RNA Kit User Guide TōTALLY RNA Kit Sample Disruption and Homogenization Animal or Plant Tissue Samples For a good yield of intact RNA, it is very important to harvest tissue quickly and to limit the time between obtaining tissue samples and inactivating RNases. 1. Harvest tissue Harvest tissue and remove as much extraneous material as possible, for example remove adipose tissue from heart, and remove gall bladder from liver. The tissue can be perfused with cold PBS if desired to eliminate some of the red blood cells. 2. Cut larger tissue into small pieces If necessary, quickly cut the tissue into pieces small enough for either storage or disruption. Weigh the tissue sample (this can be done later for samples that will be stored in RNAlater® Solution). 3. Inactive RNases by one of the following methods: • Drop the sample into 5–10 volumes of RNAlater Solution—tissue must be cut to ≤0.5 cm in at least one dimension. (See the RNAlater Solution Protocol for detailed instructions.) • Disrupt the sample in Denaturation Solution (see instructions in step 5. on page 9). This option is only appropriate for fresh tissue samples that are soft to medium consistency. • Freeze the sample in liquid nitrogen—tissue pieces must be small enough to freeze in a few seconds. When the liquid nitrogen stops churning it indicates that the tissue is completely frozen. Once frozen, remove the tissue from the liquid nitrogen and store it in an airtight container at –80°C. Very hard or fibrous tissues (e.g., bone and heart), and tissues with a high RNase content must typically be frozen in liquid nitrogen and ground to a powder for maximum RNA yield. 4. Prepare tissue disruption equipment/supplies The method used to disrupt tissue samples depends on the nature of the tissue, the storage method, and the size of the sample; the table below shows guidelines for tissue disruption methods. Tissue consistency Sample storage method Suggested disruption method All frozen samples Frozen Freeze and grind in liquid N2 Very hard Any storage method Freeze and grind in liquid N2 or use a more rigorous method like disruption in a bead mill or a freezer mill Hard or RNase-rich Freshly dissected or stored in RNAlater Solution Freeze and grind in liquid N2 Soft to medium Freshly dissected or stored in RNAlater Solution Electric or manual homogenizer Soft, small pieces (<0.5 cm3) Frozen Electric or manual homogenizer 5. Thoroughly homogenize sample in the greater of 200 µL or 10 µL/mg of tissue Denaturation Solution TōTALLY RNA Kit User Guide 9 TōTALLY RNA Kit Sample Disruption and Homogenization Thoroughly homogenize sample in the greater of 200 µL or 10 µL/mg of tissue Denaturation Solution following the instructions below for samples stored in RNAlater® Solution, fresh, or frozen samples. a. Preparing samples stored in Ambion RNAlater® Solution or RNAlater-ICE Solutions Samples in RNAlater Solution can usually be homogenized by following the instructions for fresh tissue in the next step. Extremely tough/fibrous tissues in RNAlater Solution may need to be frozen and pulverized according to the instructions for frozen tissue (below) in order to achieve good cell disruption. If the samples were immersed in RNAlater Solution and then frozen at – 80°C, simply thaw samples at room temperature before starting. Blot excess RNAlater Solution from samples, and weigh them before following the instructions for fresh tissue below. b. Fresh animal or plant tissue sample preparation (soft to medium consistency tissues) i. If the sample weight is unknown, weigh the sample. ii. Aliquot the greater of 200 µL or 10 µL/mg of tissue Denaturation Solution into the homogenization vessel. For example, if your sample weighs 500 mg, use 5 mL Denaturation Solution. For very small samples use at least 200 µL of Denaturation Solution; this will be >10 volumes. iii. Mince large samples (≥2 cm2) rapidly in cold PBS, then remove the PBS before proceeding to the next step (PBS recipe in section “Recipes” on page 27). iv. Drop samples into the Denaturation Solution, and process to homogeneity. If available, use a motorized rotor-stator homogenizer (e.g., Polytron). Some tissues may need to be frozen in liquid nitrogen and powdered in a mortar and pestle before homogenization to obtain maximum RNA yield and quality. c. Frozen, hard- consistency, or RNase-rich tissue sample preparation After removing the tissue from the freezer, it is important to process it immediately without allowing any thawing. This is necessary because as cells thaw, ice crystals rupture cellular compartments, releasing RNase. By processing samples before they thaw, RNases can be inactivated by the Denaturation Solution before they are released from their cellular compartments. i. If the sample weight is unknown, weigh the sample. ii. Aliquot the greater of 200 µL or 10 µL/mg of tissue Denaturation Solution into a a wide-mouth container. (After grinding the tissue in liquid nitrogen, the frozen powder will be transferred to this container—we find that plastic weigh boats work well.) For example, if your sample weighs 500 mg, use 5 mL Denaturation Solution. For very small samples use at least 200 µL of Denaturation Solution; this will be >10 volumes. 10 TōTALLY RNA Kit User Guide TōTALLY RNA Kit Sample Disruption and Homogenization Note: Using an electronic rotor-stator homogenizer, small pieces of relatively soft frozen tissues (i.e. <0.5 cm3) can often be added directly to the Denaturation Solution without first grinding it in a mortar and pestle. iii. Grind frozen tissue to a powder with liquid nitrogen in a pre-chilled mortar and pestle. Some researchers grind frozen tissue in a coffee grinder with dry ice. Also, sample fragments larger than ~100 mg can be shattered with a hammer. iv. Using a pre-chilled metal spatula, scrape the powdered tissue into the premeasured Denaturation Solution, then mix rapidly. v. Transfer the slush to a vessel for homogenization and process the mixture to homogeneity. If available, use a motorized rotor-stator homogenizer. Once homogenized, lysates can be processed immediately or stored frozen at –80°C for several months. Cultured Eukaryotic Cells 1. Collect the cells and remove the culture medium Suspension cells: pellet the cells at low speed, and discard the culture medium. Adherent cells: Do one of the following: • Aspirate and discard the culture medium from the culture vessel. • Trypsinize cells to detach them from the growing surface (following the method employed in your lab for the cell type). 2. Add 10 mL Denaturation Solution to 5 x 107 to 5 x 108 cells and lyse the cells a. Add 10 mL Denaturation Solution to 5 x 107 to 5 x 108 cells and vortex or pipette the lysate up and down several times to completely lyse the cells and to obtain a homogenous lysate. Cells will lyse immediately upon exposure to the solution. b. For adherent cells, collect the lysate with a rubber spatula. 3. Frozen cell pellets IMPORTANT! Instead of using frozen cell pellets, lyse fresh cells as described above if possible, and freeze the lysate. Grind frozen cell pellets in liquid nitrogen in a mortar and pestle as described for frozen tissue. This is necessary because as cells thaw, ice crystals rupture both interior and exterior cellular compartments, releasing RNase. Once homogenized, lysates can be processed immediately or stored frozen at – 80°C for several months. Bacteria 1. Harvest 108–109 cells by centrifugation. (For many strains, this corresponds to ~3 mL of an overnight culture.) Remove as much of the culture medium as possible. 2. (optional) Enzyme pre-treatment: • Resuspend cells in 100 µL TE (10 mM Tris-HCl, 1 mM EDTA) with 1 mg/mL lysozyme or lysostaphin. • Incubate 5 min at room temperature for lysozyme, or 15 min at 37°C for lysostaphin to degrade cell envelopes. TōTALLY RNA Kit User Guide 11 TōTALLY RNA Kit RNA Isolation 3. Add 300 µL Denaturation Solution for up to 3 mL of culture starting volume. (The exact volume of Denaturation Solution is usually not critical and can be adjusted according to user experience.) 4. Vortex vigorously to thoroughly lyse cells. Yeast 1. Start with a logarithmically growing culture (A600~1–2); pellet cells and rinse once with nuclease-free water. Cells that are grown past the logarithmic phase will usually be more difficult to lyse. 2. Add 300 µL Denaturation Solution per up to 3 mL of culture starting volume. 3. Remove a 2 µL aliquot as a baseline for an absorbance reading at 260 nm. Yeast are difficult to lyse, and an easy way to monitor lysis is by looking for an increase in the A260 resulting from the release of nucleic acids. Note: The glass beads will not interfere with RNA isolation, they will remain with the lower, organic phase in the next step of the procedure. 4. Add ~150–200 µL of 0.4–0.5 mm glass beads, and vortex vigorously for 1 min intervals, checking the A260 of 2 µL samples after each interval. (Dilute the samples of lysate in 1 mL water to read their absorbance). Lysis will normally be complete after 2–4 rounds of vortexing (1 min each). The A260 reading should increase sharply as lysis begins and then level off indicating that lysis is complete. RNA Isolation Preparation of Lysate for RNA Isolation 1. Reduce the viscosity of the lysate if necessary Lysate should be somewhat viscous, but if the solution is very viscous, or contains gelatinous material (which is probably genomic DNA), then it should be sonicated, homogenized in a rotor-stator homogenizer, or passed through a 25 gauge syringe needle several times until the viscosity is reduced. It may be necessary to reduce viscosity by adding more Denaturation Solution and homogenizing with an electronic tissue disrupter. To continue with the procedure the lysate should be about as viscous as a typical enzyme solution in 50% glycerol. 2. (optional) Centrifuge 2–3 min at top speed in a microcentrifuge to remove debris This centrifugation removes any debris that may be present in the lysate. Most preparations will not have any insoluble material after thorough homogenization. Phenol:Chloroform :IAA Extraction 1. Measure the Starting Volume of the lysate Measure the volume of the lysate. This volume will be referred to as the Starting Volume. Transfer lysate to a vessel resistant to organic solvents with a capacity that is slightly more than twice the volume of the lysate. 2. Extract with 1 Starting Volume Phenol: Chloroform: IAA a. Add 1 Starting Volume of Phenol:Chloroform:IAA to the lysate. 12 TōTALLY RNA Kit User Guide TōTALLY RNA Kit RNA Isolation Be sure to use the organic phase of the Phenol:Chloroform:IAA which lies under the thin upper layer of aqueous buffer. CAUTION! Phenol is highly corrosive. Wear protective clothing, gloves and safety glasses when handling phenol. b. Shake or vortex vigorously for 1 minute. c. Store lysate + phenol on ice for the following times depending on the amount of solution, then centrifuge at 10,000–12,000 x g for the indicated time preferably at 4°C: Tube Size Incubation on ice Centrifugation at 10,000–12,000 x g 1.5–2 mL 5 min 5 min 10–15 mL 10 min 15 min larger containers 15 min 15 min 3. Transfer the aqueous phase to a new vessel a. Transfer the upper, aqueous phase to new vessel of the same size being careful to avoid the material at the aqueous/organic interface. b. Measure the volume of the aqueous phase. Do not be concerned if the volume of the lysate is slightly increased compared to the Starting Volume. c. (optional) To thoroughly recover the remainder of the aqueous phase from the organic phase and the interface, withdraw the remaining aqueous material from the top of the interphase into a pipet tip; it is okay if some of the interface material enters the pipet tip also. Let the phases separate in the pipet tip for several seconds, and allow as much of the viscous interface material as possible to drip back into the tube with the phenol before transferring the aqueous portion to a fresh tube. Centrifuge the recovered aqueous material for ~1 min to separate the phases. Then, using a clean pipet tip transfer the aqueous portion to the container with the rest of the prep. Acid Phenol Extraction 1. Add 1/10 volume Sodium Acetate Solution and mix well Add 1/10 Aqueous Phase volume of Sodium Acetate Solution to the phenol extracted lysate. Mix by shaking or inversion for about 10 sec. 2. Extract with 1 Starting Volume Acid-Phenol: Chloroform a. Add 1 Starting Volume of Acid-Phenol:Chloroform. Be sure to use the organic phase of the Acid-Phenol:Chloroform which lies under the aqueous buffer floating on top. Do not add more than 1 Starting Volume even if the volume of the lysate is greater than the starting volume. b. Shake or vortex vigorously for 1 min. c. Store lysate + phenol on ice and centrifuge as in step 2.c on page 10. 3. Transfer the aqueous phase to a new vessel TōTALLY RNA Kit User Guide 13 TōTALLY RNA Kit RNA Isolation IMPORTANT! Vessels used at this step must be RNase-free because the protective effect of the Denaturation Solution will be removed after the RNA is precipitated out in the next steps. a. Transfer the upper, aqueous phase to a new RNase-free vessel with a capacity of at least twice the volume of the lysate at this point in the procedure. Be careful to avoid material from the interface of the aqueous and organic phases. b. Measure the volume of the aqueous phase. Do not be concerned if the volume of the lysate is slightly different than the Starting Volume. c. (optional) Recover the remainder of the aqueous phase from the organic phase and the interface as described in 3c. on page 14. Isopropanol Precipitation of the RNA 1. Add an equal volume of isopropanol, mix well Add an equal volume of isopropanol to the RNA prep and mix well. Note: A fine white precipitate that forms immediately upon addition of the isopropanol may indicate the presence of undesirable material which can be removed before storing the prep at –20°C. (See section “Protein contamination” on page 22 for further details.) 2. Place at –20°C for ≥ 30 min to 1 hr Place the preparation at –20°C for at least 30 min for 1.5–2 mL tubes, or at least 1 hr for larger tubes. A white flocculent precipitate may form, indicating precipitation of the RNA. At any time after a visible precipitate forms, or after overnight storage at –20°C (even if no precipitate is visible), proceed to the next step. This is a stopping point in the procedure, and the preparation may be stored at this stage for a period of up to several days if desired. STOPPING POINT. 3. Recover the RNA by centrifugation Centrifuge the precipitation mixture at 10,000–12,000 x g for 15 min in microfuge tubes, or for 20 min if the mixture is in larger tubes. 4. Carefully remove and discard the supernatant Carefully remove the supernatant solution. Pellets may not adhere tightly to the walls of some tubes, so be careful if you decant to remove the supernatant. For 1.5–2 mL tubes, we suggest removing the supernatant by gentle aspiration with a very fine pipette tip or a drawn-out Pasteur pipette and bulb. Re-spin the tube briefly and remove any residual fluid by aspiration with a finetipped pipette. 5. (optional) Ethanol wash Residual salts can be removed by washing the pellet in 70% ethanol as follows: a. Add room temperature 70% ethanol to the RNA pellet; use ~300 µL for microfuge tubes or 2–3 mL for RNA pellets in larger tubes. b. Gently vortex or flick the tube with a finger for ~0.5–3 min. 14 TōTALLY RNA Kit User Guide TōTALLY RNA Kit Assessing RNA Yield and Quality c. Recover the RNA by centrifugation for 5–10 min at low speed (~3,000 x g: 7,500 rpm in a microcentrifuge, or 5,500 rpm in an SS34 rotor) at room temp or 4°C. d. Carefully and thoroughly remove ethanol supernatant; pellets may not adhere tightly to the tubes. Re-spin tubes briefly and remove residual ethanol by aspiration with a fine-tipped pipette. 6. Resuspend RNA in DEPC Water/ EDTA Resuspend pellets in the desired volume of DEPC Water/ EDTA. Typically 100 µL–1 mL per 100 mg of starting material (or ~107 cells) will yield a reasonable RNA concentration. If necessary, heat the solution to 55–70°C, and vortex occasionally to dissolve the RNA. Typically RNA from small scale RNA preparations will go into solution after a few minutes, but RNA pellets from large scale preps may require more time to dissolve. Long Term Storage of RNA It is unwise to subject RNA to repeated cycles of freeze-thawing (RNA Methodologies, a Laboratory Guide, 1992). To avoid repeated freeze-thawing, total RNA samples should be stored in small aliquots at –70°C or –80°C. A small amount of EDTA (0.1 mM) should be present during storage to chelate divalent cations, which can catalyze RNA breakage. If degradation problems are encountered upon prolonged storage, it may be desirable to store the RNA as an ethanol precipitate (i.e., add 2 volumes of ethanol to the prep in aqueous solution). The RNA can be recovered by centrifugation, after adjusting the salt concentration to 0.25 M with potassium acetate (use the 5 M Potassium Acetate provided with the kit). Alternatively, RNA can be precipitated with LiCl (see section “(optional) LiCl Precipitation” on page 23), or with potassium acetate and ethanol and resuspended in formamide for long term storage at –20°C (Chomczynski, 1992). Assessing RNA Yield and Quality Assessing RNA Yield and Purity RNA yield • Spectrophotometry The concentration of an RNA solution can be determined by measuring its absorbance at 260 nm (A260) using a spectrophotometer. With a traditional spectrophotometer, dilute an aliquot of the RNA 1:50–1:100 in TE (10 mM TrisHCl pH 8, 1 mM EDTA), and read the absorbance. (Be sure to zero the spectrophotometer with the TE used for sample dilution.) The buffer used for dilution need not be RNase-free, since slight degradation of the RNA will not significantly affect its absorbance. NanoDrop spectrophotometers are more convenient—no dilutions or cuvettes are needed, just measure 1.5 µL of the RNA sample directly. To determine the RNA concentration in µg/mL, multiply the A260 by the dilution factor and the extinction coefficient (1 A260 = 40 µg RNA/mL). A260 x dilution factor x 40 = µg RNA/mL Be aware that any contaminating DNA in the RNA prep will lead to an overestimation of yield, since all nucleic acids absorb at 260 nm. TōTALLY RNA Kit User Guide 15 TōTALLY RNA Kit Assessing RNA Yield and Quality • Fluorometry If a fluorometer or a fluorescence microplate reader is available, Molecular Probes’ RiboGreen® fluorescence-based assay for RNA quantitation is a convenient and sensitive way to measure RNA concentration. Follow the manufacturer’s instructions for using RiboGreen. RNA quality • Microfluidic analysis Microfluidic systems such as the Agilent® 2100 Bioanalyzer™instrument with Caliper's RNA LabChip® Kits provide better quantitative data than conventional gel analysis for characterizing RNA. When used with Ambion RNA 6000 Ladder (Part no. AM7152), this system can provide a fast and accurate size distribution profile of RNA samples. Follow the manufacturer’s instructions for performing the assay. The 28S to 18S rRNA ratio is often used as an indicator of RNA integrity. Total RNA isolated from fresh and frozen mammalian tissues using this kit usually has a 28S to 18S rRNA ratio of >1.2. Using a Bioanalyzer™instrument, the RIN (RNA Integrity Number) can be calculated to further evaluate RNA integrity. A metric developed by Agilent, the RIN analyzes information from both rRNA bands, as well as information contained outside the rRNA peaks (potential degradation products) to provide a fuller picture of RNA degradation states. Search for “RIN” at Agilent’s website for information: www.chem.agilent.com • Spectrophotometry An effective measure of RNA purity is the ratio of absorbance readings at 260 and 280 nm; it should fall in the range of 1.8 to 2.1. Even if an RNA prep has a ratio outside of this range, it may function well in common applications such as RTPCR, Northern blotting, and RNase protection assays. Denaturing Agarose Gel Electrophoresis The overall quality of an RNA preparation may be assessed by electrophoresis on a denaturing agarose gel; this will also give some information about RNA yield. A denaturing gel system is suggested because most RNA forms extensive secondary structure via intramolecular base pairing, and this prevents it from migrating strictly according to its size. Be sure to include a positive control RNA on the gel so that unusual results can be attributed to a problem with the gel or a problem with the RNA under analysis. RNA molecular weight markers, an RNA sample known to be intact, or both, can be used for this purpose. Ambion NorthernMax® reagents for Northern Blotting include everything needed for denaturing agarose gel electrophoresis. These products are optimized for ease of use, safety, and low background, and they include detailed instructions for use. An alternative to using the NorthernMax reagents is to use the procedure described below. This denaturing agarose gel method for RNA electrophoresis is modified from “Current Protocols in Molecular Biology”, Section 4.9 (Ausubel et al., eds.). It is more time-consuming than the NorthernMax method, but it gives similar results. 1. Prepare the gel a. Heat 1 g agarose in 72 mL water until dissolved, then cool to 60°C. 16 TōTALLY RNA Kit User Guide TōTALLY RNA Kit Assessing RNA Yield and Quality b. Add 10 mL 10X MOPS running buffer (see table below for preparation), and 18 mL 37% formaldehyde (12.3 M). CAUTION! Formaldehyde is toxic through skin contact and inhalation of vapors. Manipulations that involve formaldehyde should be done in a chemical fume hood Concentration Component 0.4 M MOPS, pH 7.0 0.1 M sodium acetate 0.01 M EDTA c. Pour the gel using a comb that will form wells large enough to accommodate at least 25 µL. d. Assemble the gel in the tank, and add enough 1X MOPS running buffer to cover the gel by a few millimeters. Then remove the comb. 2. Prepare the RNA sample a. To 1–3 µg RNA, add 0.5–3 volumes Formaldehyde Load Dye (included in kit). • To simply check the RNA on a denaturing gel, as little as 0.5 volumes Formaldehyde Load Dye can be used, but to completely denaturate the RNA, e.g. for Northern blots, use 3 volumes of Formaldehyde Load Dye. • Ethidium bromide can be added to the Formaldehyde Load Dye at a final concentration of 10 µg/mL. Some size markers may require significantly more than 10 µg/mL ethidium bromide for visualization. To accurately size your RNA, however, it is important to use the same amount of ethidium bromide in all the samples (including the size marker) because ethidium bromide concentration affects RNA migration in agarose gels. b. Heat denature samples at 65–70°C for 5–15 min. Denaturation for 5 min is typically sufficient for simply assessing RNA on a gel, but a 15 min denaturation is recommended when running RNA for a Northern blot. The longer incubation may be necessary to completely denature the RNA. 3. Electrophoresis Load the gel and electrophorese at 5–6 V/cm until the bromophenol blue (the faster-migrating dye) has migrated at least 2–3 cm into the gel, or as far as 2/3 the length of the gel. 4. Results Visualize the gel on a UV transilluminator. (If ethidium bromide was not added to the Formaldehyde Load Dye, the gel will have to be post-stained and destained.) The following figure shows a typical denaturing agarose gel containing RNA isolated with the ToTALLY RNA Kit. The 28S and 18S ribosomal RNA bands should be fairly sharp, intense bands (size is dependent on the organism from which the RNA was obtained). The intensity of the upper band should be about TōTALLY RNA Kit User Guide 17 TōTALLY RNA Kit Troubleshooting twice that of the lower band. Smaller, more diffuse bands representing low molecular weight RNAs (tRNA and 5S ribosomal RNA) may be also present. It is normal to see a diffuse smear of ethidium bromide staining material migrating between the 18S and 28S ribosomal bands, probably comprised of mRNA and other heterogeneous RNA species. DNA contamination of the RNA preparation (if present) will be evident as a high molecular weight smear or band migrating above the 28S ribosomal RNA band, or sometimes as ethidium bromide staining material that does not migrate out of the well. Degradation of the RNA will be reflected by smearing of ribosomal RNA bands. Figure 1 Total RNA isolated using the TōTALLY RNA Kit. Total RNA was isolated from the indicated sources, and approximately 2 µg was run on a 1% denaturing agarose gel stained with ethidium bromide. The smaller rRNA bands in the lanes with leaf samples are derived from organelles such as chloroplasts and other plastids. ) m µg m f t ste lea nius (4 liver h ou n s r pr cco cco i f s illerke use a M a br ean ba oba M ze t to b mo 9 6 5 4 3 2.5 2 1.5 1 0.5 Troubleshooting The most common problems in RNA isolation are low yields, recovery of degraded RNA, and copurification of undesirable contaminants. Each of these problems is discussed below. Low RNA Yield Sample disruption was suboptimal Inadequate sample disruption is a frequent source of problems in RNA isolation, especially for tough tissues such as muscle and kidney. Tissue disruption can be improved by snap freezing tissue in liquid nitrogen, then grinding it to a powder in a mortar and pestle under liquid nitrogen. The powder should then be quickly mixed with Denaturation Solution and homogenized with a rotor/stator homogenizer. For some tissues even grinding in liquid nitrogen is not sufficient to disrupt the tissue; these extremely tough tissues will require harsher disruption techniques for example in a bead mill or in a freezer mill. 18 TōTALLY RNA Kit User Guide TōTALLY RNA Kit Troubleshooting RNA is not abundant in the tissue from which it was isolated Expected yields of RNA vary widely between tissues. If you are accustomed to working with tissues such as liver or kidney where RNA is plentiful, you may have unrealistically high expectations of RNA yields from tissues such as muscle or brain. The RNA pellet was not completely solubilized Centrifuge the preparation briefly and look for particles of undissolved material, which may appear as small amber gelatinous flecks. Solubilization may be improved by further incubation of the preparation at 55–70°C, with intermittent vortexing. Do not allow the RNA pellet to dry out completely after removing the supernatant fluid subsequent to precipitation, or solubilization will become difficult, if not impossible. The sample was too dilute in the Denaturation Solution Poor RNA yield may be improved in some cases by keeping the preparation more concentrated. If a certain sample type routinely gives low RNA yield, try using only 5 volumes of Denaturation Solution per unit mass of tissue, and scale down the subsequent reagents accordingly. Significant errors in weighing small tissue samples (less than about 50 mg) are introduced if the samples are extremely wet, so it is a good idea to blot tissues dry just before weighing them. Loss of RNA at the interface during organic extractions Poor RNA yield can also be caused by excessive loss of material at the aqueous/phenol interface during the 2 organic extraction steps. A rapid “interface respin” procedure may be used to maximize recovery of the aqueous phase. This is described in step 3c. on page 14. Problems During Denaturing Gel Electrophoresis Problems with gel electrophoresis can cause RNA to appear to be degraded. Consider these suggestions if you suspect that electrophoresis was not optimal. IMPORTANT! Gel problems can be ruled out by running an aliquot of “positive control” RNA, i.e. an archived, intact RNA sample, on the same gel as the RNA preparation being evaluated for the first time. Ribosomal RNA (rRNA) is overloaded Running more than about 5 µg of RNA in a single lane may cause smiling and/or smearing of the rRNA bands. rRNA comprises about 80% of total RNA, so if 5 µg of total RNA is loaded in a gel lane, there will be about 1 µg and 3 µg of RNA in the 18S and 28S rRNA bands respectively. Samples are incompletely denatured To completely denature RNA, the sample should be diluted with at least 3 volumes of Formaldehyde Load Dye and then incubated in a 65°C water bath for at least 15 min. A 65°C cabinet type incubator works well, but somewhat longer incubation times may be required due to the lower heat transfer capacity of air. After incubation, transfer the TōTALLY RNA Kit User Guide 19 TōTALLY RNA Kit Troubleshooting samples immediately to an ice bath. Samples are stable on ice for at least 20 min, or long enough to add ethidium bromide to the samples if desired and load them on the gel. If an interruption occurs, the samples may be incubated at 65°C again without ill effects. Gel was run too fast Smearing may occur if gels are run at more than 5–6 volts/cm as measured between the electrodes. For example, if the distance between the electrode wires in the electrophoresis chamber measures 15 cm, the gel should be run at a constant 75 volts. Electrophoresis buffer was depleted For long runs (>3 hr) the buffer may be circulated to avoid the formation of pH gradients in the gel. This can be accomplished in various ways: manual circulation of the buffer every 15–30 min throughout the run (be sure samples have migrated into the gel first), continuous circulation of the buffer from one chamber to the other with a pump, or continuous circulation of the buffer using magnetic stir bars placed in both chambers. Gel or gel apparatus was contaminated with RNase RNase contamination of the gel running equipment, reagents, or supplies can cause RNA degradation while the gel is running. To decontaminate equipment, we recommend using Ambion RNAZap® RNase Decontamination Solution following the instructions provided. RNA Degradation Improper handling of tissue It is extremely important to inactive RNases as quickly as possible after sample collection to avoid RNA degradation. When samples are obtained from sacrificed animals or cadavers, it is also important to limit the time between death and sample collection for the best yield of high quality RNA. Frozen tissue thawed before immersion in Denaturation Solution It is essential that frozen tissue stays frozen until it is disrupted in Denaturation Solution. If the tissue is frozen in small pieces (i.e. <0.5 cm3), and it will be processed with an electronic rotor-stator homogenizer (Polytron type), it can often be dropped directly in a vessel containing Denaturation Solution and processed before it has a chance to thaw or to freeze the Denaturation Solution. This shortcut generally only works for relatively soft tissues. When powdering tissue in a mortar and pestle, it is important to pre-chill the mortar and pestle, and to keep adding small amounts of liquid nitrogen during grinding so that the tissue never thaws, even partially. Once the tissue is completely powdered, it should be mixed with the Denaturation Solution quickly before any of the powder can thaw. It may be convenient to scrape the frozen powder into a plastic weigh boat containing the volumes of Denaturation Solution. 20 TōTALLY RNA Kit User Guide TōTALLY RNA Kit Troubleshooting Samples that are very high in RNase may require procedure modification for isolation of intact RNA • Extra organic extractions can be included using the Phenol:Chloroform:IAA and Acid-Phenol:Chloroform provided with the kit, followed by a final extraction with an equal volume of chloroform:isoamyl alcohol(24:1 or 49:1). • Degradation may be minimized by ultracentrifugation of the RNA through a cesium chloride cushion, as an alternative to alcohol precipitation (Groppe and Morse, 1993). • Degradation can sometimes be minimized by reducing the quantity of starting material (tissue or cells) used for the isolation (Kamdar and Evens, 1992). In such cases it may help to also use a lower mass of tissue relative to volume of Denaturation Solution, for example use 0.5–0.75 gm tissue per 10 mL Denaturation Solution. • For problematic tissues, greater care may be needed to avoid contamination with interface material during the organic extraction steps (in other words if may be advisable to sacrifice yield to avoid degradation). Include a radiolabeled “tracer RNA” to troubleshoot degradation problems For troubleshooting RNA degradation problems, it may be useful to add a radiolabeled synthetic RNA transcript to the Denaturation Solution along with the sample. If intact 32P labeled RNA is recovered at the final step (as assessed by TCA precipitation or autoradiography), then the Denaturation Solution was adequate to inactivate RNases released from the disrupted cells, and the degradation probably occurred before starting the ToTALLY RNA procedure. Material that precipitates immediately after adding isopropanol can cause RNA degradation Occasionally a degradation problem that seems to be associated with a contaminant in lung tissue is observed, which results in formation of a precipitate immediately upon addition of the isopropanol at step 1. on page 14. The material differs from RNA in the following ways: • It forms immediately after the isopropanol is added. • It is a finer, less flocculent precipitant than RNA. • Upon vortexing and briefly spinning the tube, the material forms a sheet on the side of the tube, instead of a pellet at the bottom (like RNA). If this precipitate forms in a prep immediately after the isopropanol is added, remove it by spinning the tube briefly (~10 seconds). Transfer the prep (in isopropanol) to a fresh tube and continue with the procedure. Although some RNA may be lost along with the contaminant, a lower yield of intact RNA is preferable to a good yield of degraded RNA. Contaminants in RNA; RNA Inhibits Enzymatic Reactions DNA contamination • Digest the prep with DNase I (RNase-free) Instructions for digesting RNA with DNase I are shown in section “Removal of Trace DNA Contamination with DNase I” on page 23. Alternatively, the DNA-free™ kit can be used for this application. • Precipitate the RNA with LiCl TōTALLY RNA Kit User Guide 21 TōTALLY RNA Kit Troubleshooting IMPORTANT! LiCl precipitation is not efficient when the RNA concentration is below 0.2 µg/µL. LiCl precipitation will selectively precipitate RNAs longer than 200 nt, and will leave behind most of the DNA, carbohydrates, and protein. • High molecular weight band on a gel that is not DNA Some RNA preps show faint bands of ethidium bromide staining material migrating just above the 28S ribosomal RNA band, which is not comprised of DNA, as it is not removed by DNase treatment. The origin of this material is unclear, but it does not seem to adversely affect the quality of the RNA. Other contaminants Any of the following suggestions will further purify RNA after the ToTALLY RNA procedure. 1. Precipitate the RNA with LiCl LiCl precipitation will selectively precipitate RNAs ≥200 nt, and will leave behind DNA, carbohydrates, and protein. 2. Salts Wash RNA pellets with 75% ethanol to remove residual salts which could interfere with enzymatic reactions. 3. Protein contamination Protein contamination is suspected if the RNA exhibits an A260/280 ratio below ~1.7. To remove protein, extract the RNA with an equal volume of Phenol:Chloroform:IAA (See 2. on page 13). The resultant RNA may be extracted with an equal volume of chloroform:isoamyl alcohol (24:1 or 49:1) to remove any residual phenol. Despite these efforts, the A260:A280 ratio may sometimes remain below 1.7, especially for RNA isolated from tissues such as liver and kidney. For most applications, a low A260:A280 ratio will probably not affect the results. We have used total RNA with A260:A280 ratios ranging from 1.4 to 1.8 with good results in RNase protection assays, Northern blots, in vitro translation experiments, and RT-PCR. 4. Intractable contaminants For removal of intractable contaminants, it may be desirable to recover the RNA from the Denaturation Solution by centrifugation through a cesium chloride cushion (Chirgwin et al. 1979, Molecular Cloning p. 7.19–7.22, Groppe and Morse 1993). 22 TōTALLY RNA Kit User Guide TōTALLY RNA Kit Optional RNA Clean-up Procedures Optional RNA Clean-up Procedures (optional) LiCl Precipitation Lithium chloride precipitation removes carbohydrates and gross DNA contamination. IMPORTANT! The concentration of RNA should be at least 0.2 µg/µL to assure efficient precipitation. Also, LiCl precipitation will not quantitatively precipitate small RNAs such as tRNA and 5S ribosomal RNA. 1. Mix the RNA well with one-half volume LiCl Precipitation Solution. 2. Incubate at –20°C for at least 30 min. 3. Microcentrifuge 15 min at top speed. 4. Carefully remove and discard the supernatant. 5. Wash pellet with cold 70% ethanol, re-centrifuge, aspirate away the supernatant. 6. Air dry the pellet. Removal of Trace DNA Contamination with DNase I Trace DNA contamination can be enzymatically removed using DNase I. Note that under optimal conditions, PCR can detect a single DNA molecule, so even DNase treatment cannot always guarantee removal of genomic DNA below the level detectable by PCR. Ambion TURBO DNA-free™ Kit The Ambion TURBO DNA-free™ Kit (Part no. AM1907) includes Ambion TURBO DNase™, the first DNase I enzyme engineered for superior DNA digestion. The TURBO DNA-free kit also simplifies the process of inactivating the DNase without the risk incurred by heating the RNA, or the inconvenience of extracting with phenol/ chloroform. The TURBO DNA-free kit is the method of choice for eliminating contaminating DNA prior to RT-PCR; to use the TURBO DNA-free kit, follow the instructions provided with the product. Using your own DNase treatment reagents • DNase digestion buffer DNase treatment can be carried out in the buffer supplied or in a buffer recommended by the manufacturer of the enzyme; most restriction enzyme buffers can also be used. DNase I works well in a large range of salt and pH conditions. The enzyme requires magnesium (~5 mM) for optimal activity. • Amount of DNase – To remove small amounts of DNA, DNase I should be used at approximately 10 Units/mL RNA. – To treat severe DNA contamination, use 1 Unit DNase I per µg of contaminating DNA. • Incubation conditions Incubate DNase digestions at 37°C for 30 min. • Inactivate DNase I by one of the following methods: – Add EDTA to 5 mM, heat to 75°C for 10 min TōTALLY RNA Kit User Guide 23 TōTALLY RNA Kit Optional RNA Clean-up Procedures – Add EDTA to 20 mM – Extract with phenol/chloroform, and alcohol precipitate the RNA. 24 TōTALLY RNA Kit User Guide A Materials Not Provided With the Kit Required Materials Not Provided With the Kit Apparatus for tissue grinding and homogenization Liquid nitrogen, mortar and pestle, dry ice • For grinding tissue: not all samples will need to be ground in liquid nitrogen, see Table 1 on page 6. Tissue homogenizer • Electronic rotor-stator tissue homogenizers are recommended if available. • Manual tissue homogenizers or grinders: conical ground glass tissue grinders work well, dounce-type smooth glass homogenizers may also be acceptable. Reagents • Isopropanol: ACS grade or better • (Optional) Ethanol: ACS grade or better 95–100% ethanol • (Optional) Liquid nitrogen (for disruption of frozen and/or hard tissue) Centrifugation equipment • Centrifuge capable of 10,000 x g for large-scale preps, or a microcentrifuge capable of 12,000 x g for small-scale preps. • Centrifuge tubes compatible with phenol/chloroform (glass, polypropylene, or polyallomer), capable of withstanding centrifugal forces of 12,000 x g, with securely fitting closures. Materials for analysis of RNA • Spectrophotometer or Agilent Bioanalyzer™ instrument (http://www.chem.agilent.com/Scripts/PDS.asp?lPage=51) • Reagents and apparatus for preparation and electrophoresis of denaturing agarose gels. TōTALLY RNA Kit User Guide 25 A Appendix A Materials Not Provided With the Kit Related Products Available from Life Technologies RNAlater® Solution Part nos. AM7020, AM7021 RNaseZap® Solution Part nos. AM9780, AM9782, AM9784 Millennium™ Markers and BrightStar® Biotinylated Millennium™ Markers Part nos. AM7150 and AM7170 DNA-free™ Reagents Part no. AM1906 Electrophoresis Reagents 26 RNAlater Tissue Collection: RNA Stabilization Solution is an aqueous sample collection solution that stabilizes and protects cellular RNA in intact, unfrozen tissue and cell samples. RNAlater Solution eliminates the need to immediately process samples or to freeze samples in liquid nitrogen. Samples can be submerged in RNAlater Solution for storage at RT, 4°C, or –20°C without jeopardizing the quality or quantity of RNA that can be obtained. RNaseZap RNase Decontamination Solution is simply sprayed, poured, or wiped onto surfaces to instantly inactivate RNases. Rinsing twice with distilled water will eliminate all traces of RNase and RNaseZap Solution. Ambion’s Millennium™ Markers are designed to provide very accurate size determination of single-stranded RNA transcripts from 0.5 to 9 kb and can be used in any Northern protocol. They are a mixture of 10 easy-to-remember sizes of in vitro transcripts: 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6 and 9 kb. DNase treatment and removal reagents. This product contains Life Technologies’s ultra-high quality RNase-free DNase I and reaction buffer for degrading DNA. It is ideal for removing contaminating DNA from RNA preparations. A novel reagent for removing the DNase without the hassles or hazards of phenol extraction or alcohol precipitation is also included. Life Technologies offers gel loading solutions, agaroses, acrylamide solutions, powdered gel buffer mixes, nuclease-free water, and RNA and DNA molecular weight markers for electrophoresis. TōTALLY RNA Kit User Guide B 10X TBE Recipes TBE is generally used at 1X final concentration for preparing gels and/or for gel running buffer. IMPORTANT! Do not treat TBE with diethylpyrocarbonate (DEPC). Concentration Component for 1 L 0.9 M Tris base 109 g 0.9 M Boric Acid 55 g 20 mM 0.5 M EDTA solution 40 mL Dissolve with stirring in about 850 mL nuclease-free water. Adjust the final volume to 1 L. Alternatively, Life Technologies offers nuclease-free solutions of 10X TBE (Part nos. AM9863, AM9865) and ready-to-resuspend powdered 10X TBE packets (Part no. AM9864). Both are made from of ultrapure molecular biology grade reagents. RNase-free water a. Add DEPC to 0.05% to double-distilled, deionized water (i.e. add 0.5 mL per liter of water). b. Stir well, incubate several hours to overnight at 37°C or 42°C. c. Autoclave 2 L or smaller volumes for at least 45 min. After autoclaving, the scent of DEPC should be either undetectable or only very slightly detectable. Phosphate Buffered Saline (PBS) Concentration Component 137 mM NaCl 2.7 mM KCl 10 mM 1.8 mM for 1 L 8 g 0.2 g Na2HPO4 1.42 g KH2PO4 0.25 g Dissolve the components in about 800 ml dH2O. Adjust the pH to 7.4 with HCl. Adjust the volume to 1 L. Sterilize by autoclaving. Store at room temperature. TōTALLY RNA Kit User Guide 27 B 28 Appendix B Recipes TōTALLY RNA Kit User Guide C Safety General Safety WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document. · Before using an instrument or device, read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device. · Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use appropriate personal protective equipment (gloves, gowns, eye protection, etc). To obtain SDSs, see the “Documentation and Support” section in this document. Chemical safety WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below, and consult the relevant SDS for specific precautions and instructions: Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the “Documentation and Support” section in this document. · Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). · Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). · Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer's cleanup procedures as recommended in the SDS. · Handle chemical wastes in a fume hood. · Ensure use of primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.). · After emptying a waste container, seal it with the cap provided. · Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory. · Ensure that the waste is stored, transferred, transported, and disposed of TōTALLY RNA Kit User Guide 29 C Appendix C Safety Biological hazard safety · according to all local, state/provincial, and/or national regulations. IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal limitations may apply. Biological hazard safety WARNING! Potential Biohazard. Depending on the samples used on this instrument, the surface may be considered a biohazard. Use appropriate decontamination methods when working with biohazards. WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective equipment, which includes but is not limited to: protective eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices). Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially infectious materials. Read and follow the applicable guidelines and/or regulatory requirements in the following: In the U.S.: · U.S. Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at: www.cdc.gov/biosafety · Occupational Safety and Health Standards, Bloodborne Pathogens (29 CFR§1910.1030), found at: www.access.gpo.gov/nara/cfr/waisidx_01/ 29cfr1910a_01.html · Your company’s/institution’s Biosafety Program protocols for working with/ handling potentially infectious materials. · Additional information about biohazard guidelines is available at: www.cdc.gov In the EU: · Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization (WHO) Laboratory Biosafety Manual, third edition, found at: www.who.int/ csr/resources/publications/biosafety/WHO_CDS_CSR_LYO_2004_11/en/ 30 TōTALLY RNA Kit User Guide Bibliography Chirgwin, JM, Przybyla, AE, MacDonald, RJ, and Rutter, WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 24: 5294–5299. Chomczynski, P and Sacchi, N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159. Chomczynski, P, (1992) Solubilization in Formamide Protects RNA From Degradation. Nucleic Acids Res 20: 3791. Groppe, J, and Morse, D (1993) Isolation of full-length RNA templates for reverse transcription from tissues rich in RNase and proteoglycans. Anal Biochem 210:337–343. Kamdar, S and Evans, R (1992) Modifications of the Guanidine Hydrochloride Procedure for the Extraction of RNA: Isolation From a Variety of Tissues and Adherent/Non-adherent Cell Types. BioTechniques 12: 632–637. Nolan C, Editor (1989) Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Springs Harbor Laboratory Press. Puissant, C and Houdebine, LM (1990) An improvement of the single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. BioTechniques 8: 148–149. Robert E Farrell Jr, Editor (1993) RNA Methodologies, A Laboratory Guide for Isolation and Characterization Academic Press, Inc. Monstein, HJ, Nylander, G, and Chen, D (1995) RNA extraction from gastrointestinal tract and pancreas by a modified Chomczynski and Sacchi method. BioTechniques 19:339–344 TōTALLY RNA Kit User Guide 31 Bibliography 32 TōTALLY RNA Kit User Guide Documentation and Support Obtaining SDSs Safety Data Sheets (SDSs) are available from www.lifetechnologies.com/sds Note: For the SDSs of chemicals not distributed by Life Technologies, contact the chemical manufacturer. Obtaining support For the latest services and support information for all locations, go to: www.lifetechnologies.com At the website, you can: • Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities • Search through frequently asked questions (FAQs) • Submit a question directly to Technical Support • Search for user documents, SDSs, vector maps and sequences, application notes, formulations, handbooks, certificates of analysis, citations, and other product support documents • Obtain information about customer training • Download software updates and patches TōTALLY RNA Kit User Guide 33 Documentation and Support Obtaining support 34 TōTALLY RNA Kit User Guide Headquarters 5791 Van Allen Way | Carlsbad, CA 92008 USA | Phone +1 760 603 7200 | Toll Free in USA 800 955 6288 For support visit www.lifetechnologies.com/support www.lifetechnologies.com ">

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Key features
- RNA Isolation
- Tissue Disruption
- Sample Homogenization
- RNase Inactivation
- High RNA Yield
- Remove Impurities
Frequently asked questions
The TOTALLY RNA Kit can be used for isolating total RNA from animal and plant tissues, cultured cells, bacteria and yeast. Refer to the user manual for detailed instructions on sample preparation and lysis.
The kit is designed to isolate RNA from up to 10 g of tissue or about 109 cultured cells. The actual yield depends on the type and condition of the sample.
Most components should be stored at 4°C. Refer to the user manual for specific storage temperatures for each component.