RNeasy® Protect Saliva Mini Handbook
Contents
1. To ensure maximal RNA yields store the stabilized saliva sample for at least 24 hours before starting RNA purification RNeasy Protect Saliva Mini Handbook 09 2010 11 L Z gt i a EL Q fe E c d o 2 4 az EJ a lt z 7 Protocol Purification of RNA from Stabilized Saliva Using the RNeasy Micro Kit Important points before starting If working with RNA for the first time read the appendix page 20 Saliva samples generally contain very low amounts of RNA To ensure maximal RNA yields store the stabilized saliva sample for at least 24 hours before starting RNA purification Due to the heterogenous nature of saliva shorter storage times may be sufficient for some samples However we still recommend storage for at least 24 hours Buffer RIT may form a precipitate during storage If necessary redissolve by warming and then place at room temperature 15 25 C Buffer RIT 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 including centrifugation at room temperature During the procedure work quickly Things to do before starting Before using the RNeasy Micro Kit for the first time prepare 80 ethanol by mixing 24 ml ethanol 96 100 with 6 ml RNase free water supplied with the RNeasy Micro Kit Buffer RPE is supplied as a concentrate Before using2. for the first time add 4 volumes of ethanol 96 100 as indicated on the bottle to obtain a working solution Prepare DNase stock solution before using the RNase Free DNase Set for the first time Dissolve the lyophilized DNase 1500 Kunitz units in 550 pl of the RNase free water provided To avoid loss of DNase do not open the vial Inject RNase free water into the vial using an RNase free needle and syringe Mix gently by inverting the vial Do not vortex For long term storage of DNase remove the stock solution from the glass vial divide it into single use aliquots and store at 20 C for up to 9 months Thawed aliquots can be stored at 2 8 C for up to 6 weeks Do not refreeze the aliquots after thawing RNeasy Protect Saliva Mini Handbook 09 2010 Procedure l Centrifuge the mix of saliva and RNAprotect Saliva Reagent for 10 min at 10 000 x g in a microcentrifuge Note The stabilized saliva sample must be stored for at least 24 hours prior to centrifugation see Important points before starting page 12 Note If the sample was stored at below room temperature e g 2 8 C or 20 C thaw it completely and equilibrate it to room temperature 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 L z gt y ch Ed A Q z 5 3 Loosen the pellet by fl
3. profile in saliva samples which can then be stored and transported at ambient temperature Note RNA in saliva is already degrading prior to collection and stabilization with RNAprotect Saliva Reagent This means that RNA purified from saliva samples will be of lower quality than RNA purified from other samples types such as cultured cells and animal tissues RNA purification using the RNeasy Micro Kit RNeasy Micro technology combines the selective binding properties of a silica based membrane with the speed of microspin technology Guanidine thiocyanate containing lysis buffer and ethanol are added to the sample to create conditions that promote selective binding of RNA to the RNeasy MinElute membrane The sample is then applied to the RNeasy MinElute spin column RNA binds to the silica membrane Traces of DNA that may copurify are removed by a DNase treatment on the spin column DNase and any contaminants are washed away and high quality total RNA is eluted in RNase free water see flowchart next page With the RNeasy Micro procedure all RNA molecules longer than 200 nucleotides are purified The procedure enriches for mRNA since most RNAs lt 200 nucleotides such as 5 8S rRNA 5S rRNA and tRNAs which together make up 15 2076 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 8 RN
4. quantitative multiplex real time one step RT PCR using sequence specific probes QuantiTect Multiplex For 200 x 50 pl reactions 3 x 1 7 ml 204643 RT PCR Kit 200 2x Master Mix contains ROX dye 100 pl RT Mix 2 x 2 ml RNase Free Water QuantiTect Multiplex For 200 x 50 pl reactions 3 x 1 7 ml 204843 RT PCR NR Kit 200 2x Master Mix contains no ROX dye 100 yl RT Mix 2 x 2 ml RNase Free Water For up to date licensing information and product specific disclaimers see the respective GIAGEN kit handbook or user manual GIAGEN 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 Visit www giagen com GeneGlobe to search for and order primer probe sets Recommended for ABI PRISM and Applied Biosystems cyclers Recommended for all other cyclers 26 RNeasy Protect Saliva Mini Handbook 09 2010 Notes RNeasy Protect Saliva Mini Handbook 09 2010 27 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 3
5. simple keyword search or by specifying the application research area title etc For a complete list of references visit the QIAGEN Reference Database online at www qgiagen com RefDB search asp or contact QIAGEN Technical Services or your local distributor 22 RNeasy Protect Saliva Mini Handbook 09 2010 Ordering Information Product Contents Cat No RNeasy Protect Saliva RNAprotect Saliva Reagent 50 ml 74324 Mini Kit 50 and RNeasy Micro Kit 50 Accessories Collection Tubes 2 ml 1000 x 2 ml Collection Tubes 19201 Buffer RLT 220 ml 220 ml Buffer RLT 79216 RNase Free DNase Set 50 1500 units RNase Free DNase 79254 RNase Free Buffer RDD and RNase Free Water RNeasy Micro Kit 50 50 RNeasy MinElute Spin Columns 74004 Collection Tubes RNase Free DNase Carrier RNA RNase Free Reagents and Buffers Related products for RNA stabilization and purification RNAlater RNA Stabilization Reagent for immediate stabilization of the gene expression profile in harvested tissues RNAlater RNA For stabilization of RNA in 76104 Stabilization Reagent 50 ml 25 x 200 mg tissue samples 50 ml RNAlater RNA Stabilization Reagent RNAlater TissueProtect Tubes for collecting harvested tissues and immediate stabilization of the gene expression profile and subsequent transport and storage RNAlater TissueProtect For stabilization of RNA in 76154 Tubes 50 x 1 5 ml 50 x 150 mg tissue samples 50 screw top tubes cont
6. the flow through and collection tube Note Prepare 80 ethanol using ethanol 96 100 and the RNase free water supplied with the kit Note After centrifugation carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur 13 Place the RNeasy MinElute spin column in a new 2 ml collection tube supplied Open the lid of the spin column and centrifuge at full speed for 5 min Discard the flow through and collection tube To avoid damage to their lids place the spin columns into the centrifuge with at least one empty position between columns Orient the lids so that they point in a direction opposite to the rotation of the rotor e g if the rotor rotates clockwise orient the lids counterclockwise Flow through contains Buffer RW1 and is therefore not compatible with bleach See page 6 for safety information 14 RNeasy Protect Saliva Mini Handbook 09 2010 14 Note It is important to dry the spin column membrane since residual ethanol may interfere with downstream reactions Centrifugation with the lids open ensures that no ethanol is carried over during RNA elution Place the RNeasy MinElute spin column in a new 1 5 ml collection tube supplied Add 14 yl RNase free water directly to the center of the spin column membrane Close the lid gently and centrifuge for 1 min at full speed to elute the RNA The dead v
7. 865 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 800 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 639 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
8. DNA PREP 800 362 7737 WWW QIAGEN COM QIAGEN 1064942 09 2010
9. September 2010 RNeasy Protect Saliva Mini Handbook For immediate stabilization of total RNA in saliva and subsequent RNA purification 26086 00000 QIAGEN Trademarks QIAGEN MinElute Omniscript Quantiscript QuantiTect RNAprotect RNeasy Sensiscript QIAGEN Group ABI PRISM Applied Biosystems Applera Corporation or its subsidiaries LightCycler Roche Group PAXgene PreAnalytiX GmbH SYBR Molecular Probes Inc RNAlater is a trademark of AMBION Inc Austin Texas and is covered by various U S and foreign patents The PAXgene Blood RNA Kit cat nos 762164 and 762174 is for in vitro diagnostic use Not available in all countries Purchase of QIAGEN products for PCR containing HotStarTag DNA Polymerase is accompanied by a limited license to use them in the polymerase chain reaction PCR process for research and development activities in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up front license fee either by payment to Applied Biosystems or as purchased i e an authorized thermal cycler The PCR process is covered by the foreign counterparts of U S Patents Nos 4 683 202 and 4 683 195 owned by F Hoffmann La Roche Ltd The 5 nuclease process is covered by patents owned by Roche Molecular Systems Inc and F Hoffmann La Roche Ltd Patents of third parties in certain countries may cover the process of multiple
10. a Mini Handbook 09 2010 13 8 Add 10 pl DNase I stock solution to 70 pl Buffer RDD Mix by gently inverting the tube Note DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube Do not vortex 9 Add the DNase incubation mix 80 pl directly to the RNeasy MinElute spin column membrane and incubate on the benchtop 20 30 C for 15 min Note Be sure to add the DNase incubation mix directly to the RNeasy MinElute spin column membrane DNase digestion will be incomplete if part of the mix sticks to the walls or the O ring of the spin column 10 Add 350 pl Buffer RW1 to the RNeasy MinElute spin column Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm Discard the flow through and collection tube c d o 2 4 az E 5 a lt z 7 11 Place the RNeasy MinElute spin column in a new 2 ml collection tube supplied Add 500 yl Buffer RPE to the spin column Close the lid gently and centrifuge for 15 s at z8000 x g 210 000 rpm to wash the spin column membrane Discard the flow through Reuse the collection tube in step 12 Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use see Things to do before starting 12 Add 500 pl of 80 ethanol to the RNeasy MinElute spin column Close the lid gently and centrifuge for 2 min at 28000 x g 210 000 rpm to dry the spin column membrane Discard
11. aining 1 5 ml RNAlater RNA Stabilization Reagent each RNeasy Protect Mini Kit for immediate stabilization of the gene expression profile in animal 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 Larger kit size and or format available see www giagen com RNeasy Protect Saliva Mini Handbook 09 2010 23 Ordering Information Product Contents RNAprotect Bacteria Reagent for in vivo stabilization of the gene expression profile in bacteria RNAprotect Bacteria Reagent RNAprotect Bacteria Reagent 2 x 100 ml RNeasy Protect Bacteria Mini Kit for in vivo stabilization of the gene expression profile in bacteria and subsequent RNA purification RNeasy Protect RNeasy Mini Kit 50 and RNAprotect Bacteria Mini Kit 50 Bacteria Reagent 2 x 100 ml 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 50 PAXgene Shredder Spin Columns Processing Tubes RNase Free DNase RNase Free Reagents and Buffers To be used in conjunction with PAXgene Blood RNA Tubes Related products for downstream applications Omniscript RT Kit for reverse transcription using 50 ng to 2 pg RNA per reaction Omniscript RT Kit 50 For 50 x 20 yl reactio
12. cware 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 preparing 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 T Plastics used for some electrophoresis tanks are not resistant to ethanol Take p
13. easy Protect Saliva Mini Handbook 09 2010 RNeasy Protect Saliva Procedure Saliva Mix with RNAprotect Saliva Reagent 24 h incubation Dissolve pellet in Buffer RLT Add ethanol os V ind total RNA xm gt f gt d Concentrated RNA solution RNeasy Protect Saliva Mini Handbook 09 2010 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 For saliva collection and RNA stabilization B ice E Vessels for collecting saliva e g 50 ml polypropylene tubes B 2 ml microcentrifuge tubes Hl Vortexer For RNA purification Ethanol 70 and 96 100 Sterile RNase free pipet tips Microcentrifuge with rotor for 2 ml tubes Vortexer Disposable gloves Do not use denatured alcohol which contains other substances such as methanol or methylethylketone 10 RNeasy Protect Saliva Mini Handbook 09 2010 Protocol Stabilization of RNA in Saliva Using RNAprotect Saliva Reagent Important points before starting The subject should refrain from eating drinking or oral hygiene procedures for at least 1 hour prior to collection of saliva Optionally the mouth can be rinsed with water without swallowing 5 minutes before saliva collection Collect saliva using a collection vessel wit
14. f Insufficient incubation time after mixing saliva with RNAprotect Saliva Reagent Comments and suggestions Centrifugation at low temperatures e g under refrigeration can cause the formation of precipitates that can clog the RNeasy MinElute spin column Perform all steps of the procedure including centrifugation at room temperature Be sure to completely dissolve the saliva derived pellet in Buffer RLT see step 4 of the second protocol Be sure to purify RNA from 200 pl saliva per RNeasy MinElute spin column Repeat RNA elution but incubate the RNeasy MinElute spin column on the bench top for 10 min with RNase free water before centrifuging After washing with 80 ethanol be sure to dry the RNeasy MinElute spin column membrane by centrifuging at full speed for 5 min see step 13 of the second protocol After centrifugation carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur RNases can be introduced if the water used to dilute the ethanol is not RNase free Prepare 80 ethanol using ethanol 96 100 and the RNase free water supplied with the kit as described in Things to do before starting page 12 After mixing the saliva sample with RNAprotect Saliva Reagent store the sample for at least 24 hours before starting the RNA purification procedure RNeasy Protect Saliva Mini Handb
15. h an appropiate volume and a sufficiently large opening e g a 50 ml polypropylene tube The subject should spit into the collection vessel but should not cough up mucus During saliva collection keep the collection vessel on ice to minimize changes in the gene expression pattern After saliva collection the sample should be immediately mixed with RNAprotect Saliva Reagent RNA is not stabilized until the sample is treated with RNAprotect Saliva Reagent Perform the procedure described below as quickly as possible Procedure 1 Collect 200 pl saliva in a collection vessel placed on ice Proceed immediately to step 2 If processing larger volumes of saliva collect several samples of 200 yl saliva Note The RNA in the saliva sample is not stabilized until the sample is treated with RNAprotect Saliva Reagent in step 2 Add 200 yl saliva to 1 ml RNAprotect Saliva Reagent in a 2 ml microcentrifuge tube not supplied Mix well by vortexing The RNA in the saliva sample is now stabilized Store the mix of saliva and RNAprotect Saliva Reagent for up to 1 day at 37 C up to 14 days at room temperature 15 25 C or up to 28 days 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 37 C Note A precipitate may form during storage especially at lower temperatures This does not affect RNA purification Note
16. icking the tube Loosening the pellet facilitates dissolving in Buffer RLT in step 4 4 Add 350 pl Buffer RLT Dissolve the pellet completely by vortexing Note Be sure to dissolve the pellet completely This can take about 1 min 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 5 Add 1 volume 350 pl of 70 ethanol and mix well by pipetting or vortexing Do not centrifuge Proceed immediately to step 6 A precipitate may form after addition of ethanol but this will not affect the procedure 6 Transfer the sample to an RNeasy MinElute spin column 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 7 7 Add 350 pl Buffer RW1 to the RNeasy MinElute 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 8 Flow through contains Buffer RIT or Buffer RW1 and is therefore not compatible with bleach See page 6 for safety information RNeasy Protect Saliv
17. ins potentially infectious agents clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite The following risk and safety phrases apply to the components of the RNeasy Protect Saliva Mini Kit RNAprotect Saliva Reagent Contains tetradecyltrimethylammonium oxalate irritant dangerous for the environment Risk and safety phrases R43 51 53 S536 37 39 61 Buffer RLT Contains guanidine thiocyanate harmful Risk and safety phrases R20 21 22 32 13 26 36 46 Buffer RW1 Contains ethanol flammable Risk phrase R10 RNase free DNase Contains deoxyribonuclease sensitizer Risk and safety phrases R42 43 S22 24 26 36 37 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 R42 43 May cause sensitization by inhalation and skin contact 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 S22 Do not breathe dust 524 Avoid contact with skin S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 36 Wear suitable pr
18. ion Clogged column a Sample not completely dissolved b Too much sample Comments and suggestions RNA purified from saliva is usually less intact than total RNA purified from other samples such as cells blood and tissues To check for RNA degradation purify RNA from a stabilized saliva sample and an unstabilized saliva sample and compare the intactness of the purified RNA Mix the saliva sample immediately with RNAprotect Saliva Reagent Collect saliva on ice and as quickly as possible Saliva mixed with RNAprotect Saliva Reagent can be stored for up to 1 day at 37 C up to 14 days at 15 25 C or up to 28 days 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 free RNases can be introduced during use Be careful not to introduce any RNases during RNA purification or later handling See the appendix page 20 for general remarks on handling RNA Be sure to completely dissolve the saliva derived pellet in Buffer RLT see step 4 of the second protocol Be sure to purify RNA from 200 pl saliva per RNeasy MinElute spin column RNeasy Protect Saliva Mini Handbook 09 2010 c Centrifugation temperature too low Low RNA yield a Sample not completely dissolved b Too much sample c RNA still bound to spin column membrane d Ethanol carryover e 8076 ethanol not made with RNase free water
19. ns Omniscript Reverse Transcriptase 10x Buffer RT dNTP Mix RNase Free Water Sensiscript RT Kit for reverse transcription using less than 50 ng RNA per reaction Sensiscript RT Kit 50 For 50 x 20 pl reactions Sensiscript Reverse Transcriptase 10x Buffer RT dNTP Mix RNase Free Water Larger kit size or format available see www qiagen com e Canada and USA only Rest of the world kit not available in all countries Cat No 76506 74524 762164 7621745 205111 205211 PAXgene Blood RNA Tubes can be ordered from BD and BD authorized distributors www bd com 24 RNeasy Protect Saliva Mini Handbook 09 2010 Ordering Information Product Contents Cat No QIAGEN OneStep RT PCR Kit for fast and successful one step RT PCR QIAGEN OneStep For 25 x 50 pl reactions QIAGEN 210210 RT PCR Kit 25 OneStep RT PCR Enzyme Mix 5x QIAGEN OneStep RT PCR Buffer dNTP Mix 5x Q Solution RNase Free Water 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 SYBR Green PCR Kit for quantitative real time two step RT PCR using SYBR Green QuantiTect SYBR Green For 200 x 50 yl reactions 3 x 1 7 ml 204143 PCR Kit 200 2x Mas
20. olume of the RNeasy MinElute spin column is 2 pl elution with 14 pl RNase free water results in a 12 pl eluate Elution with smaller volumes of RNase free water leads to higher total RNA concentrations but lower RNA yields RNA yield will be reduced by approximately 20 if RNA is eluted in 8 pl RNase free water We do not recommend eluting RNA in less than 8 pl RNase free water as the spin column membrane may not be sufficiently hydrated Note When performing RT PCR with the purified RNA we recommend using the QIAGEN OneStep RT PCR Kit This kit contains a specially formulated blend of Omniscript Reverse Transcriptase designed for gt 50 ng RNA and Sensiscript Reverse Transcriptase designed for lt 50 ng RNA For quantitative real time RT PCR we recommend QIAGEN QuantiTeci Kits See page 23 for ordering information RNeasy Protect Saliva Mini Handbook 09 2010 15 b z Y ch cue e E E 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 molecular biology applications see back cover for contact information RNA degraded a RNA in saliva already degraded prior to collection b Saliva sample not immediately stabilized c Prolonged storage d RNA degradation during RNA purificat
21. ons or experience any difficulties regarding the RNeasy Protect Saliva 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 product performance or new applications and techniques For technical assistance and more information please call one of the QIAGEN Technical Service Departments or local distributors see back cover 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 ts msds asp where you can find view and print the MSDS for each GIAGEN kit and kit component 6 RNeasy Protect Saliva Mini Handbook 09 2010 CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Buffer RLT contains guanidine thiocyanate and Buffer RW1 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 conta
22. ontact the flow through Otherwise carryover of ethanol will occur RNeasy Protect Saliva Mini Handbook 09 2010 b Salt carryover during elution c Reverse transcription with too small an amount of RNA d Too much sample e Insufficient incubation time after mixing saliva with RNAprotect Saliva Reagent Comments and suggestions Ensure that Buffer RPE is at room temperature and ethanol is added as described on the bottle Most reverse transcriptases are intended for use with approximately 1 pg RNA When performing reverse transcription with very small amounts of RNA we recommend using the QIAGEN Sensiscript RT Kit which is specially designed for highly sensitive reverse transcription using 50 ng RNA For one step RT PCR and quantitative real time RT PCR we recommend the QIAGEN OneStep RT PCR Kit and QuantiTect RT PCR Kits respectively These kits contain a specially formulated blend of Omniscript and Sensiscript Reverse Transcriptases for amplification of a wide range of RNA amounts from as little as 1 pg per reaction Be sure to purify RNA from 200 pl saliva per RNeasy MinElute spin column After mixing the saliva sample with RNAprotect Saliva Reagent store the sample for at least 24 hours before starting the RNA purification procedure RNeasy Protect Saliva Mini Handbook 09 2010 19 Appendix General Remarks on Handling RNA Handling RNA Ribonucleases RNases are very stable and active en
23. ook 09 2010 17 Low or no recovery of RNA a RNase free water incorrectly dispensed b Ethanol carryover c 80 ethanol not made with RNase free water Comments and suggestions Pipet RNase free water to the center of the RNeasy MinElute spin column membrane to ensure that the membrane is completely covered After washing with 80 ethanol be sure to dry the RNeasy MinElute spin column membrane by centrifuging at full speed for 5 min see step 13 of the second protocol After centrifugation carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur RNases can be introduced if the water used to dilute the ethanol is not RNase free Prepare 80 ethanol using ethanol 96 100 and the RNase free water supplied with the kit as described in Things to do before starting page 12 DNA contamination in downstream experiments No DNase treatment Be sure to perform the on column DNase digestion as described in steps 8 9 of the second protocol RNA does not perform well in downstream experiments a Ethanol carryover After washing with 80 ethanol be sure to dry the RNeasy MinElute spin column membrane by centrifuging at full speed for 5 min see step 13 of the second protocol After centrifugation carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not c
24. otective clothing 36 37 Wear suitable protective clothing and gloves S36 37 39 Wear suitable protective clothing gloves and eye face protection S46 If swallowed seek medical advice immediately and show the container or label S61 Avoid release to the environment Refer to special instructions safety data sheets RNeasy Protect Saliva Mini Handbook 09 2010 7 Introduction The RNeasy Protect Saliva Mini procedure provides a complete solution for the stabilization and purification of total RNA from saliva RNA in collected saliva is immediately stabilized using RNAprotect Saliva Reagent and then rapidly purified and concentrated using the RNeasy Micro Kit Principle and procedure RNA stabilization using RNAprotect Saliva Reagent Immediate stabilization of RNA in a saliva sample is a prerequisite for reliable gene expression analysis using microarray real time RT PCR or other nucleic acid based technology This is because changes in the gene expression pattern occur immediately after saliva collection due to unspecific and specific RNA degradation as well as to transcriptional induction Also since saliva contains bacteria in addition to cells and free circulating molecules e g nucleic acids of human origin it is important to prevent the uncontrolled growth of bacteria which can also affect the gene expression profile RNAprotect Saliva Reagent uses a novel patentpending technology to immediately stabilize the gene expression
25. oughly rinsed with 0 1 M NaOH 1 mM EDTA followed by RNase free water see Solutions page 21 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 20 RNeasy Protect Saliva Mini Handbook 09 2010 Glassware Glassware should be 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 plasti
26. pplicable guidelines RNeasy Protect Saliva Mini Handbook 09 2010 5 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 GIAGEN 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 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 GIAGEN 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 Technical Assistance At GIAGEN 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 molecular biology and the use of GIAGEN products If you have any questi
27. roper care and check the supplier s instructions RNeasy Protect Saliva Mini Handbook 09 2010 21 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 DNA contamination No currently available purification method can guarantee that RNA is completely free of DNA even when it is not visible on an agarose gel While the RNeasy Protect Saliva 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 ABI PRISM and LightCycler instruments we recommend designing primers that anneal at intron splice junctions so that genomic DNA will not be amplified QuantiTect Assays from QIAGEN are designed for real time RT PCR analysis of RNA sequences without detection of genomic DNA where possible To search for and order assays online visit www giagen com GeneGlobe References QIAGEN maintains a large up to date online database of scientific publications utilizing QIAGEN products Comprehensive search options allow you to find the articles you need either by a
28. t compatible with disinfectants containing bleach See page 6 for safety information Before using for the first time add 4 volumes of ethanol 96 100 as indicated on the bottle to obtain a working solution RNeasy Protect Saliva Mini Handbook 09 2010 Quality Control In accordance with GIAGEN s ISO certified Quality Management System each lot of RNeasy Protect Saliva Mini Kit is tested against predetermined specifications to ensure consistent product quality Shipping and Storage Store RNAprotect Saliva Reagent at room temperature 15 25 C The reagent is stable for at least 12 months under these conditions The RNeasy Micro Kit is shipped at room temperature 15 25 C Store the RNeasy MinElute spin columns and the RNase Free DNase Set box containing RNase free DNase Buffer RDD and RNase free water immediately upon receipt at 2 8 C Store the remaining components of the RNeasy Micro Kit dry at room temperature All components of the RNeasy Micro Kit are stable for at least 9 months under these conditions Product Use Limitations The RNeasy Protect Saliva 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 a
29. ter Mix 2 x 2 ml RNase Free Water QuantiTect SYBR Green RT PCR Kit for quantitative real time one step RT PCR using SYBR Green I QuantiTect SYBR Green For 200 x 50 pl reactions 3 x 1 7 ml 204243 RT PCR Kit 200 1 2x Master Mix 100 pl RT Mix 2 x 2 ml RNase Free Water QuantiTect Probe PCR Kit for quantitative real time two step RT PCR using sequence specific probes QuantiTect Probe PCR For 200 x 50 pl reactions 3 x 1 7 ml 204343 Kit 200 2x Master Mix 2 x 2 ml RNase Free Water Larger kit size available see www giagen com t Visit www giagen com GeneGlobe to search for and order primer sets or primer probe sets RNeasy Protect Saliva Mini Handbook 09 2010 25 Ordering Information Product Contents Cat No QuantiTect Probe RT PCR Kit for quantitative real time one step RT PCR using sequence specific probes QuantiTect Probe For 200 x 50 yl reactions 3 x 1 7 ml 204443 RT PCR Kit 200 1 2x Master Mix 100 pl RT Mix 2 x 2 ml RNase Free Water QuantiTect Multiplex PCR Kits for quantitative multiplex real time two step RT PCR using sequence specific probes QuantiTect Multiplex For 200 x 50 pl reactions 3 x 1 7 ml 204543 PCR Kit 200 2x Master Mix contains ROX dye 2 x 2 ml RNase Free Water QuantiTect Multiplex For 200 x 50 pl reactions 3 x 1 7 ml 204743 PCR NoROX Kit 200 8 2x Master Mix contains no ROX dye 2 x 2 ml RNase Free Water QuantiTect Multiplex RT PCR Kits for
30. x PCR or of certain applications 2006 2010 QIAGEN all rights reserved Contents Kit Contents 4 Quality Control 5 Shipping and Storage 5 Product Use Limitations 5 Product Warranty and Satisfaction Guarantee 6 Technical Assistance 6 Safety Information 6 Introduction 8 Principle and procedure 8 Equipment and Reagents to Be Supplied by User 10 Protocols L1 Stabilization of RNA in Saliva Using RNAprotect Saliva Reagent 11 E Purification of RNA from Stabilized Saliva Using the RNeasy Micro Kit 12 Troubleshooting Guide 16 Appendix General Remarks on Handling RNA 20 References 22 Ordering Information 23 QIAGEN Distributors 27 RNeasy Protect Saliva Mini Handbook 09 2010 3 Kit Contents RNeasy Protect Saliva Mini Kit Catalog no Number of preps RNAprotect Saliva Reagent box 1 of 2 E RNAprotect Saliva Reagent E RNeasy Protect Saliva Mini Handbook RNeasy Micro Kit box 2 of 2 B RNeasy MinElute Spin Columns each in a 2 ml Collection Tube 50 Collection Tubes 1 5 ml 50 Collection Tubes 2 ml 100 Buffer RLT 45 ml Buffer RWT1 45 ml Buffer RPE concentrate 11 ml RNase Free Water 10 ml Carrier RNA poly A 1 vial 300 pg RNase Free DNase Set E RNase free DNase lyophilized 1500 units E Buffer RDD 2x2ml E RNase Free Water 1 5 ml RNeasy Micro Handbook 1 Follow the instructions in the RNeasy Protect Saliva Mini Handbook when stabilizing and purifying RNA from saliva Contains a guanidine salt No
31. zymes 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 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 thor
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