RNAprotect® Cell Reagent Handbook
Contents
1. DNase RNase Free Reagents and Buffers To be used in conjunction with PAXgene Blood RNA Tubes PAXgene Bone Marrow RNA System for storage and transport of bone marrow samples and stabilization and purification of intracellular RNA PAXgene Bone Marrow 50 Bone Marrow Collection Tubes 764114 RNA Tubes 50 To be used in conjunction with the PAXgene Bone Marrow RNA Kit PAXgene Bone Marrow For 30 RNA preps 30 PAXgene 764133 RNA Kit 30 Spin Columns 30 PAXgene Shredder Spin Columns Processing Tubes RNase Free DNase RNase Free Reagents and Buffers To be used in conjunction with PAXgene Bone Marrow RNA Tubes Larger kit format available see www giagen com RNA PAXgene Blood RNA Tubes can be ordered from BD and BD authorized distributors www bd com Canada and USA only Rest of the world kit not available in all countries 38 RNAprotect Cell Reagent Handbook 10 2010 Ordering Information Product Contents Cat no QuantiTect Reverse Transcription Kit for fast cDNA synthesis for sensitive real time two step RT PCR QuantiTect Reverse For 50 x 20 yl reactions gDNA 205311 Transcription Kit 50 Wipeout Buffer Quantiscript Reverse Transcriptase Quantiscript RT Buffer RT Primer Mix RNase Free Water QuantiTect Primer Assays for use in real time RT PCR with SYBR Green detection search for and order assays at www giagen com GeneGlobe QuantiTect Primer Assay 200 For 200 x 50 pl2. High quality RNA is then eluted in 30 pl or more of RNase free water With the RNeasy Plus Mini Kit 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 15 20 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 The RNeasy Plus Mini Kit provides RNA purification from up to 1 x 10 cells For very small cell samples we recommend using the RNeasy Plus Micro Kit see page 36 for ordering information which enables RNA purification from up to 5 x 105 cells with an elution volume of as low as 10 yl Description of protocols Stabilization of Nucleic Acids in Sorted or Cultured Cells Using RNAprotect Cell Reagent This protocol describes how to add RNAprotect Cell Reagent to sorted or cultured cells for immediate simultaneous stabilization of RNA and DNA Purification of RNA from RNAprotect Stabilized Cells Using the RNeasy Plus Mini Kit This protocol describes how to purify RNA from sorted or cultured cells that have been stabilized in RNAprotect Cell Reagent The protocol requires use of the RNeasy Plus Mini Kit included in the RNeasy Protect Cell Mini Kit If purifying RNA from very small cell samples using the RNeasy Plus Micro Kit see pag
3. 2010 Storage Store RNAprotect Cell Reagent at room temperature 15 25 C The reagent is stable for at least 12 months under these conditions The RNeasy Plus Mini Kit should be stored dry at room temperature 15 25 C and is stable for at least 9 months under these conditions Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of RNAprotect Cell Reagent and RNeasy Protect Cell Mini Kit is tested against predetermined specifications to ensure consistent product quality Product Use Limitations The RNAprotect Cell Reagent and the RNeasy Protect Cell Mini Kit are intended for molecular biology applications These products are 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 GIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines Product Warranty and Satisfaction Guarantee GIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its
4. 50 minipreps DNeasy Mini 69504 Spin Columns Proteinase K Buffers Collection Tubes DNeasy Blood amp Tissue For 250 minipreps DNeasy Mini 69506 Kit 250 Spin Columns Proteinase K Buffers Collection Tubes GlAamp DNA Mini Kit for purification of genomic mitochondrial bacterial parasite or viral DNA from human samples GlAamp DNA Mini Kit 50 For 50 minipreps GlAamp Mini 51304 Spin Columns Proteinase K Buffers Collection Tubes 36 RNAprotect Cell Reagent Handbook 10 2010 Ordering Information Product Contents Cat no GlAamp DNA Mini Kit 250 For 250 minipreps GlAamp Mini 51306 Spin Columns Proteinase K Buffers Collection Tubes Related products for sample stabilization and purification Allprotect Tissue Reagent for immediate stabilization of DNA RNA and protein in animal and human tissues Allprotect Tissue Reagent 100 ml Allprotect Tissue Reagent 76405 100 ml Allprotect Reagent Pump RNAlater RNA Stabilization Reagent for immediate stabilization of the gene expression profile in harvested tissues RNAlater RNA Stabilization For stabilization of RNA in 76104 Reagent 50 ml 25 x 200 mg tissue samples 50 ml RNAlater RNA Stabilization Reagent RNAlater TissueProtect Tubes for collecting harvested tissues with immediate stabilization of the gene expression profile and subsequent transport and storage RNAlater TissueProtect Tubes For stabilization of RNA in 76154 50 x 1 5 ml 50 x 150 mg tissue
5. RLT Plus or Buffer RW1 and is therefore not compatible with bleach See page 6 for safety information 20 RNAprotect Cell Reagent Handbook 10 2010 13 Place the RNeasy spin column in a new 1 5 ml collection tube supplied Add 30 50 pl RNase free water directly to the spin column membrane Close the lid gently and centrifuge for 1 min at 28000 x g 210 000 rpm to elute the RNA 14 If the expected RNA yield is gt 30 pg repeat step 13 using another 30 50 pl of RNase free water or using the eluate from step 13 if high RNA concentration is required Reuse the collection tube from step 13 If using the eluate from step 13 the RNA yield will be 15 30 less than that obtained using a second volume of RNase free water but the final RNA concentration will be higher For RT PCR and real time RT PCR with the purified RNA QIAGEN offers a range of optimized ready to use kits that provide highly specific and sensitive results For details visit www giagen com PCR ro z Y e E EI RNAprotect Cell Reagent Handbook 10 2010 21 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www qiagen com Comments and suggest
6. RNAprotect Cell Reagent can be added directly to cells adherent or suspension in medium or storage solution e g PBS Alternatively the reagent can be added to cells not in medium or storage solution such as pelleted cells n e a E Q fo E Important points before starting BE f using RNAprotect Cell Reagent for the first time read Important Notes page 13 If DNA or DNA RNA will be purified refer also to the relevant section in the DNeasy GlAamp or AllPrep handbook on determining the amount of starting material M Nucleic acids in cells are not protected until the cells are mixed with a sufficient volume of RNAprotect Cell Reagent The reagent should be added immediately to pelleted cells or should be added directly to cells in medium or storage solution E Be sure to mix cells with a sufficient volume of RNAprotect Cell Reagent as described in the procedure below Smaller volumes may lead to nucleic acid degradation during storage and reduced RNA and DNA yields Larger volumes can be used if necessary or desired E if adding RNAprotect Cell Reagent directly to cells that are in a large volume of medium or storage solution the pelleting of the cells for nucleic acid purification page 17 31 or 33 may be incomplete leading to reduced RNA and DNA yields When processing high numbers of cells we recommend a volume of 1 ml or less for the medium or storage solution In general we recommed lower volumes e
7. at www giagen com MyQlAcube a m o 10 RNAprotect Cell Reagent Handbook 10 2010 RNeasy Protect Cell Procedure Cells Mix with RNAprotect Cell Reagent Dissolve pellet in Buffer RLT Plus Remove genomic DNA Genomic DNA Total RNA 1 T ET l 1 Total Bind total RNA RNA Wash Elute e 1 e Te RNAprotect Cell Reagent Handbook 10 2010 11 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier M Sterile RNase free pipet tips E Centrifuge and appropriately sized centrifuge tubes for centrifugation of stabilized cells E Microcentrifuge with rotor for 2 ml tubes Bb Vortexer E Ethanol 70 and 96 100 E Disposable gloves E Equipment for cell homogenization we recommend either GlAshredder homogenizers or the TissueRuptor with TissueRuptor Disposable Probes see ordering information page 35 E Optional 14 3 M B mercaptoethanol ME commercially available solutions are usually 14 3 M Do not use denatured alcohol which contains other substances such as methanol or methylethylketone 12 RNAprotect Cell Reagent Handbook 10 2010 Important Notes Determining the amount of starting material It is essential to use the correct amount of starting material in orde
8. cause RNA degradation Proceed to step 5 Table 3 Volumes of Buffer RLT Plus for lysing pelleted cells Number of pelleted cells Volume of Buffer RLT Plus 5 x 10 350 yl 5 x 10 1 x 10 600 pl 5 Proceed immediately with homogenization and DNA and RNA purification accord 32 ing to the cell protocol in the AllPrep handbook RNAprotect Cell Reagent Handbook 10 2010 Appendix D Preparing RNAprotect Stabilized Cells for DNA Purification This specialized protocol describes how to prepare cultured or sorted cells stabilized in RNAprotect Cell Reagent for the purification of DNA The purification procedure requires the use of the DNeasy Blood amp Tissue Kit QlAamp DNA Mini Kit or other similar GIAGEN kit for genomic DNA purification from animal or human cells see page 36 for ordering information Important points before starting E f using the DNeasy Kit or GlAamp Kit for the first time carefully read the handbook supplied with the kit especially the Safety Information and Important Notes sections and determine the appropriate number of cells to be processed E Buffer AL supplied with the DNeasy or GlAamp Kit contains a guanidine salt and is therefore not compatible with disinfecting reagents containing bleach See the DNeasy or GlAamp handbook for safety information BP The first step of the procedure requires the use of PBS 50 mM potassium phosphate 150 mM NaCl pH 7 2 E The fourth step of
9. or buffer providing instantaneous RNA stabilization The reagent can also be used to treat cells not in medium or storage solution such as pelleted cells After stabilization in RNAprotect Cell Reagent cells can be conveniently stored and transported at ambient temperature with the cellular RNA remaining intact and undegraded RNAprotect Cell Reagent also enables stabilization of DNA providing simultaneous stabilization of RNA and DNA in the same cell sample RNA purification using the RNeasy Plus Mini Kit The RNeasy Plus Mini Kit integrates QIAGEN s patented technology for selective removal of double stranded DNA with well established RNeasy technology Efficient purification of high quality RNA is guaranteed without the need for additional DNase digestion 8 RNAprotect Cell Reagent Handbook 10 2010 Cells stabilized in RNAprotect Cell Reagent are centrifuged and the resulting pellet is lysed and homogenized in a highly denaturing guanidine thiocyanate containing buffer which immediately inactivates RNases to ensure isolation of intact RNA The lysate is then passed through a gDNA Eliminator spin column This column in combination with the optimized high salt buffer allows efficient removal of genomic DNA Ethanol is added to the flow through to provide appropriate binding conditions for RNA and the sample is then applied to an RNeasy spin column where total RNA binds to the membrane and contaminants are efficiently washed away
10. performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www qiagen com RNAprotect Cell Reagent Handbook 10 2010 5 Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of GIAGEN products If you have any questions or experience any difficulties regarding RNAprotect Cell Reagent the RNeasy Protect Cell Mini Kit or QIAGEN products in general please do not hesitate to contact us GIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about produ
11. pg ml When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 28 RNAprotect Cell Reagent Handbook 10 2010 Total amount concentration x volume in milliliters 440 pg ml x 0 1 ml 44 pg of RNA Purity of RNA The ratio of the readings at 260 nm and 280 nm A 4 A provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV spectrum such as protein However the A Azs ratio is influenced considerably by pH Since water is not buffered the pH and the resulting A A ratio can vary greatly Lower pH results in a lower A o A so ratio and reduced sensitivity to protein contamination For accurate values we recommend measuring absorbance in 10 mM Tris Cl pH 7 5 Pure RNA has an A 4 Asso ratio of 1 9 2 1 in 10 mM Tris Cl pH 7 5 Always be sure to calibrate the spectrophotometer with the same solution used for dilution For determination of RNA concentration however we recommend dilution of the sample in a buffer with neutral pH since the relationship between absorbance and concentration Azs reading of 1 44 pg ml RNA is based on an extinction coefficient calculated for RNA at neutral pH see Spectrophotometric quantification of RNA page 28 DNA contamination No currently available purification method can guarantee
12. samples 50 screw top tubes containing 1 5 ml RNAlater RNA Stabilization Reagent each RNeasy Protect Mini Kit for immediate stabilization of the gene expression profile in tissues and subsequent RNA purification RNeasy Protect Mini Kit 50 RNAlater RNA Stabilization Reagent 74124 50 ml 50 RNeasy Mini Spin Columns Collection Tubes RNase Free Reagents and Buffers RNAprotect Bacteria Reagent for in vivo stabilization of the gene expression profile in bacteria RNAprotect Bacteria Reagent RNAprotect Bacteria Reagent 76506 2 x 100 ml Larger kit size and or format available see www qiagen com RNA RNAprotect Cell Reagent Handbook 10 2010 37 Ordering Information Product Contents Cat no RNeasy Protect Bacteria Mini Kit for in vivo stabilization of the gene expression profile in bacteria and subsequent RNA purification RNeasy Protect Bacteria RNeasy Mini Kit 50 and 74524 Mini Kit 50 RNAprotect Bacteria Reagent 2 x 100 ml RNeasy Protect Saliva Mini Kit for immediate stabilization of RNA in saliva and subsequent total RNA purification RNeasy Protect Saliva RNAprotect Saliva Reagent 50 ml 74324 Mini Kit 50 and RNeasy Micro Kit 50 PAXgene Blood RNA Kit for isolation and purification of intracellular RNA from whole blood stabilized in PAXgene Blood RNA Tubes PAXgene Blood RNA Kit 50 50 PAXgene Spin Columns 762164 50 PAXgene Shredder Spin Columns 762174 Processing Tubes RNase Free
13. should appear as sharp bands or peaks The apparent ratio of 28S rRNA to 18S RNA should be approximately 2 1 If the ribosomal bands or peaks of a specific sample are not sharp but appear as a smear towards smaller sized RNAs it is likely that the sample suffered major degradation either before or during RNA purification When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 30 RNAprotect Cell Reagent Handbook 10 2010 Appendix C Preparing RNAprotect Stabilized Cells for Simultaneous DNA and RNA Purification This specialized protocol describes how to prepare cultured or sorted cells stabilized in RNAprotect Cell Reagent for the simultaneous purification of DNA and RNA The purification procedure requires the use of an AllPrep Kit such as the AllPrep DNA RNA Micro Kit or AllPrep DNA RNA Mini Kit see page 36 for ordering information Separate eluates of RNA and DNA are simultaneously purified from the same sample Important points before starting E f using the AllPrep Kit for the first time carefully read the handbook supplied with the kit especially the Safety Information and Important Notes sections and determine the appropriate number of cells to be processed M Buffer RIT Plus supplied with the AllPrep Kit contains a guanidine salt and is therefore not c
14. that RNA is completely free of DNA even when it is not visible on an agarose gel While the RNeasy Plus Mini Kit will remove the vast majority of cellular DNA trace amounts may still remain depending on the amount and nature of the sample For analysis of very low abundance targets any interference by residual DNA contamination can be detected by performing real time RT PCR control experiments in which no reverse transcriptase is added prior to the PCR step To prevent any interference by DNA in real time RT PCR applications such as with Applied Biosystems and LightCycler instruments we recommend designing primers that anneal at intron splice junctions so that genomic DNA will not be amplified QuantiTect Primer Assays from QIAGEN are designed for SYBR Green based real time RT PCR analysis of RNA sequences without detection of genomic DNA where possible Wilfinger W W Mackey M and Chomczynski P 1997 Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity BioTechniques 22 474 Values up to 2 3 are routinely obtained for pure RNA in 10 mM Tris Cl pH 7 5 with some spectrophotometers RNAprotect Cell Reagent Handbook 10 2010 29 Integrity of RNA The integrity and size distribution of total RNA purified with RNeasy Kits can be checked by denaturing agarose gel electrophoresis and ethidium bromide staining or by using an Agilent 2100 bioanalyzer The respective ribosomal RNAs
15. 0 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 080 000 7146 Fax 02 2626 5703 Technical 080 000 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1 122 330 Technical 01 800 7742 436 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 1800 742 4362 Fax 65 6854 8184 Technical 1800 742 4368 Spain Orders 91 630 7050 Fax 91 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN Sample amp Assay Technologies
16. A below For small amounts of RNA however it may be difficult to determine amounts photometrically Small amounts of RNA can be accurately quantified using an Agilent 2100 bioanalyzer quantitative RT PCR or fluorometric quantification Spectrophotometric quantification of RNA To ensure significance Aygo readings should be greater than 0 15 An absorbance of 1 unit at 260 nm corresponds to 44 pg of RNA per ml Ayo 1 44 pg ml This relation is valid only for measurements at a neutral pH Therefore if it is necessary to dilute the RNA sample this should be done in a buffer with neutral pH As discussed below see Purity of RNA page 29 the ratio between the absorbance values at 260 and 280 nm gives an estimate of RNA purity When measuring RNA samples be certain that cuvettes are RNase free especially if the RNA is to be recovered after spectrophotometry This can be accomplished by washing cuvettes with 0 1 M NaOH 1 mM EDTA followed by washing with RNase free water see Solutions page 27 Use the buffer in which the RNA is diluted to zero the spectrophotometer An example of the calculation involved in RNA quantification is shown below Volume of RNA sample 100 pl Dilution 10 pl of RNA sample 490 pl of 10 mM Tris Cl pH 7 0 1 50 dilution Measure absorbance of diluted sample in a 1 ml cuvette RNase free A 503 Concentration of RNA sample 44 pg ml x Azo x dilution factor 44 pg ml x 0 2 x 50 440
17. A 26 Appendix B Storage Quantification and Determination of Quality of RNA 28 Appendix C Preparing RNAprotect Stabilized Cells for Simultaneous DNA and RNA Purification 31 Appendix D Preparing RNAprotect Stabilized Cells for DNA Purification 33 Ordering Information 35 RNAprotect Cell Reagent Handbook 10 2010 3 Kit Contents RNAprotect Cell Reagent 250 ml Catalog no 76526 RNAprotect Cell Reagent 250 ml RNAprotect Cell Reagent Handbook 1 RNeasy Protect Cell Mini Kit 50 Catalog no 74624 Number of preps 50 RNAprotect Cell Reagent box 1 of 2 RNAprotect Cell Reagent 50 ml RNAprotect Cell Reagent Handbook 1 RNeasy Plus Mini Kit box 2 of 2 B gDNA Eliminator Mini Spin Columns uncolored each in a 2 ml Collection Tube 50 E RNeasy Mini Spin Columns pink each in a 2 ml Collection Tube 50 HM Collection Tubes 1 5 ml 50 E Collection Tubes 2 ml 50 BE Buffer RIT Plus 45 ml E Buffer RW1 45 ml Buffer RPE concentrate 11 ml E RNase Free Water 10 ml M RNeasy Plus Mini Handbook 1 Follow the instructions in the RNAprotect Cell Reagent Handbook when stabilizing and purifying RNA from cells 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 6 100 as indicated on the bottle to obtain a working solution 4 RNAprotect Cell Reagent Handbook 10
18. NA Eliminator spin column is exceeded the purified RNA will be contaminated with DNA Although the gDNA Eliminator spin column can bind more than 100 pg DNA we recommend using samples containing less than 20 pg DNA to ensure removal of virtually all genomic DNA Counting cells is the most accurate way to quantitate the amount of starting material As a guide Table 1 shows the number of Hela cells obtained in various culture vessels after confluent growth RNAprotect Cell Reagent Handbook 10 2010 13 Table 1 Growth area and number of HeLa cells in various culture vessels Cell culture vessel Growth area cm Number of cells Multiwell plates B 96well 0 32 0 6 4 5 x 104 48 well eae B 24 well 2 2 5 x 105 B 12 well 4 Sx OP B well 9 5 1x 10 Dishes E 35mm 8 1 x 10 E 60mm 21 2 S9 IO B 100mm 56 7 x 106 M 145 150 mm 145 2o Flasks B 40 50 ml 25 3 x 10 B 250 300 ml TS Jd ss OA B 650 750 ml 162 175 2 x 10 Per well if multiwell plates are used varies slightly depending on the supplier t Cell numbers are given for HeLa cells approximate length 15 pm assuming confluent growth Cell numbers will vary for different kinds of animal cells which vary in length from 10 to 30 pm 14 RNAprotect Cell Reagent Handbook 10 2010 Protocol Stabilization of Nucleic Acids in Sorted or Cultured Cells Using RNAprotect Cell Reagent This protocol describes how to stabilize RNA and DNA in different cell formats
19. Second Edition October 2010 RNAprotect Cell Reagent Handbook RNAprotect Cell Reagent For immediate stabilization of nucleic acids in cells RNeasy Protect Cell Mini Kit For immediate stabilization of nucleic acids in cells and subsequent total RNA purification QIAGEN Trademarks QIAGEN GlAamp GlAcube AllPrep Allprotect DNeasy MinElute Quantiscript QuantiTect RNAprotect RNeasy TissueRuptor QIAGEN Group Applied Biosystems Applera Corporation or its subsidiaries Agilent Agilent Technologies Inc LightCycler Roche Group PAXgene PreAnalytiX GmbH SYBR Molecular Probes Inc RNA ater is a trademark of AMBION Inc Austin Texas and is covered by various U S and foreign patents 2006 2010 QIAGEN all rights reserved Contents Kit Contents 4 Storage 5 Quality Control 5 Product Use Limitations 5 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 6 Safety Information 6 Introduction 8 Principle and procedure 8 Description of protocols 9 Automated purification 10 Equipment and Reagents to Be Supplied by User 12 Important Notes 13 Determining the amount of starting material 13 Protocols Stabilization of Nucleic Acids in Sorted or Cultured Cells Using RNAprotect Cell Reagent 15 H Purification of RNA from RNAprotect Stabilized Cells Using the RNeasy Plus Mini Kit 17 Troubleshooting Guide 22 Appendix A General Remarks on Handling RN
20. arryover during elution Ensure that Buffer RPE is at 20 30 C When reusing collection tubes between washing steps remove residual flow through from the rim by blotting on clean paper towels 24 RNAprotect Cell Reagent Handbook 10 2010 Comments and suggestions b Ethanol carryover During the second wash with Buffer RPE be sure to centrifuge at 28000 x g 210 000 rpm for 2 min at 20 25 C to dry the RNeasy spin column membrane After centrifugation carefully remove the column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur Perform the optional centrifugation to dry the RNeasy spin column membrane if any flow through is present on the outside of the column step 12 of the second protocol page 17 RNAprotect Cell Reagent Handbook 10 2010 25 Appendix A General Remarks on Handling RNA Handling RNA Ribonucleases RNases are very stable and active enzymes that generally do not require cofactors to function Since RNases are difficult to inactivate and even minute amounts are sufficient to destroy RNA do not use any plasticware or glassware without first eliminating possible RNase contamination Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure In order to create and maintain an RNase free environment the following precautions must be taken during pretreatment and use of
21. bes for use with the TissueRuptor Buffer AL 216 ml 216 ml lysis buffer for use in DNA 19075 purification GIAGEN Proteinase K 2 ml 2 ml proteinase K solution 19131 2600 mAU ml for use in DNA purification QIAGEN Proteinase K 10 ml 10 ml proteinase K solution 19133 gt 600 mAU ml for use in DNA purification RNAprotect Cell Reagent Handbook 10 2010 35 Ordering Information Product Contents Cat no RNase A 17 500 U 2 5 ml RNase A solution 19101 100 mg ml 7000 units ml Related products for sample purification RNeasy Plus Micro Kit for purification of total RNA from small cell and tissue samples using gDNA Eliminator columns RNeasy Plus Micro Kit 50 For 50 micropreps RNeasy 74034 MinElute Spin Columns gDNA Eliminator Spin Columns Collection Tubes Carrier RNA RNase Free Water and Buffers AllPrep DNA RNA Kits for simultaneous purification of genomic DNA and total RNA from the same cell or tissue sample AllPrep DNA RNA For 50 micropreps AllPrep DNA 80284 Micro Kit 50 Spin Columns RNeasy MinElute Spin Columns Collection Tubes Carrier RNA RNase Free Water and Buffers AllPrep DNA RNA For 50 minipreps AllPrep DNA 80204 Mini Kit 50 Spin Columns RNeasy Mini Spin Columns Collection Tubes RNase Free Water and Buffers DNeasy Blood amp Tissue Kit for purification of total DNA from animal blood and tissues and from cells yeast bacteria or viruses DNeasy Blood amp Tissue Kit 50 For
22. ct performance or new applications and techniques For technical assistance and more information please see our Technical Support Center at www giagen com goto TechSupportCenter or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www qiagen com J Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www giagen com Support MSDS aspx where you can find view and print the MSDS for each GIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Buffer RIT Plus contains guanidine thiocyanate and Buffer RW contains a small amount of guanidine thiocyanate This chemical can form highly reactive compounds when combined with bleach If liquid containing these buffers is spilt 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 176 v v sodium hypochlorite 6 RNAprotect Cell Reagent Handbook 10 2010 The following risk and safety phrases apply to RNAprotect Cell Reagent and the components of the RNeasy Protect Cell Mini Kit RNAprotect Cell Reagent Contai
23. disposable and nondisposable vessels and solutions while working with RNA General handling Proper microbiological aseptic technique should always be used when working with RNA Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin or from dusty laboratory equipment Change gloves frequently and keep tubes closed whenever possible Keep purified RNA on ice when aliquots are pipetted for downstream applications Disposable plasticware The use of sterile disposable polypropylene tubes is recommended throughout the procedure These tubes are generally RNase free and do not require pretreatment to inactivate RNases Nondisposable plasticware Nondisposable plasticware should be treated before use to ensure that it is RNase free Plasticware should be thoroughly rinsed with 0 1 M NaOH 1 mM EDTA followed by RNase free water see Solutions page 27 Alternatively chloroform resistant plasticware can be rinsed with chloroform to inactivate RNases When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 26 RNAprotect Cell Reagent Handbook 10 2010 Glassware Glassware should b
24. e 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 emply the collection tube completely 10 Add 500 pl Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm to wash the spin column membrane Discard the flow through Reuse the collection tube in step 11 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 Add 500 pl Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 2 min at gt 8000 x g 210 000 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 12 Optional Place the RNeasy spin column in a new 2 ml collection tube supplied and discard the old collection tube with the flow through 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 11 Flow through contains Buffer
25. e 36 for ordering information follow the cell protocol in the RNeasy Plus Micro Handbook RNAprotect Cell Reagent Handbook 10 2010 9 Specialized protocols The appendices contain specialized protocols for preparing sorted or cultured cells stabilized in RNAprotect Cell Reagent for subsequent DNA purification Appendix C page 31 describes how to prepare RNAprotect stabilized cells for simultaneous purification of DNA and RNA using an AllPrep Kit Appendix D page 33 describes how to prepare RNAprotect stabilized cells for DNA purification using a DNeasy Kit or GlAamp Kit Automated purification Purification of RNA can be fully automated on the GlAcube The innovative QlAcube 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 QlAcube follows the same steps as the manual procedure i e lyse bind wash and elute enabling you to continue using the RNeasy Plus 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 MyGlAcube The QlAcube 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
26. e treated before use to ensure that it is RNase free Glassware used for RNA work should be cleaned with a detergent thoroughly rinsed and oven baked at 240 C for at least 4 hours overnight if more convenient before use Autoclaving alone will not fully inactivate many RNases Alternatively glassware can be treated with DEPC diethyl pyrocarbonate Fill glassware with 0 1 DEPC 0 1 in water allow to stand overnight 12 hours at 37 C and then autoclave or heat to 100 C for 15 minutes to eliminate residual DEPC Electrophoresis tanks Electrophoresis tanks should be cleaned with detergent solution e g 0 5 SDS thoroughly rinsed with RNase free water and then rinsed with ethanol and allowed to dry Solutions Solutions water and other solutions should be treated with 0 1 DEPC DEPC is a strong but not absolute inhibitor of RNases It is commonly used at a concentration of 0 1 to inactivate RNases on glass or plasticware or to create RNase free solutions and water DEPC inactivates RNases by covalent modification Add 0 1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution Let the solution incubate for 12 hours at 37 C Autoclave for 15 minutes to remove any trace of DEPC DEPC will react with primary amines and cannot be used directly to treat Tris buffers DEPC is highly unstable in the presence of Tris buffers and decomposes rapidly into ethanol and CO When preparin
27. ential that the sample and Buffer AL are mixed immediately and thoroughly by vortexing or pipetting to yield a homogeneous solution 6 Proceed with adding ethanol 200 pl and applying the sample to the DNeasy or QlAamp spin column according to the blood protocol in the DNeasy or GlAamp handbook 34 RNAprotect Cell Reagent Handbook 10 2010 Ordering Information Product Contents Cat no RNAprotect Cell Reagent 250 ml RNAprotect Cell Reagent 76526 250 ml RNeasy Protect Cell Mini RNAprotect Cell Reagent 50 ml 74624 Kit 50 and RNeasy Plus Mini Kit 50 RNeasy Plus Mini Kit 50 50 RNeasy Mini Spin Columns 74134 50 gDNA Eliminator Mini Spin Columns Collection Tubes RNase Free Reagents and Buffers Accessories Collection Tubes 2 ml 1000 x 2 ml Collection Tubes 19201 QlAshredder 50 50 disposable cell lysate 79654 homogenizers QlAshredder 250 250 disposable celllysate 79656 homogenizers TissueRuptor 115 V 50 60 Hz Handheld rotor stator homogenizer 9001271 115 V 50 60 Hz for North America and Japan TissueRuptor 235 V 50 60 Hz Handheld rotor stator homogenizer 9001272 235 V 50 60 Hz for Europe excluding UK and Ireland TissueRuptor 235 V 50 60 Hz Handheld rotor stator homogenizer 9001273 235 V 50 60 Hz for UK and Ireland TissueRuptor 235 V 50 60 Hz Handheld rotor stator homogenizer 9001274 235 V 50 60 Hz for Australia TissueRuptor Disposable 25 nonsterile plastic disposable 990890 Probes 25 pro
28. entrifuge tube for use in step 1 of the protocol on page 17 31 or 33 after adding the reagent 1b Pelleted cells or other cells not covered with solution e g smears Add 300 yl RNAprotect Cell Reagent to the cell pellet in a centrifuge tube not supplied Resuspend the cells completely by vortexing Note Be sure to add at least 5 volumes of RNAprotect Cell Reagent to the cell pellet If residual culture medium or buffer increases the overall volume to over 60 yl increase the amount of RNAprotect Cell Reagent accordingly Note Larger volumes of RNAprotect Cell Reagent can be added if desired 2 Store the mix of cells and RNAprotect Cell Reagent for up to 1 day at 30 C up to 7 days at room temperature 15 25 C or up to 4 weeks at 2 8 C or archive at 20 C or 80 C Note We recommend lower storage temperatures whenever possible e g 2 8 C instead of room temperature or room temperature instead of 30 C Note A precipitate may form during storage especially at lower temperatures This does not affect RNA and DNA purification 16 RNAprotect Cell Reagent Handbook 10 2010 Protocol Purification of RNA from RNAprotect Stabilized Cells Using the RNeasy Plus Mini Kit Disrupting and homogenizing starting material Efficient 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 Compl
29. ete disruption of plasma membranes of cells and organelles is absolutely required to release all the RNA contained in the sample 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 With the RNeasy procedure disruption of cultured cells is achieved by thorough vortexing in Buffer RLT Plus Homogenization is then carried out using either QlAshredder homogenizers or the TissueRuptor QlAshredder homogenizers provide a fast and efficient way to homogenize cell lysates without cross contamination of samples Up to 700 yl of lysate is loaded onto a QlAshredder spin column placed in a 2 ml collection tube and spun for 2 minutes at full speed in a microcentrifuge The lysate is homogenized as it passes through the spin column The TissueRuptor is a rotor stator homogenizer that can be used to homogenize cell lysates The blade of the TissueRuptor disposable probe rotates at a very high speed causing the sample to be homogenized by a combination of turbulence and mechanical shearing Be sure to use a suitably sized vessel to hold the sample and to kee
30. g Tris buffers treat water with DEPC first and then dissolve Tris to make the appropriate buffer Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation Carbethoxylated RNA is translated with very low efficiency in cell free systems However its ability to form DNA RNA or RNA RNA hybrids is not seriously affected unless a large fraction of the purine residues have been modified Residual DEPC must always be eliminated from solutions or vessels by autoclaving or heating to 100 C for 15 minutes Note RNeasy buffers are guaranteed RNase free without using DEPC treatment and are therefore free of any DEPC contamination When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier Plastics used for some electrophoresis tanks are not resistant to ethanol Take proper care and check the supplier s instructions RNAprotect Cell Reagent Handbook 10 2010 27 Appendix B Storage Quantification and Determination of Quality of RNA Storage of RNA Purified RNA may be stored at 20 C or 70 C in RNase free water Under these conditions no degradation of RNA is detectable after 1 year Quantification of RNA The concentration of RNA should be determined by measuring the absorbance at 260 nm A 4 in a spectrophotometer see Spectrophotometric quantification of RN
31. g in an appropriately sized centrifuge tube IF transferring the sample to the centrifuge tube from a storage vessel be sure to resuspend any material deposited at the bottom of the vessel by vortexing or by pipetting up and down It is important to transfer all sample material to the centrifuge tube otherwise DNA and RNA yields will be reduced Note If the sample was stored at below room temperature e g 20 C thaw it completely before starting centrifugation Note A precipitate may form during storage especially at lower temperatures This does not affect DNA and RNA purification Remove the supernatant completely by pipetting Loosen the pellet by flicking the tube Loosening the pellet facilitates dissolving in Buffer RLT Plus in step 4 Add 350 pl or 600 pl Buffer RLT Plus see Table 3 Dissolve the pellet completely by vortexing If necessary add ME to Buffer RIT Plus before use see Things to do before starting Note Be sure to dissolve the pellet completely This can take about 1 min Incomplete dissolving may lead to inefficient lysis and reduced DNA and RNA yields Note The dissolved pellet may be turbid This does not affect DNA and RNA purification The dissolved pellet can be stored at 70 C for several months After removal from storage incubate the dissolved pellet at room temperature or at 37 C in a water bath until completely thawed and salts are dissolved Avoid prolonged incubation at 37 C which can
32. he second protocol page 17 Be sure to remove any excess RNAprotect Cell Reagent to prevent significant dilution of Buffer RIT Plus Sample lysis will be impaired if the lysis buffer is diluted 23 Comments and suggestions g Cells in too large a volume of In subsequent preparations reduce the medium or storage solution volume of medium or storage solution e g PBS Low Aygo Aogo value Water used to dilute RNA for Use 10 mM Tris Cl pH 7 5 not RNase free Asso A280 measurement water to dilute the sample before measuring purity see Appendix B page 28 DNA contamination in downstream experiments a Cell number too high For some cell types the efficiency of DNA removal may be reduced when processing very high cell numbers containing more than 20 pg genomic DNA If the eluted RNA contains substantial DNA contamination try processing smaller cell numbers b Incomplete removal of Be sure to remove any excess RNAprotect RNAprotect Cell Reagent Cell Reagent to prevent significant dilution of Buffer RLT Plus The gDNA Eliminator spin column will not work effectively if the lysis buffer is diluted RNA concentration too low Elution volume too high Elute RNA with less than 2 x 50 pl of water Do not use less than 1 x 30 pl of water Although eluting with less than 2 x 50 pl of water results in increased RNA concentrations RNA yields may be reduced RNA does not perform well in downstream experiments a Salt c
33. ions Nucleic acids degraded a Cells not immediately stabilized b Prolonged storage c RNA degradation during RNA purification Clogged gDNA Eliminator spin column a Inefficient disruption and or homogenization b Too much starting material 22 Mix the cells immediately with RNAprotect Cell Reagent Cells mixed with RNAprotect Cell Reagent can be stored for up to 1 day at 30 C up to 7 days at 15 25 C or up to 4 weeks at 2 8 C or archived at 20 C or 80 C We recommend lower storage temperatures whenever possible Although all RNeasy buffers have been tested and are guaranteed RNase ree RNases can be introduced during use Be careful not to introduce any RNases during RNA purification or later handling See Appendix A page 26 for general remarks on handling RNA See Disrupting and homogenizing starting material page 17 for details on disruption and homogenization Increase g force and centrifugation time if necessary In subsequent preparations reduce the amount of starting material see page 13 and or increase the homogenization time Reduce the amount of starting material It is essential to use the correct amount of starting material see page 13 RNAprotect Cell Reagent Handbook 10 2010 Comments and suggestions c Centrifugation temperature too low Low RNA yield a Insufficient disruption and homogenization b Too much starting material c Ethanol added to ly
34. lection tube supplied Centrifuge for 30 s at 28000 x g 210 000 rpm Discard the column and save the flow through Note Make sure that no liquid remains on the column membrane after centrifugation If necessary repeat the centrifugation until all liquid has passed through the membrane Add 1 volume of 70 ethanol to the flow through and mix well by pipetting Do not centrifuge The volume of flow through may be less than 350 pl or 600 pl due to loss during homogenization and DNA removal Note When purifying RNA from certain cell lines precipitates may be visible after addition of ethanol This does not affect the procedure RNAprotect Cell Reagent Handbook 10 2010 19 L z gt y ch Ed 2 Q z 5 8 Transfer up to 700 pl 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 28000 x g 210 000 rpm Discard the flow through Reuse the collection tube in step 9 If the sample volume exceeds 700 yl centrifuge successive aliquots in the same RNeasy spin column Discard the flow through after each centrifugation 9 Add 700 pl Buffer RW1 to the RNeasy spin column Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm to wash the spin column membrane Discard the flow through Reuse the collection tube in step 10 c d o 3 4 az 5 a lt z 7 Not
35. ns tetradecyltrimethylammonium oxalate irritant dangerous for the environment Risk and safety phrases R43 51 53 36 37 39 61 Buffer RLT Plus Contains guanidine thiocyanate harmful Risk and safety phrases R20 21 22 32 13 26 36 46 Buffer RW1 Contains ethanol flammable Risk phrase R10 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 6131 19240 R10 Flammable R20 21 22 Harmful by inhalation in contact with skin and if swallowed R32 Contact with acids liberates very toxic gas R43 May cause sensitization by skin contact R51 53 Toxic to aquatic organisms may cause long term adverse effects in the aquatic environment 13 Keep away from food drink and animal feedingstuffs S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 36 Wear suitable protective clothing S36 37 39 Wear suitable protective clothing gloves and eye face protection 46 If swallowed seek medical advice immediately and show the container or label 561 Avoid release to the environment Refer to special instructions safety data sheets RNAprotect Cell Reagent Handbook 10 2010 7 Introduction The RNeasy Protect Cell procedure provides a complete solution for the stabilization and purification of total RNA from sorted or cultured cells RNA in cells is immediately stabili
36. ompatible with disinfecting reagents containing bleach See the AllPrep handbook for safety information E Perform all steps of the procedure at room temperature 15 25 C During the procedure work quickly E Perform all spin column centrifugation steps at 20 25 C in a standard microcentrifuge Ensure that the centrifuge does not cool below 20 C E Should any problems arise refer to Nucleic acids degraded on page 22 and also to the troubleshooting guide in the AllPrep handbook Things to do before starting E Buffer RIT Plus may form a precipitate during storage If necessary redissolve by warming and then place at room temperature 15 25 C E Many common cell lines such as Hela and Jurkat cells do not require p mercaptoethanol B ME for RNA purification However B ME may help to avoid RNA degradation during RNA purification from some cell lines In this case add 10 pl B ME per 1 ml Buffer RLT Plus before use Dispense in a fume hood and wear appropriate protective clothing Buffer RIT Plus containing B ME can be stored at room temperature 15 25 C for up to 1 month When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier RNAprotect Cell Reagent Handbook 10 2010 31 Procedure Centrifuge the mix of cells and RNAprotect Cell Reagent for 5 min at 5000 x
37. otherwise DNA yields will be reduced Note If the sample was stored at below room temperature e g 20 C thaw it completely before starting centrifugation Note A precipitate may form during storage especially at lower temperatures This does not affect DNA purification 2 Remove the supernatant completely by pipetting 3 Loosen the pellet by flicking the tube Loosening the pellet facilitates dissolving in PBS in step 4 4 Add 200 pl PBS and dissolve the pellet completely by vortexing Add 20 yl proteinase K supplied with the DNeasy or GlAamp Kit Ensure that an appropriate number of cells is used in the procedure For cell lines with a high degree of ploidy e g HeLa cells we recommend using less than the maximum number of cells specified in the DNeasy or GlAamp handbook Note Be sure to dissolve the pellet completely This can take about 1 min Incomplete dissolving may lead to inefficient lysis and reduced DNA yields Note The dissolved pellet may be turbid This does not affect DNA purification Optional If RNA free genomic DNA is required add 4 yl RNase A 100 mg ml mix by vortexing and incubate for 2 min at room temperature 15 25 C before continuing with step 5 5 Add 200 yl Buffer AL without added ethanol Mix thoroughly by vortexing and incubate at 56 C for 10 min Ensure that ethanol has not been added to Buffer AL Buffer AL can be purchased seperately see page 35 for ordering information It is ess
38. p the tip of the probe submerged during homogenization Generally round bottomed tubes allow more efficient homogenization than conicalbottomed tubes For guidelines on homogenization using the TissueRuptor refer to the TissueRuptor Handbook For other rotor stator homogenizers refer to suppliers guidelines Important points before starting if If using the RNeasy Protect Cell Mini Kit for the first time read Important Notes page 13 If working with RNA for the first time read Appendix A page 26 If using the TissueRuptor ensure that you are familiar with operating it by referring to the TissueRuptor User Manual and TissueRuptor Handbook RNAprotect Cell Reagent Handbook 10 2010 17 L z gt y ch Ed A Q z 5 c d o 3 4 az EJ a lt z 7 Buffer RLT Plus may form a precipitate during storage If necessary redissolve by warming and then place at room temperature 15 25 C Buffer RIT Plus 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 spin column centrifugation steps at 20 25 C in a standard microcentrifuge Ensure that the centrifuge does not cool below 20 C Things to do before starting Buffer RPE is supplied as a concentrate Before using for the fir
39. r to obtain optimal RNA yield and purity The maximum amount that can be used is limited by The volume of RNAprotect Cell Reagent required for cell stabilization The cell type and its RNA content The volume of Buffer RIT Plus required for efficient cell lysis The DNA removal capacity of the gDNA Eliminator spin column The RNA binding capacity of the RNeasy spin column Effective cell stabilization requires the mixing of 5 volumes of RNAprotect Cell Reagent with 1 volume of cell culture or buffer or 1 volume of pelleted cells but the cell number should not be too high In addition the maximum volume of Buffer RIT Plus that can be used limits the maximum amount of starting material to 1 x 107 cells Since RNA content can vary greatly between cell types we recommend starting with no more than 3 4 x 10 cells Depending on RNA integrity yield and purity it may be possible to increase the cell number up to 1 x 10 cells in subsequent preparations The amount of starting material must not exceed the maximum of 1 x 10 cells even if the RNA binding capacity of the RNeasy spin column 100 pg RNA will not be reached 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 If the binding capacity of the RNeasy spin column is exceeded RNA yields will not be consistent and may also be reduced IF the DNA removal capacity of the gD
40. reactions or Varies 400 x 25 yl reactions 10x QuantiTect Primer Assay lyophilized For up to date licensing information and productspecific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www qiagen com or can be requested from QIAGEN Technical Services or your local distributor Larger kit size available see www giagen com PCR RNAprotect Cell Reagent Handbook 10 2010 39 www qiagen com Australia Orders 1 800 243 800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 86 21 3865 3865 Fax 86 21 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 800 789 544 Fax 02 334304 826 Technical 80
41. sate before DNA removal d RNA still bound to RNeasy spin column membrane e Ethanol carryover Incomplete removal of RNAprotect Cell Reagent RNAprotect Cell Reagent Handbook 10 2010 The centrifugation temperature should be 20 25 C Some centrifuges may cool to below 20 C even when set at 20 C This can cause formation of precipitates that can clog the gDNA Eliminator spin column If this happens set the centrifugation temperature to 25 C Warm the lysate to 37 C before transferring it to the gDNA Eliminator spin column See Disrupting and homogenizing starting material page 17 for details on disruption and homogenization In subsequent preparations reduce the amount of starting material see page 13 and or increase the volume of lysis buffer and the homogenization time Overloading the RNeasy spin column significantly reduces RNA yield Reduce the amount of starting material see page 13 Pass the lysate through the gDNA Eliminator spin column before adding ethanol to it Repeat RNA elution but incubate the RNeasy spin column on the benchtop for 10 min with RNase free water before centrifuging During the second wash with Buffer RPE be sure to centrifuge at 28000 x g 210 000 rpm for 2 min at 20 25 C to dry the RNeasy spin column membrane Perform the optional centrifugation to dry the RNeasy spin column membrane if any flow through is present on the outside of the column step 12 of t
42. specially for low numbers of cells E Standard cell lines such as Hela cells Jurkat cells and macrophages do not lyse in RNAprotect Cell Reagent However some cell lines may lyse in the reagent releasing nucleic acids into solution Although the released RNA and DNA remain stabilized they need to be collected in a cell pellet prior to nucleic acid purification page 17 31 or 33 To maximize recovery of the released RNA and DNA we recommend using lower volumes of medium or storage solution if possible MH Only fresh unfrozen cells can be stabilized using RNAprotect Cell Reagent E Perform the procedure described below as quickly as possible RNAprotect Cell Reagent Handbook 10 2010 15 Procedure 1 AddRNAprotect Cell Reagent to cells according to step 1a or 1b la Cells in solution or cells covered with solution Add 5 volumes of RNAprotect Cell Reagent to 1 volume of cell culture medium or storage solution Mix by shaking pipetting or vortexing Note The medium or storage solution must not exceed 1 ml We recommend lower volumes especially if the cells are susceptible to lysis in RNAprotect Cell Reagent gc O re ds B D Adherent cells will detach after addition of RNAprotect Cell Reagent No trypsin treatment is necessary During storage of the stabilized cells material may sink to the bottom of the storage vessel To avoid having to resuspend this material we recommend transferring the cells to a c
43. st time add 4 volumes of ethanol 96 100 as indicated on the bottle to obtain a working solution E Many common cell lines such as Hela and Jurkat cells do not require B mercaptoethanol B ME for RNA purification However B ME may help to avoid RNA degradation during RNA purification from some cell lines In this case add 10 pl B ME per 1 ml Buffer RIT Plus before use Dispense in a fume hood and wear appropriate protective clothing Buffer RLT Plus containing B ME can be stored at room temperature 15 25 C for up to 1 month Procedure l Centrifuge the mix of cells and RNAprotect Cell Reagent for 5 min at 5000 x g in an appropriately sized centrifuge tube If transferring the sample to the centrifuge tube from a storage vessel be sure to resuspend any material deposited at the bottom of the vessel by vortexing or by pipetting up and down It is important to transfer all sample material to the centrifuge tube otherwise RNA yields will be reduced Note If the sample was stored at below room temperature e g 20 C thaw it completely before starting centrifugation Note A precipitate may form during storage especially at lower temperatures This does not affect RNA purification 2 Remove the supernatant completely by pipetting 3 Loosen the pellet by flicking the tube Loosening the pellet facilitates dissolving in Buffer RLT Plus in step 4 4 Add 350 pl or 600 pl Buffer RLT Plus see Table 2 Dissolve the pellet completely by
44. the procedure requires the optional use of RNase A see page 36 for ordering information BE Perform all steps of the procedure at room temperature 15 25 C During the procedure work quickly E Perform all spin column centrifugation steps at 20 25 C in a standard microcentrifuge Ensure that the centrifuge does not cool below 20 C E Vortexing should be performed by pulse vortexing for 5 10 s E Should any problems arise refer to Nucleic acids degraded on page 22 and also to the troubleshooting guide in the DNeasy or GlAamp handbook Things to do before starting E Buffer AL may form a precipitate during storage If necessary warm to 56 C until the precipitate has fully dissolved E Preheat a thermomixer shaking water bath or rocking platform to 56 C for use in step 5 When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier RNAprotect Cell Reagent Handbook 10 2010 33 Procedure l Centrifuge the mix of cells and RNAprotect Cell Reagent for 5 min at 5000 x g in an appropriately sized centrifuge tube IF transferring the sample to the centrifuge tube from a storage vessel be sure to resuspend any material deposited at the bottom of the vessel by vortexing or by pipetting up and down It is important to transfer all sample material to the centrifuge tube
45. vortexing and proceed immediately to step 5 18 RNAprotect Cell Reagent Handbook 10 2010 5a 5b If necessary add ME to Buffer RIT Plus before use see Things to do before starting Note Be sure to dissolve the pellet completely This can take about 1 min Incomplete dissolving may lead to inefficient lysis and reduced RNA yields Note The dissolved pellet may be turbid This does not affect RNA purification The dissolved pellet can be stored at 70 C for several months After removal from storage incubate the dissolved pellet at room temperature or at 37 C in a water bath until completely thawed and salts are dissolved Avoid prolonged incubation at 37 C which can cause RNA degradation Proceed to step 5 Table 2 Volumes of Buffer RLT Plus for lysing cells Number of cells Volume of Buffer RLT Plus yl lt 5 x 10 350 SOIN Og 600 Homogenize the lysate according to step 5a or 5b See Disrupting and homogenizing starting material page 17 for more details on homogenization Note Incomplete homogenization leads to significantly reduced RNA yields and can cause clogging of the RNeasy spin column Pipet the lysate directly into a QlAshredder spin column placed in a 2 ml collection tube and centrifuge for 2 min at full speed Proceed to step 6 Homogenize the lysate for 30 s using the TissueRuptor Proceed to step 6 Transfer the homogenized lysate to a gDNA Eliminator spin column placed in a 2 ml col
46. zed using RNAprotect Cell Reagent and then rapidly purified using the RNeasy Plus Mini Kit which includes gDNA Eliminator columns for rapid effective removal of genomic DNA contamination The stabilized cells can be shipped at ambient temperature prior to RNA purification no dry ice is required RNAprotect Cell Reagent also stabilizes DNA in cells The stabilized DNA can be purified using QIAGEN kits for DNA purification such as DNeasy Kits and GlAamp Kits Alternatively stabilized DNA and RNA can be purified simultaneously from the same cell sample using an AllPrep Kit Principle and procedure RNA stabilization using RNAprotect Cell Reagent Immediate stabilization of RNA in sorted or cultured cells is generally a prerequisite for reliable gene expression analysis using microarray realtime RT PCR or other nucleic acid based technology This is because changes in the gene expression pattern occur immediately after harvesting due to unspecific and specific RNA degradation as well as fo transcriptional induction RNAprotect Cell Reagent uses a novel patentpending technology to immediately stabilize the gene expression profile in cells With other technologies it is necessary to wash or process the cells e g trypsin treatment prior to adding RNA stabilization reagent However changes in gene expression may occur during the washing or processing steps In contrast RNAprotect Cell Reagent can be added directly to cells in culture medium
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