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QIAGEN RNeasy mini kit 74104
Fourth EditionJune 2012RNeasy? Mini HandbookRNeasy Mini Kit For purification of total RNA from animal cells, animal tissues, bacteria, and yeast, and for RNA cleanup RNeasy Protect Mini Kit For immediate stabilization of RNA in harvested animal tissues and subsequent total RNA purification RNeasy Plant Mini Kit For purification of total RNA from plants and filamentous fungiSample & Assay TechnologiesQIAGEN Sample and Assay TechnologiesQIAGEN is the leading provider of innovative sample and assay technologies, enabling the isolation and detection of contents of any biological sample. Our advanced, high-quality products and services ensure success from sample to result. QIAGEN sets standards in:I I I IPurification of DNA, RNA, and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologiesOur mission is to enable you to achieve outstanding success and breakthroughs. For more information, visit www.qiagen.com.ContentsKit Contents Storage Intended Use Safety Information Quality Control Introduction Principle and procedure Description of protocols Equipment and Reagents to Be Supplied by User Important Notes Determining the amount of starting material Handling and storing starting material Disrupting and homogenizing starting material Eliminating genomic DNA contamination Protocols Purification of Total RNA from Animal Cells using Spin Technology Purification of Total RNA from Animal Cells using Vacuum/ Spin Technology Stabilization of RNA in Harvested Animal Tissues Purification of Total RNA from Animal Tissues Purification of Total RNA from Yeast Purification of Total RNA from Plant Cells and Tissues and Filamentous Fungi RNA Cleanup Troubleshooting Guide Appendix A: General Remarks on Handling RNA Appendix B: Storage, Quantification, and Determination of Quality of RNA Appendix C: Formaldehyde Agarose Gel Electrophoresis Appendix D: Optional On-Column DNase Digestion with the RNase-Free DNase Set Appendix E: DNase Digestion of RNA before RNA Cleanup Appendix F: Acetone Precipitation of Protein from Buffer RLT Lysates Appendix G: RT-PCR and Real-Time RT-PCR Ordering Information 23 29 34 37 43 50 54 56 61 63 65 67 69 70 71 72 4 5 5 6 6 7 8 10 13 16 16 18 18 21RNeasy Mini Handbook 06/20123Kit ContentsRNeasy Mini Kit Catalog no. Number of preps RNeasy Mini Spin Columns (pink) Collection Tubes (1.5 ml) Collection Tubes (2 ml)* Buffer RLT*? ? ?(50)
50 50 45 ml 45 ml 11 ml 10 ml 1(250) 0 250 250 220 ml 220 ml 65 ml 50 ml 1Buffer RW1Buffer RPE (concentrate) RNase-Free Water Quick-Start ProtocolRNeasy Protect Mini Kit Catalog no. Number of preps RNAlater RNA Stabilization Reagent*?(50)
ml 50 50 50 45 ml 45 ml 11 ml 10 ml 1(250) 0 ml 250 250 250 220 ml 220 ml 65 ml 50 ml 1RNeasy Mini Spin Columns (pink) Collection Tubes (1.5 ml) Collection Tubes (2 ml)* Buffer RLT*? Buffer RW1? ?Buffer RPE (concentrate) RNase-Free Water Quick-Start Protocol* Also available separately. See page 72 for ordering information.?Contains a guanidine salt. Not compatible with disinfectants containing bleach. See page 6 for safety information. Before using for the first time, add 4 volumes of ethanol (96C100%) as indicated on the bottle to obtain a working solution.?4RNeasy Mini Handbook 06/2012RNeasy Plant Mini Kit Catalog no. Number of preps RNeasy Mini Spin Columns (pink) QIAshredder Spin Columns (lilac) Collection Tubes (1.5 ml) Collection Tubes (2 ml)* Buffer RLT*? Buffer RLC? ?(20)
20 20 20 18 ml 18 ml 18 ml 5 ml 10 ml 1(50)
50 50 50 45 ml 45 ml 45 ml 11 ml 10 ml 1Buffer RW1Buffer RPE? (concentrate) RNase-Free Water Handbook* Also available separately. See page 72 for ordering information.?Contains a guanidine salt. Not compatible with disinfectants containing bleach. See page 6 for safety information. Before using for the first time, add 4 volumes of ethanol (96C100%) as indicated on the bottle to obtain a working solution.?StorageThe RNeasy Mini Kit, RNeasy Protect Mini Kit (including RNAlater RNA Stabilization Reagent), and RNeasy Plant Mini Kit should be stored dry at room temperature (15C25°C) and are stable for at least 9 months under these conditions. Storage of RNAlater Reagent at lower temperatures may cause precipitation. Before use, redissolve the precipitate by heating to 37°C with agitation.Intended UseThe RNeasy Mini Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease. All due care and attention should be exercised in the handling of the products. We recommend all users of QIAGEN? products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments, or to other applicable guidelines.RNeasy Mini Handbook 06/20125Safety InformationWhen working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate safety data sheets (SDSs). These are available online in convenient and compact PDF format at www.qiagen.com/safety where you can find, view, and print the SDS for each QIAGEN kit and kit component.CAUTION: DO NOT add bleach or acidic solutions directly to the sample preparation waste.Buffer RLT contains guanidine thiocyanate, Buffer RLC contains guanidine hydrochloride, and Buffer RW1 contains a small amount of guanidine thiocyanate. Guanidine salts can form highly reactive compounds when combined with bleach. If liquid containing these buffers is split, clean with suitable laboratory detergent and water. If the spilt liquid contains potentially infectious agents, clean the affected area first with laboratory detergent and water, and then with 1% (v/v) sodium hypochlorite. 24-hour emergency information Emergency medical information in English, French, and German can be obtained 24 hours a day from: Poison Information Center Mainz, Germany Tel: +49-Quality ControlIn accordance with QIAGEN’s ISO-certified Quality Management System, each lot of the RNeasy Mini Kit, RNeasy Protect Mini Kit, and RNeasy Plant Mini Kit is tested against predetermined specifications to ensure consistent product quality.6RNeasy Mini Handbook 06/2012IntroductionThe RNeasy Mini Handbook provides protocols for use with the following kits: RNeasy Mini Kit ― for purification of total RNA from animal cells, animal tissues, and yeast, and for cleanup of RNA from crude RNA preps and enzymatic reactions (e.g., DNase digestion, proteinase digestion, RNA ligation, and labeling reaction) RNeasy Protect Mini Kit ― for immediate stabilization of RNA in harvested animal tissues and subsequent purification of total RNA RNeasy Plant Mini Kit ― for purification of total RNA from plant cells and tissues and filamentous fungi The RNeasy Mini Kit can also be used to purify total RNA from bacteria. In this case, we strongly recommend using the kit in combination with RNAprotect? Bacteria Reagent (available separately), which provides in vivo stabilization of RNA in bacteria to ensure reliable gene expression analysis. Various protocols for stabilizing and purifying RNA from different bacteria species are included in the RNAprotect Bacteria Reagent Handbook. The RNeasy Mini Kit and RNAprotect Bacteria Reagent can also be purchased together as the RNeasy Protect Bacteria Mini Kit. For ordering information, see pages 74C75. It is also possible to use the RNeasy Mini Kit to purify cytoplasmic RNA from animal cells. The protocol can be downloaded at www.qiagen.com/literature/protocols/RNeasyMini.aspx. The RNeasy Kits are designed to purify RNA from small amounts of starting material. They provide a fast and simple method for preparing up to 100 ?g total RNA per sample. The purified RNA is ready for use in downstream applications such as: RT-PCR and real-time RT-PCR Differential display cDNA synthesis Northern, dot, and slot blot analyses Primer extension Poly A+ RNA selection RNase/S1 nuclease protection Microarrays RNA-Seq The RNeasy Kits allow the parallel processing of multiple samples in less than 30 minutes. Time-consuming and tedious methods, such as CsCl step-gradient ultracentrifugation and alcohol precipitation, or methods involving the use of toxic substances, such as phenol and/or chloroform, are replaced by the RNeasy procedure.RNeasy Mini Handbook 06/20127Principle and procedureRNA purification using RNeasy technology The RNeasy procedure represents a well-established technology for RNA purification. This technology combines the selective binding properties of a silica-based membrane with the speed of microspin technology. A specialized high-salt buffer system allows up to 100 ?g of RNA longer than 200 bases to bind to the RNeasy silica membrane. Biological samples are first lysed and homogenized in the presence of a highly denaturing guanidine-thiocyanateCcontaining buffer, which immediately inactivates RNases to ensure purification of intact RNA. Ethanol is added to provide appropriate binding conditions, and the sample is then applied to an RNeasy Mini spin column, where the total RNA binds to the membrane and contaminants are efficiently washed away. High-quality RNA is then eluted in 30C100 ?l water. With the RNeasy procedure, all RNA molecules longer than 200 nucleotides are purified. The procedure provides an enrichment for mRNA since most RNAs &200 nucleotides (such as 5.8S rRNA, 5S rRNA, and tRNAs, which together comprise 15C20% of total RNA) are selectively excluded. The size distribution of the purified RNA is comparable to that obtained by centrifugation through a CsCl cushion, where small RNAs do not sediment efficiently. Protocols for purification of small RNA using RNeasy Kits are available at www.qiagen.com/goto/microRNAprotocols. In this handbook, different protocols are provided for different starting materials. The protocols differ primarily in the lysis and homogenization of the sample and in the adjustment of the conditions for binding RNA to the RNeasy membrane. Once the sample is bound to the membrane, the protocols are similar (see flowchart, next page). RNA stabilization using RNAlater technology RNA stabilization is an absolute prerequisite for reliable gene expression analysis. Immediate stabilization of RNA in biological samples is necessary because, directly after harvesting the samples, changes in the gene expression pattern occur due to specific and nonspecific RNA degradation as well as to transcriptional induction. Such changes need to be avoided for all reliable quantitative gene expression analyses, such as microarray analyses, quantitative RT-PCR, such as TaqMan? and LightCycler? technology, and other nucleic acid-based technologies.8RNeasy Mini Handbook 06/2012RNeasy Mini ProcedureRNeasy Protect Mini ProcedureRNeasy Plant Mini ProcedureRNeasy Mini Handbook 06/20129The RNeasy Protect Mini Kit is supplied with RNAlater RNA Stabilization Reagent, which represents a novel technology for the immediate preservation of the gene expression pattern in animal tissues, enabling reliable gene expression analysis. After harvesting, tissues are immediately submerged in RNAlater RNA Stabilization Reagent, which rapidly permeates the tissues to stabilize and protect cellular RNA in situ. The reagent preserves RNA for up to 1 day at 37°C, 7 days at room temperature (15C25°C), or 4 weeks at 2C8°C, allowing transportation, storage, and shipping of samples without ice or dry ice. Alternatively, the samples can be archived at C20°C or C80°C. During storage or transport in RNAlater RNA Stabilization Reagent, even at elevated temperatures (e.g., room temperature or 37°C), the cellular RNA remains intact and undegraded. RNAlater technology allows large numbers of samples to be easily processed and replaces inconvenient, dangerous, and equipment-intensive methods, such as snap-freezing of samples in liquid nitrogen, storage at C80°C, cutting and weighing on dry ice, or immediate processing of harvested samples. Note: RNAlater RNA Stabilization Reagent is not for stabilization of RNA in animal cells, whole blood, plasma, or serum.Description of protocolsPurification of Total RNA from Animal Cells Using Spin Technology Up to 1 x 107 cells, depending on the cell line, are disrupted in Buffer RLT and homogenized. An overview of disruption and homogenization methods is given on pages 18C21. Ethanol is then added to the lysate, creating conditions that promote selective binding of RNA to the RNeasy membrane. The sample is then applied to the RNeasy Mini spin column. Total RNA binds to the membrane, contaminants are efficiently washed away, and high-quality RNA is eluted in RNase-free water. All bind, wash, and elution steps are performed by centrifugation in a microcentrifuge. Purification of Total RNA from Animal Cells Using Vacuum/Spin Technology Up to 1 x 106 cells, depending on the cell line, are disrupted in Buffer RLT and homogenized. An overview of disruption and homogenization methods is given on pages 18C21. Ethanol is then added to the lysate, creating conditions that promote selective binding of RNA to the RNeasy membrane. The sample is then applied to the RNeasy Mini spin column. Total RNA binds to the membrane, contaminants are efficiently washed away, and high-quality RNA is eluted in RNase-free water. The bind and wash steps are performed on a QIAvac 24 or QIAvac 24 Plus manifold, and the final elution step is performed by centrifugation in a microcentrifuge.10RNeasy Mini Handbook 06/2012Stabilization of RNA in Harvested Animal Tissues This protocol describes how to stabilize RNA in harvested animal tissues using RNAlater RNA Stabilization Reagent. Purification of total RNA from the stabilized tissues can be subsequently carried out according to “Protocol: Purification of Total RNA from Animal Tissues” (page 37). Purification of Total RNA from Animal Tissues Fresh, frozen, or RNAlater stabilized tissue (up to 30 mg, depending on the tissue type) is disrupted in Buffer RLT and homogenized. An overview of disruption and homogenization methods is given on pages 18C21. Ethanol is then added to the lysate, creating conditions that promote selective binding of RNA to the RNeasy membrane. The sample is then applied to the RNeasy Mini spin column. Total RNA binds to the membrane, contaminants are efficiently washed away, and high-quality RNA is eluted in RNase-free water. Purification of Total RNA from Yeast This protocol is for the purification of total RNA from up to 5 x 107 yeast cells. Two alternative methods of disrupting yeast cell walls are provided: enzymatic lysis or mechanical disruption. In general, both methods function equally well. For some applications, enzymatic lysis might be preferable since no additional laboratory equipment is required. Mechanical disruption, however, is well-suited for time-course experiments where enzymatic digestion incubations are not practical. The enzymatic lysis method uses zymolase or lyticase digestion of the cell walls to convert cells to spheroplasts, which are then used in the RNeasy procedure. For samples of up to 5 x 107 yeast cells, spheroplasts are separated from the digestion mixture by centrifugation before being lysed. For samples of up to 2 x 107 yeast cells, the digestion mixture is used directly in the RNeasy procedure without prior separation of the spheroplasts. After addition of Buffer RLT and ethanol, samples are loaded onto the RNeasy Mini spin column. Total RNA binds to the RNeasy membrane, contaminants are efficiently washed away, and high-quality RNA is eluted in RNase-free water. Using the mechanical disruption method, yeast cells are lysed and homogenized by high-speed agitation in the TissueLyser LT, TissueLyser II, or other bead mill in the presence of glass beads and Buffer RLT. Ethanol is added to the lysate, creating conditions that promote selective binding of RNA to the RNeasy membrane. The sample is then applied to the RNeasy Mini spin column. Total RNA binds to the membrane, contaminants are efficiently washed away, and high-quality RNA is eluted in RNase-free water.RNeasy Mini Handbook 06/201211Purification of Total RNA from Plant Cells and Tissues and Filamentous Fungi Up to 100 mg of sample is first ground in liquid nitrogen and then lysed under highly denaturing conditions. The RNeasy Plant Mini Kit provides a choice of lysis buffers: Buffer RLT and Buffer RLC, which contain guanidine thiocyanate and guanidine hydrochloride, respectively. The higher cell disruption and denaturing properties of Buffer RLT frequently make it the buffer of choice. However, some tissues, such as milky endosperm of maize or mycelia of filamentous fungi, solidify in Buffer RLT, making the extraction of RNA impossible. In these cases, Buffer RLC should be used instead. After lysis with either buffer, samples are centrifuged through a QIAshredder homogenizer. This simultaneously removes insoluble material and reduces the viscosity of the lysates by disrupting gelatinous material often formed in plant and fungal lysates. Ethanol is added to the cleared lysate, creating conditions which promote selective binding of RNA to the RNeasy membrane. The sample is then applied to the RNeasy Mini spin column. Total RNA binds to the membrane, contaminants are efficiently washed away, and high-quality RNA is eluted in RNase-free water. RNA Cleanup This protocol can be used to purify RNA from enzymatic reactions (e.g., DNase digestion, RNA labeling) or to desalt RNA samples (up to 100 ?g RNA). Buffer RLT and ethanol are added to the sample to create conditions that promote selective binding of RNA to the RNeasy membrane. The sample is then applied to the RNeasy Mini spin column. Total RNA binds to the membrane, contaminants are efficiently washed away, and high-quality RNA is eluted in RNase-free water. Automated purification Purification of RNA can be fully automated on the QIAcube?. The innovative QIAcube uses advanced technology to process QIAGEN spin columns, enabling seamless integration of automated, low-throughput sample prep into your laboratory workflow. Sample preparation using the QIAcube follows the same steps as the manual procedure (i.e., lyse, bind, wash, and elute), enabling you to continue using the RNeasy Mini Kit for purification of high-quality RNA. For more information about the automated procedure, see the relevant protocol sheet available at www.qiagen.com/MyQIAcube. The QIAcube is preinstalled with protocols for purification of plasmid DNA, genomic DNA, RNA, viral nucleic acids, and proteins, plus DNA and RNA cleanup. The range of protocols available is continually expanding, and additional QIAGEN protocols can be downloaded free of charge at www.qiagen.com/MyQIAcube.12RNeasy Mini Handbook 06/2012Equipment and Reagents to Be Supplied by UserWhen working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier. For all protocols14.3 M β-mercaptoethanol (β-ME) (commercially available solutions are usually 14.3 M) Sterile, RNase-free pipet tips Microcentrifuge (with rotor for 2 ml tubes) 96C100% ethanol* Disposable gloves Equipment for sample disruption and homogenization (see pages 18C21). Depending on the method chosen, one or more of the following are required: Trypsin and PBS QIAshredder homogenizer (see ordering information, page 73 Blunt needle and syringe Mortar and pestle TissueLyser LT or TissueLyser II (see ordering information, page 73) RotorCstator homogenizerFor RNA purification from animal cells 70% ethanol* For RNA purification from animal cells using vacuum technology QIAvac 24 (no longer available); QIAvac 24 Plus (cat. no. 19413); or other vacuum manifold with luer connectors and capable of dealing with vacuum pressures of C800 to C900 mbar QIAGEN Vacuum Pump (see page 73 for ordering information); or other vacuum pump capable of generating a vacuum pressure of C800 to C900 mbar and with a capacity of 18C20 liter/min Note: Use of insufficient vacuum pressure may reduce RNA yield and purity. The RNeasy procedure requires higher vacuum pressures compared with other QIAGEN procedures. Most water pumps or house vacuums do not provide sufficient vacuum pressure.* Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone.RNeasy Mini Handbook 06/201213Optional: Vacuum Regulator (cat. no. 19530) to measure the pressure difference between the inside and outside of a vacuum system A vacuum pressure of C800 to C900 mbar should develop when RNeasy Mini spin columns are used on the vacuum manifold. Vacuum pressures exceeding C900 mbar should be avoided. The vacuum pressure is the pressure difference between the inside of the manifold and the atmosphere (standard atmospheric pressure: 1013 mbar or 760 mm Hg) and can be regulated and measured using a pressure gauge or vacuum regulator. Vacuum recommendations are given in negative units to indicate the required reduction in pressure with respect to the atmosphere. Optional: VacConnectors (cat. no. 19407) These disposable connectors fit between the RNeasy Mini spin columns and the luer extensions on the QIAvac 24 or QIAvac 24 Plus. They prevent direct contact between the RNeasy Mini spin columns and luer connectors during RNA purification, avoiding any cross-contamination between samples. VacConnectors are discarded after single use. For RNA purification from animal tissues 70% ethanol* Optional: Dithiothreitol (DTT) For RNA purification from yeast using enzymatic lysis 70% ethanol* Buffer for enzymatic lysis In most cases, Buffer Y1 (containing sorbitol, EDTA, β-ME, and lyticase or zymolase) can be used. See the protocol on page 45 for details on preparing Buffer Y1. For RNA purification from yeast using mechanical disruption 70% ethanol* Glass beads, 0.45C0.55 mm diameter Concentrated nitric acid, deionized water, and baking oven TissueLyser LT, TissueLyser II, or other bead-mill homogenizer* Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone.14RNeasy Mini Handbook 06/2012For RNA purification from plants and fungi Liquid nitrogen Mortar and pestle (alternatively, TissueLyser LT, TissueLyser II, or other bead-mill homogenizer) Suppliers of equipment for disruption and homogenization* RotorCstator homogenizers can be purchased from: BioSpec Products, Inc. (www.biospec.com): Tissue-Tearor? homogenizer Charles Ross & Son Company (www.mixers.com) IKA (www.ika.de): ULTRA-TURRAX? dispersers KINEMATICA AG (www.kinematica.ch) or Brinkmann Instruments, (www.brinkmann.com): POLYTRON? laboratory dispersing devices Omni International, Inc. (www.omni-inc.com) Silverson (www.silverson.com) VirTis (www.virtis.com) Bead-mill homogenizers and stainless steel and tungsten carbide beads can be purchased from: QIAGEN (TissueLyser system, see page 73 for ordering information) Glass, stainless steel, and tungsten carbide beads can be purchased from: Retsch (www.retsch.de) Inc.* This is not a complete list of suppliers and does not include many important vendors of biological supplies.RNeasy Mini Handbook 06/201215Important NotesDetermining the amount of starting materialIt is essential to use the correct amount of starting material to obtain optimal RNA yield and purity. The maximum amount that can be used is determined by: The type of sample and its RNA content The volume of Buffer RLT required for efficient lysis The RNA binding capacity of the RNeasy spin column When processing samples containing high amounts of RNA, less than the maximum amount of starting material shown in Table 1 should be used, so that the RNA binding capacity of the RNeasy spin column is not exceeded. When processing samples containing average or low amounts of RNA, the maximum amount of starting material shown in Table 1 can be used. However, even though the RNA binding capacity of the RNeasy spin column is not reached, the maximum amount of starting material must not be exceeded. Otherwise, lysis will be incomplete and cellular debris may interfere with the binding of RNA to the RNeasy spin column membrane, resulting in lower RNA yield and purity. More information on using the correct amount of starting material is given in each protocol. Table 2 shows expected RNA yields from various sources. Table 1. RNeasy Mini spin column specifications Maximum binding capacity Maximum loading volume RNA size distribution Minimum elution volume Maximum amount of starting material Animal cells Animal tissues Yeast Plant tissues Filamentous fungi 1 x 107* 30 mg* 5 x 107* 100 mg 100 mg 100 ?g RNA 700 ?l RNA &200 nucleotides 30 ?l* For larger sample sizes, RNeasy Kits and RNeasy Protect Kits are available in midi and maxi formats. For details, visit www.qiagen.com/RNA.16RNeasy Mini Handbook 06/2012Note: If the binding capacity of the RNeasy spin column is exceeded, RNA yields will not be consistent and may be reduced. If lysis of the starting material is incomplete, RNA yields will be lower than expected, even if the binding capacity of the RNeasy spin column is not exceeded. Table 2. Typical yields of total RNA with RNeasy Mini spin columns Source Cell cultures (1 x 10 cells)6Yield of total RNA* (?g) 10 15 35 12 15NIH/3T3 HeLa COS-7 LMH Huh Mouse/rat tissues (10 mg) Embryo (13 day) Kidney Liver Spleen Thymus Lung Yeast (1 x 107 cells) S. cerevisiae Plants (100 mg leaves) Arabidopsis Maize Tomato Tobacco25 20C30 40C60 30C40 40C50 10C202535 25 65 60* Amounts can vary due to factors such as species, developmental stage, and growth conditions. Since the RNeasy procedure enriches for mRNA and other RNA species &200 nucleotides, the total RNA yield does not include 5S rRNA, tRNA, and other low-molecular-weight RNAs, which make up 15C20% of total cellular RNA.RNeasy Mini Handbook 06/201217Handling and storing starting materialRNA in animal and plant tissues is not protected after harvesting until the sample is treated with RNAlater RNA Stabilization Reagent (animal tissues only), flash-frozen, or disrupted and homogenized in the presence of RNase-inhibiting or denaturing reagents. Otherwise, unwanted changes in the gene expression profile will occur. It is therefore important that tissue samples are immediately frozen in liquid nitrogen and stored at C70°C, or immediately immersed in RNAlater RNA Stabilization Reagent. Alternatively, use Allprotect Tissue Reagent, which provides immediate stabilization of DNA, RNA, and protein in tissues samples at room temperature. The procedures for tissue harvesting and RNA protection should be carried out as quickly as possible. Frozen tissue samples should not be allowed to thaw during handling or weighing. After disruption and homogenization in Buffer RLT (lysis buffer), samples can be stored at C70°C for months. Animal and yeast cells can be pelleted and then stored at C70°C until required for RNA purification. However, if performing RNA purification from yeast cells with enzymatic lysis, only freshly harvested samples can be used.Disrupting and homogenizing starting materialEfficient disruption and homogenization of the starting material is an absolute requirement for all total RNA purification procedures. Disruption and homogenization are 2 distinct steps: Disruption: Complete disruption of cell walls and plasma membranes of cells and organelles is absolutely required to release all the RNA contained in the sample. Different samples require different methods to achieve complete disruption. Incomplete disruption results in significantly reduced RNA yields. Homogenization: Homogenization is necessary to reduce the viscosity of the lysates produced by disruption. Homogenization shears high-molecular-weight genomic DNA and other high-molecular-weight cellular components to create a homogeneous lysate. Incomplete homogenization results in inefficient binding of RNA to the RNeasy spin column membrane and therefore significantly reduced RNA yields. Some disruption methods simultaneously homogenize the sample, while others require an additional homogenization step. Table 3 (page 19) gives an overview of different disruption and homogenization methods, and is followed by a detailed description of each method. This information can be used as a guide to choose the appropriate methods for your starting material. Note: After storage in RNAlater RNA Stabilization Reagent, tissues become slightly harder than fresh or thawed tissues. Disruption and homogenization of these tissues, however, is usually not a problem.18RNeasy Mini Handbook 06/2012Table 3. Disruption and homogenization methods Disruption method Addition of lysis buffer Homogenization method TissueRuptor or QIAshredder homogenizer* or syringe and needle TissueLyser LT or TissueLyser IISample Animal cellsCommentsIf processing ≤1 x 105 cells, lysate can be homogenized by vortexing The TissueLyser LT or TissueLyser II gives results comparable to using the TissueRuptor Simultaneously disrupts and homogenizes TissueRuptor usually gives higher yields than mortar and pestleAnimal tissuesTissueLyser LT or TissueLyser IITissueRuptor Mortar and pestleTissueRuptor QIAshredder homogenizer* or syringe and needle VortexingYeastEnzymatic digestion of cell wall followed by lysis of spheroplasts TissueLyser LT or TissueLyser II with glass beadsTissueLyser LT or TissueLyser II with glass beadsTissueLyser LT or TissueLyser II simultaneously disr cannot be replaced by vortexing Mortar and pestle cannot be replaced by the TissueRuptorPlants and filamentous fungiMortar and pestleQIAshredder homogenizer** QIAshredder homogenizers are supplied in the RNeasy Plant Mini Kit and can be purchased separately for use with the RNeasy Mini Kit or RNeasy Protect Mini Kit. See page 74 for ordering information.RNeasy Mini Handbook 06/201219Disruption and homogenization using the TissueLyser system In bead-milling, cells and tissues can be disrupted by rapid agitation in the presence of beads and lysis buffer. Disruption and simultaneous homogenization occur by the shearing and crushing action of the beads as they collide with the cells. Disruption efficiency is influenced by: Size and composition of beads Ratio of buffer to beads Amount of starting material Speed and configuration of the TissueLyser LT or TissueLyser II Disintegration time For animal tissues, the optimal beads are 3C7 mm diameter stainless steel beads, and for yeast cells, the optimal beads are 0.5 mm diameter glass beads. It is essential that glass beads are prewashed in concentrated nitric acid. All other disruption parameters must be determined empirically for each application. The protocol for RNA purification from yeast (page 43) describes how to perform mechanical disruption of yeast cells with glass beads. For guidelines on disruption and homogenization of animal tissues using the TissueLyser system, refer to the TissueLyser LT Handbook or the TissueLyser Handbook. For other bead mills, please refer to suppliers’ guidelines for further details. Plant tissues can be disrupted using the TissueLyser LT or TissueLyser II, in combination with stainless steel or tungsten carbide beads. In this case, plant material, beads, and disruption vessels must all be precooled in liquid nitrogen, and disruption is performed without lysis buffer. Disruption and homogenization using the TissueRuptor or other rotorCstator homogenizers RotorCstator homogenizers thoroughly disrupt and simultaneously homogenize, in the presence of lysis buffer, single samples of animal tissues in 15C90 seconds depending on the toughness and size of the sample. RotorCstator homogenizers can also be used to homogenize cell lysates. The rotor turns at a very high speed, causing the sample to be disrupted and homogenized by a combination of turbulence and mechanical shearing. Foaming of the sample should be kept to a minimum by using properly sized vessels, keeping the tip of the homogenizer submerged, and holding the immersed tip to the side of the tube. RotorCstator homogenizers are available in different sizes and operate with differently sized probes. Probes with diameters of 5 mm and 7 mm are suitable for volumes up to 300 ?l and can be used for homogenization in microcentrifuge tubes. Probes with a diameter of 10 mm or above require larger tubes. In addition, round-bottomed tubes allow more efficient homogenization than conicalbottomed tubes.20RNeasy Mini Handbook 06/2012Disruption using a mortar and pestle For disruption using a mortar and pestle, freeze the animal or plant tissue immediately in liquid nitrogen and grind to a fine powder under liquid nitrogen. Transfer the suspension (tissue powder and liquid nitrogen) into a liquid-nitrogenCcooled, appropriately sized tube and allow the liquid nitrogen to evaporate without allowing the sample to thaw. Add lysis buffer and continue as quickly as possible with the homogenization according to one of the 2 methods below. Note: Grinding the sample using a mortar and pestle will disrupt the sample, but will not homogenize it. Homogenization must be performed afterwards. Homogenization using QIAshredder homogenizers Using QIAshredder homogenizers is a fast and efficient way to homogenize cell and tissue lysates without cross-contamination of samples. Up to 700 ?l of lysate is loaded onto a QIAshredder spin column placed in a 2 ml collection tube, and spun for 2 minutes at maximum speed in a microcentrifuge. The lysate is homogenized as it passes through the spin column. QIAshredder spin columns are supplied in the RNeasy Plant Mini Kit and can be purchased separately for use with the RNeasy Mini Kit and RNeasy Protect Mini Kit. See page 74 for ordering information. Homogenization using a syringe and needle Cell and tissue lysates can be homogenized using a syringe and needle. Lysate is passed through a 20-gauge (0.9 mm) needle attached to a sterile plastic syringe at least 5C10 times or until a homogeneous lysate is achieved. Increasing the volume of lysis buffer may be required to facilitate handling and minimize loss.Eliminating genomic DNA contaminationGenerally, DNase digestion is not required with RNeasy Kits since RNeasy silicamembrane technology efficiently removes most of the DNA without DNase treatment. However, further DNA removal may be necessary for certain RNA applications that are sensitive to very small amounts of DNA (e.g., TaqMan RT-PCR analysis with a low-abundance target). In these cases, residual DNA can be removed by optional oncolumn DNase digestion using the RNase-Free DNase Set (see Appendix D, page 67). The DNase is efficiently removed in subsequent wash steps. Alternatively, residual DNA can be removed by a DNase digestion after RNA purification (see Appendix E, page 69). The DNase digestion can then be cleaned up, if desired, using “Protocol: RNA Cleanup” (page 54). The RNeasy Plus Mini Kit, which is designed for RNA purification from animal cells and tissues, integrates unique gDNA Eliminator spin columns with RNeasy technology. Genomic DNA is effectively removed in a single, rapid centrifugation step, avoiding the need for DNase digestion. See page 74 for ordering information.RNeasy Mini Handbook 06/2012 21If the purified RNA will be used in real-time, two-step RT-PCR, we recommend using the QuantiTect? Reverse Transcription Kit. The kit provides a fast and convenient procedure, enabling cDNA synthesis and genomic DNA removal in only 20 minutes. For ordering information, see page 76.22RNeasy Mini Handbook 06/2012Protocol: Purification of Total RNA from Animal Cells using Spin TechnologyThis protocol requires the RNeasy Mini Kit. Determining the correct amount of starting material It is essential to use the correct amount of starting material to obtain optimal RNA yield and purity. The minimum amount is generally 100 cells, while the maximum amount depends on: The RNA content of the cell type The RNA binding capacity of the RNeasy spin column (100 ?g RNA) The volume of Buffer RLT required for efficient lysis (the maximum volume of Buffer RLT that can be used limits the maximum amount of starting material to 1 x 107 cells) RNA content can vary greatly between cell types. The following examples illustrate how to determine the maximum amount of starting material: COS cells have high RNA content (approximately 35 ?g RNA per 106 cells). Do not use more than 3 x 106 cells, otherwise the RNA binding capacity of the RNeasy spin column will be exceeded. HeLa cells have average RNA content (approximately 15 ?g RNA per 106 cells). Do not use more than 7 x 106 cells, otherwise the RNA binding capacity of the RNeasy spin column will be exceeded. NIH/3T3 cells have low RNA content (approximately 10 ?g RNA per 106 cells). The maximum amount of starting material (1 x 107 cells) can be used. If processing a cell type not listed in Table 2 (page 17) and if there is no information about its RNA content, we recommend starting with no more than 3C4 x 106 cells. Depending on RNA yield and purity, it may be possible to increase the cell number in subsequent preparations. Do not overload the RNeasy spin column, as this will significantly reduce RNA yield and purity. Counting cells is the most accurate way to quantitate the amount of starting material. As a guide, the number of HeLa cells obtained in various culture vessels after confluent growth is given in Table 4.Animal Cells SpinRNeasy Mini Handbook 06/201223Table 4. Growth area and number of HeLa Cells in various culture vessels Cell-culture vessel Growth area (cm2)* 0.32C0.6 1 2 4 9.5 Number of cells? 4C5 x 104 1 x 105 2.5 x 105 5 x 105 1 x 106Animal Cells SpinMultiwell-plates 96-well 48-well 24-well 12-well 6-well Dishes 35 mm 60 mm 100 mm 145C150 mm Flasks 40C50 ml 250C300 ml 650C750 ml 25 75 162C175 3 x 106 1 x 107 2 x 107 8 21 56 145 1 x 106 2.5 x 106 7 x 106 2 x 107* Per well, if multi varies slightly depending on the supplier.?Cell numbers are given for HeLa cells (approximate length = 15 ?m), assuming confluent growth. Cell numbers will vary for different kinds of animal cells, which vary in length from 10 to 30 ?m.Important points before starting If using the RNeasy Kit for the first time, read “Important Notes” (page 16). If working with RNA for the first time, read Appendix A (page 61). Cell pellets can be stored at C70°C for later use or used directly in the procedure. Determine the number of cells before freezing. Frozen cell pellets should be thawed slightly so that they can be dislodged by flicking the tube in step 2. Homogenized cell lysates from step 3 can be stored at C70°C for several months. Frozen lysates should be incubated at 37°C in a water bath until completely thawed and salts are dissolved. Avoid prolonged incubation, which may compromise RNA integrity. If any insoluble material is visible, centrifuge for 5 min at
x g. Transfer supernatant to a new RNase-free glass or polypropylene tube, and continue with step 4.24RNeasy Mini Handbook 06/2012Buffer RLT may form a precipitate upon storage. If necessary, redissolve by warming, and then place at room temperature (15C25°C). Buffer RLT and Buffer RW1 contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach. See page 6 for safety information. Perform all steps of the procedure at room temperature. During the procedure, work quickly. Perform all centrifugation steps at 20C25°C in a standard microcentrifuge. Ensure that the centrifuge does not cool below 20°C. Things to do before starting If purifying RNA from cell lines rich in RNases, we recommend adding β-mercaptoethanol (β-ME) to Buffer RLT before use. Add 10 ?l β-ME per 1 ml Buffer RLT. Dispense in a fume hood and wear appropriate protective clothing. Buffer RLT containing β-ME can be stored at room temperature (15C25°C) for up to 1 month. Buffer RPE is supplied as a concentrate. Before using for the first time, add 4 volumes of ethanol (96C100%) as indicated on the bottle to obtain a working solution. If performing optional on-column DNase digestion, prepare DNase I stock solution as described in Appendix D (page 67). Procedure 1. Harvest cells according to step 1a or 1b. 1a. Cells grown in suspension (do not use more than 1 x 107 cells): Determine the number of cells. Pellet the appropriate number of cells by centrifuging for 5 min at 300 x g in a centrifuge tube (not supplied). Carefully remove all supernatant by aspiration, and proceed to step 2. Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute the lysate, affecting the conditions for binding of RNA to the RNeasy membrane. Both effects may reduce RNA yield.Animal Cells SpinRNeasy Mini Handbook 06/201225Animal Cells Spin1b. Cells grown in a monolayer (do not use more than 1 x 107 cells): Cells can be either lysed directly in the cell-culture vessel (up to 10 cm diameter) or trypsinized and collected as a cell pellet prior to lysis. Cells grown in cell-culture flasks should always be trypsinized. To lyse cells directly: Determine the number of cells. Completely aspirate the cell-culture medium, and proceed immediately to step 2. Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute the lysate, affecting the conditions for binding of RNA to the RNeasy membrane. Both effects may reduce RNA yield. To trypsinize and collect cells: Determine the number of cells. Aspirate the medium, and wash the cells with PBS. Aspirate the PBS, and add 0.1C0.25% trypsin in PBS. After the cells detach from the dish or flask, add medium (containing serum to inactivate the trypsin), transfer the cells to an RNase-free glass or polypropylene centrifuge tube (not supplied), and centrifuge at 300 x g for 5 min. Completely aspirate the supernatant, and proceed to step 2. Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute the lysate, affecting the conditions for binding of RNA to the RNeasy membrane. Both effects may reduce RNA yield. 2. Disrupt the cells by adding Buffer RLT. For pelleted cells, loosen the cell pellet thoroughly by flicking the tube. Add the appropriate volume of Buffer RLT (see Table 5). Vortex or pipet to mix, and proceed to step 3. Note: Incomplete loosening of the cell pellet may lead to inefficient lysis and reduced RNA yields. Table 5. Volumes of Buffer RLT for lysing pelleted cells Number of pelleted cells &5 x 106 6 7Volume of Buffer RLT (?l) 350 6005 x 10 C 1 x 10For direct lysis of cells grown in a monolayer, add the appropriate volume of Buffer RLT (see Table 6) to the cell-culture dish. Collect the lysate with a rubber policeman. Pipet the lysate into a microcentrifuge tube (not supplied). Vortex or pipet to mix, and ensure that no cell clumps are visible before proceeding to step 3.26RNeasy Mini Handbook 06/2012Table 6. Volumes of Buffer RLT for direct cell lysis Dish diameter (cm) &6 6C10 3. Homogenize the lysate according to step 3a, 3b, or 3c. See “Disrupting and homogenizing starting material”, pages 18C21, for more details on homogenization. If processing ≤1 x 105 cells, homogenize by vortexing for 1 min. After homogenization, proceed to step 4. Note: Incomplete homogenization leads to significantly reduced RNA yields and can cause clogging of the RNeasy spin column. Homogenization with a rotorC stator or QIAshredder homogenizer generally results in higher RNA yields than with a syringe and needle. 3a. Pipet the lysate directly into a QIAshredder spin column placed in a 2 ml collection tube, and centrifuge for 2 min at full speed. Proceed to step 4. 3b. Homogenize the lysate for 30 s using a rotorCstator homogenizer. Proceed to step 4. 3c. Pass the lysate at least 5 times through a blunt 20-gauge needle (0.9 mm diameter) fitted to an RNase-free syringe. Proceed to step 4. 4. Add 1 volume of 70% ethanol to the homogenized lysate, and mix well by pipetting. Do not centrifuge. Note: The volume of lysate may be less than 350 ?l or 600 ?l due to loss during homogenization. Note: When purifying RNA from certain cell lines, precipitates may be visible after addition of ethanol. This does not affect the procedure. 5. Transfer up to 700 ?l of the sample, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 ml collection tube (supplied). Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-through.* Reuse the collection tube in step 6. If the sample volume exceeds 700 ?l, centrifuge successive aliquots in the same RNeasy spin column. Discard the flow-through after each centrifugation.* Optional: If performing optional on-column DNase digestion (see “Eliminating genomic DNA contamination”, page 21), follow steps D1CD4 (page 67) after performing this step.* Flow-through contains Buffer RLT or Buffer RW1 and is therefore not compatible with bleach. See page 6 for safety information.Volume of Buffer RLT (?l)*Animal Cells Spin350 600* Regardless of the cell number, use the buffer volumes indicated to completely cover the surface of the dish.RNeasy Mini Handbook 06/2012276.Add 700 ?l Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through.* Reuse the collection tube in step 7. Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Be sure to empty the collection tube completely. Skip this step if performing optional on-column DNase digestion (page 67).Animal Cells Spin7.Add 500 ?l Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through. Reuse the collection tube in step 8. Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use (see “Things to do before starting”).8.Add 500 ?l Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. The long centrifugation dries the spin column membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere with downstream reactions. Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur.9.Optional: Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flow-through. Close the lid gently, and centrifuge at full speed for 1 min. Perform this step to eliminate any possible carryover of Buffer RPE, or if residual flow-through remains on the outside of the RNeasy spin column after step 8.10. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 30C50 ?l RNase-free water directly to the spin column membrane. Close the lid gently, and centrifuge for 1 min at ≥8000 x g (≥10,000 rpm) to elute the RNA. 11. If the expected RNA yield is &30 ?g, repeat step 10 using another 30C50 ?l RNasefree water, or using the eluate from step 10 (if high RNA concentration is required). Reuse the collection tube from step 10. If using the eluate from step 10, the RNA yield will be 15C30% less than that obtained using a second volume of RNase-free water, but the final RNA concentration will be higher.* Flow-through contains Buffer RLT or Buffer RW1 and is therefore not compatible with bleach. See page 6 for safety information.28RNeasy Mini Handbook 06/2012Protocol: Purification of Total RNA from Animal Cells using Vacuum/Spin TechnologyThis protocol requires the RNeasy Mini Kit. Determining the correct amount of starting material See “Determining the correct amount of starting material”, page 16.Animal Cells Vacuum/SpinImportant points before starting If using the RNeasy Kit for the first time, read “Important Notes” (page 16). If working with RNA for the first time, read Appendix A (page 61). Cell pellets can be stored at C70°C for later use or used directly in the procedure. Determine the number of cells before freezing. Frozen cell pellets should be thawed slightly so that they can be dislodged by flicking the tube in step 2. Homogenized cell lysates from step 3 can be stored at C70°C for several months. Frozen lysates should be incubated at 37°C in a water bath until completely thawed and salts are dissolved. Avoid prolonged incubation, which may compromise RNA integrity. If any insoluble material is visible, centrifuge for 5 min at
x g. Transfer supernatant to a new RNase-free glass or polypropylene tube, and continue with step 4. Buffer RLT may form a precipitate upon storage. If necessary, redissolve by warming, and then place at room temperature (15C25°C). Buffer RLT and Buffer RW1 contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach. See page 6 for safety information. Perform all steps of the procedure at room temperature. During the procedure, work quickly. Perform all centrifugation steps at 20C25°C in a standard microcentrifuge. Ensure that the centrifuge does not cool below 20°C. Do not use more than 106 cells per prep. The cell numbers in each prep should be similar (no more than a twofold difference between the highest and lowest) to allow uniform flow rates on the vacuum manifold. Between loading steps, switch off the vacuum and ventilate the manifold to maintain uniform conditions for each sample. This can be done with a vacuum regulator inserted between the vacuum source and the vacuum manifold. Always use caution and wear safety glasses when working near a vacuum manifold under pressure. Always leave the lids of the RNeasy spin columns open while applying vacuum.RNeasy Mini Handbook 06/201229The flow-through from each vacuum step is collected in the QIAvac 24 Plus or in the QIAvac 24 base. Each can hold the waste from 24 samples. At the end of the procedure, discard the liquid waste and clean the vacuum manifold as described in the QIAvac 24 Plus Handbook or QIAvac Handbook. If using other vacuum manifolds, follow the supplier’s instructions. Things to do before startingAnimal Cells Vacuum/SpinIf purifying RNA from cell lines rich in RNases, we recommend adding β-mercaptoethanol (β-ME) to Buffer RLT before use. Add 10 ?l β-ME per 1 ml Buffer RLT. Dispense in a fume hood and wear appropriate protective clothing. Buffer RLT containing β-ME can be stored at room temperature (15C25°C) for up to 1 month. Buffer RPE is supplied as a concentrate. Before using for the first time, add 4 volumes of ethanol (96C100%) as indicated on the bottle to obtain a working solution. If performing optional on-column DNase digestion, prepare DNase I stock solution as described in Appendix D (page 67). Set up the vacuum manifold according to the supplier’s instructions. If using the QIAvac Plus 24, refer to the QIAvac 24 Plus Handbook. If using the QIAvac 24, refer to the QIAvac Handbook. Insert each RNeasy spin column into a luer connector. Procedure 1. Harvest cells according to step 1a or 1b. 1a. Cells grown in suspension (do not use more than 1 x 106 cells): Determine the number of cells. Pellet the appropriate number of cells by centrifuging for 5 min at 300 x g in a centrifuge tube (not supplied). Carefully remove all supernatant by aspiration, and proceed to step 2. Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute the lysate, affecting the conditions for binding of RNA to the RNeasy membrane. Both effects may reduce RNA yield.30RNeasy Mini Handbook 06/20121b. Cells grown in a monolayer (do not use more than 1 x 106 cells): Cells can be either lysed directly in the cell-culture vessel (up to 10 cm diameter) or trypsinized and collected as a cell pellet prior to lysis. Cells grown in cell-culture flasks should always be trypsinized. To lyse cells directly: Determine the number of cells. Completely aspirate the cell-culture medium, and proceed immediately to step 2. Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute the lysate, affecting the conditions for binding of RNA to the RNeasy membrane. Both effects may reduce RNA yield. To trypsinize and collect cells: Determine the number of cells. Aspirate the medium, and wash the cells with PBS. Aspirate the PBS, and add 0.1C0.25% trypsin in PBS. After the cells detach from the dish or flask, add medium (containing serum to inactivate the trypsin), transfer the cells to an RNase-free glass or polypropylene centrifuge tube (not supplied), and centrifuge at 300 x g for 5 min. Completely aspirate the supernatant, and proceed to step 2. Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute the lysate, affecting the conditions for binding of RNA to the RNeasy membrane. Both effects may reduce RNA yield. 2. Disrupt the cells by adding Buffer RLT. For pelleted cells, loosen the cell pellet thoroughly by flicking the tube. Add 350 ?l Buffer RLT. Vortex or pipet to mix, and proceed to step 3. Note: Incomplete loosening of the cell pellet may lead to inefficient lysis and reduced RNA yields. For direct lysis of cells grown in a monolayer, add 350 ?l Buffer RLT to the cellculture dish (if 350 ?l is not enough to cover the dish, use 600 ?l Buffer RLT be sure then to use 600 ?l of 70% ethanol in step 4). Collect the lysate with a rubber policeman. Pipet the lysate into a microcentrifuge tube (not supplied). Vortex or pipet to mix, and ensure that no cell clumps are visible before proceeding to step 3. 3. Homogenize the lysate according to step 3a, 3b, or 3c. See “Disrupting and homogenizing starting material”, pages 18C21, for more details on homogenization. If processing ≤1 x 105 cells, homogenize by vortexing for 1 min. After homogenization, proceed to step 4. Note: Incomplete homogenization leads to significantly reduced RNA yields and can cause clogging of the RNeasy spin column. Homogenization with a rotorC stator or QIAshredder homogenizer generally results in higher RNA yields than with a syringe and needle.Animal Cells Vacuum/SpinRNeasy Mini Handbook 06/2012313a. Pipet the lysate directly into a QIAshredder spin column placed in a 2 ml collection tube, and centrifuge for 2 min at full speed. Proceed to step 4. 3b. Homogenize the lysate for 30 s using a rotorCstator homogenizer. Proceed to step 4. 3c. Pass the lysate at least 5 times through a blunt 20-gauge needle (0.9 mm diameter) fitted to an RNase-free syringe. Proceed to step 4. 4. Add 1 volume of 70% ethanol to the homogenized lysate, and mix well by pipetting. Do not centrifuge. Note: The volume of lysate may be less than 350 ?l or 600 ?l due to loss during homogenization. Note: When purifying RNA from certain cell lines, precipitates may be visible after addition of ethanol. This does not affect the procedure. 5. 6. Transfer 700 ?l of each sample from step 4, including any precipitate that may have formed, to each RNeasy spin column on the vacuum manifold. Switch on the vacuum. Apply vacuum until transfer is complete. Switch off the vacuum and ventilate the vacuum manifold. Make sure that the vacuum manifold is assembled correctly before loading. The flow-through is collected in the QIAvac 24 Plus or the QIAvac 24 base.* If a spin column clogs, switch off the vacuum, ventilate, and try again. If it still clogs, continue with “Protocol: Purification of Total RNA from Animal Cells Using Spin Technology”, page 23. Note: Be sure to switch off the vacuum and ventilate the manifold between pipetting steps to maintain uniform conditions for each sample. 7. If necessary, repeat steps 5 and 6 with the remaining volume (approx. 500 ?l) of each sample. The flow-through is collected in the QIAvac 24 Plus or the QIAvac 24 base.* 8. 9. Add 700 ?l Buffer RW1 to each RNeasy spin column. Switch on the vacuum. Apply vacuum until transfer is complete. Switch off the vacuum and ventilate the vacuum manifold. The flow-through is collected in the QIAvac 24 Plus or the QIAvac 24 base.* 10. Add 500 ?l Buffer RPE to each RNeasy spin column. Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use (see “Things to do before starting”). 11. Switch on the vacuum. Apply vacuum until transfer is complete. Switch off the vacuum and ventilate the vacuum manifold. The flow-through is collected in the QIAvac 24 Plus or the QIAvac 24 base.* Flow-through contains Buffer RLT or Buffer RW1 and is therefore not compatible with bleach. See page 6 for safety information.Animal Cells Vacuum/Spin32RNeasy Mini Handbook 06/201212. Add 500 ?l Buffer RPE to each RNeasy spin column. 13. Switch on the vacuum. Apply vacuum until transfer is complete. Switch off the vacuum and ventilate the vacuum manifold. The flow-through is collected in the QIAvac 24 Plus or the QIAvac 24 base. 14. Remove the RNeasy spin columns from the vacuum manifold, and place each in a 2 ml collection tube (supplied). Close the lids gently, and centrifuge at full speed for 1 min. 15. Place each RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 30C50 ?l RNase-free water directly to each spin column membrane. Close the lids gently, and centrifuge for 1 min at ≥8000 x g (≥10,000 rpm) to elute the RNA. 16. If the expected RNA yield is &30 ?g, repeat step 15 using another 30C50 ?l RNasefree water, or using the eluate from step 15 (if high RNA concentration is required). Reuse the collection tubes from step 15. If using the eluate from step 15, the RNA yield will be 15C30% less than that obtained using a second volume of RNase-free water, but the final RNA concentration will be higher.Animal Cells Vacuum/SpinRNeasy Mini Handbook 06/201233Protocol: Stabilization of RNA in Harvested Animal TissuesThis protocol describes how to stabilize and store human and animal tissues in RNAlater RNA Stabilization Reagent, included in the RNeasy Protect Mini Kit. For RNA purification from stabilized tissues, see “Protocol: Purification of Total RNA from Animal Tissues”, page 37. Important notes about RNAlater RNA Stabilization Reagent RNA in harvested animal tissue is not protected until the tissue is completely submerged in a sufficient volume of RNAlater RNA Stabilization Reagent. After harvesting, the tissue should be immediately placed in at least 10 volumes of the reagent (or approximately 10 ?l reagent per 1 mg tissue). Larger volumes can be used if necessary or desired. Smaller volumes may lead to RNA degradation during storage. Storage containers should be wide enough so that the reagent covers the entire tissue. Storage containers or tubes with large diameters may require more reagent to completely cover the tissue. The procedures for tissue harvesting and RNA stabilization should be carried out as quickly as possible. Tissue size is critical for successful RNA stabilization with RNAlater RNA Stabilization Reagent. Immediately upon contact, the reagent diffuses into the surface layer and outer portions of solid tissues. To ensure rapid and reliable stabilization of RNA even in the inner parts of solid tissues, the sample must be cut into slices less than 0.5 cm thick. The slices can be any convenient size, provided one dimension of the sample is &0.5 cm. If the slices are thicker than 0.5 cm, the reagent will diffuse too slowly into the interior of the sample and RNA degradation will occur. Small organs such as rat kidney and spleen or most mouse organs (except liver) do not require slicing: the entire organ can be placed in RNAlater RNA Stabilization Reagent. The following guide may help you to determine the amount of RNAlater RNA Stabilization Reagent required for RNA stabilization: A cube of rat kidney with a 5 mm edge length ([5 mm]3 = 125 mm3 = 125 ?l) weighs 150C175 mg and requires at least 1.5C1.75 ml of the reagent. A 3 mm cube ([3 mm]3 = 27 mm3 = 27 ?l) of most animal tissues weighs 30C35 mg and requires at least 300C350 ?l of the reagent. Although weighing tissues is generally more accurate, RNA in unstabilized tissues will degrade during weighing. In some cases, however, it may be more convenient to quickly estimate the weight of tissue pieces. Average weights of various entire adult mouse organs and the corresponding amounts of RNAlater RNA Stabilization Reagent to use are given in Table 7.Stabilization34RNeasy Mini Handbook 06/2012Table 7. Tissue weights and amounts of RNAlater RNA Stabilization Reagent Amount of RNAlater RNA Stabilization Reagent (ml)Mouse organ Kidney Spleen Lung Heart Liver Important points before startingWeight (mg) 180C250 100C160 190C210 100C170 ≥2.5 ≥1.6 ≥2.1 ≥1.7 ≥18If using the RNeasy Protect Mini Kit for the first time, read “Important Notes” (page 16). RNAlater RNA Stabilization Reagent may form a precipitate during storage below room temperature (15C25°C). Before using the reagent, redissolve the precipitate by heating to 37°C with agitation. Only fresh, unfrozen tissues can be stabilized using RNAlater RNA Stabilization Reagent. Previously frozen tissues thaw too slowly in the reagent, preventing the reagent from diffusing into the tissues quickly enough to prevent RNA degradation. Procedure 1. 2. Before excising the tissue sample, estimate the volume (or weight) of the sample to be stabilized in RNAlater RNA Stabilization Reagent. Determine the appropriate volume of RNAlater RNA Stabilization Reagent for preserving the tissue. At least 10 volumes of the reagent (or approximately 10 ?l reagent per 1 mg of tissue) is required. Pipet the correct amount of reagent into an appropriate collection vessel. Note: Be sure to completely submerge the tissue in RNAlater RNA Stabilization Reagent. For details, see “Important notes about RNAlater RNA Stabilization Reagent”, above. 3. Excise the tissue sample from the animal and, if necessary, cut it into slices less than 0.5 cm thick. Perform this step as quickly as possible and proceed immediately to step 4. Note: For effective RNA stabilization, the tissue sample must be less than 0.5 cm thick. For details, see “Important notes about RNAlater RNA Stabilization Reagent”, above.StabilizationRNeasy Mini Handbook 06/2012354.Completely submerge the tissue piece(s) in the collection vessel containing RNAlater RNA Stabilization Reagent from step 2. Note: The tissue sample must be immediately submerged in RNAlater RNA Stabilization Reagent to protect the RNA.5.Store the tissue submerged in RNAlater RNA Stabilization Reagent for up to 4 weeks at 2C8°C, up to 7 days at 15C25°C, or up to 1 day at 37°C. For archival storage at C20°C, first incubate the tissue overnight in the reagent at 2C8°C. Then transfer the tissue, in the reagent, to C20°C for storage. For archival storage at C80°C, first incubate the tissue overnight in the reagent at 2C8°C. Then remove the tissue from the reagent, and transfer it to C80°C for storage. Note: Lower temperatures are recommended for longer storage (e.g., 2C8°C for up to 4 weeks instead of 37°C or room temperature (15C25°C); C20°C or C80°C for longer storage). Tissues stored in RNAlater RNA Stabilization Reagent at C20°C may not freeze. The low temperature may cause the formation of crystals or a precipitate in the reagent. This will not affect subsequent RNA purification. There is no need to redissolve the precipitate. RNAlater stabilized tissues stored at C20°C or C80°C can be thawed at room temperature and frozen again for up to 20 freezeCthaw cycles without affecting RNA quality or yield. If transporting tissue samples in RNAlater RNA Stabilization Reagent, ensure that the tissues always remain submerged in the reagent. Either keep the tubes upright during transport or fill the tubes completely with RNAlater RNA Stabilization Reagent.Stabilization6.After storage, continue with “Protocol: Purification of Total RNA from Animal Tissues” (page 37).36RNeasy Mini Handbook 06/2012Protocol: Purification of Total RNA from Animal TissuesThis protocol requires the RNeasy Mini Kit or RNeasy Protect Mini Kit. Determining the correct amount of starting material It is essential to use the correct amount of starting material to obtain optimal RNA yield and purity. A maximum amount of 30 mg fresh or frozen tissue or 15C20 mg RNAlater stabilized tissue (which is partially dehydrated) can generally be processed. For most tissues, the RNA binding capacity of the RNeasy spin column and the lysing capacity of Buffer RLT will not be exceeded by these amounts. Average RNA yields from various tissues are given in Table 2 (page 17). Some tissues, such as spleen, parts of brain, lung, and thymus are more difficult to lyse or tend to form precipitates during RNA purification. The volume of Buffer RLT may need to be increased to facilitate complete homogenization and to avoid significantly reduced RNA yields, DNA contamination, or clogging of the RNeasy spin column. See the procedure below for details. RNA yields from fibrous tissues, such as skeletal muscle, heart, and skin, may be low due to the abundance of contractile proteins, connective tissue, and collagen. For maximum RNA yields from these tissues, we recommend using the RNeasy Fibrous Tissue Mini Kit instead. See page 74 for ordering information. Greater RNA yields from fatty tissues, such as brain }