RNeasy® Protect Animal Blood Handbook
Contents
1. Flow through contains Buffer RBT and is therefore not compatible with bleach See page 5 for safety information 28 RNeasy Protect Animal Blood Handbook 06 2012 C8 Place the RNeasy MinElute spin column in a 1 5 ml collection tube Add 14 pl RNase free water directly to the center of the spin column membrane Close the lid gently and centrifuge for 1 min at 8000 x g 10 000 rpm to elute small RNAs If preferred Buffer REB can be used for elution instead of RNase free water RNeasy Protect Animal Blood Handbook 06 2012 29 Ordering Information Product Contents Cat no RNeasy Protect Animal For 50 preps RNeasy MinElute Spin 73224 Blood Kit 50 Columns QlAshredder Spin Columns Collections Tubes Proteinase K RNase Free DNase and RNase Free Reagents and Buffers to be used with RNAprotect Animal Blood Tubes RNAprotect Animal 50 tubes for collection storage and 76544 Blood Tubes 50 x 100 pl transport of 100 pl animal blood samples with RNA stabilization to be used with the RNeasy Protect Animal Blood Kit RNAprotect Animal 50 tubes for collection storage and 76554 Blood Tubes 50 x 500 pl transport of 500 pl animal blood samples with RNA stabilization to be used with the RNeasy Protect Animal Blood Kit Buffer RWT For purification of total RNA including 1067933 miRNA Supplied as a concentrate 80 ml Related products Quantilect Reverse For 50 x 20 pl reverse transcription 205311 Transcription Kit 52. RNeasy Rotor Gene QIAGEN Group Applied Biosystems Applied Biosystems LCC Affymetrix GeneChip Affymetrix Inc Agilent Agilent Technologies Inc Eppendorf Eppendorf AG Ovation NUGEN Technologies Inc SYBR Molecular Probes Inc Limited License Agreement for RNeasy Protect Animal Blood Kit Use of this product signifies the agreement of any purchaser or user of the product to the following terms 1 The product may be used solely in accordance with the protocols provided with the product and this handbook and for use with components contained in the kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product this handbook and additional protocols available at www giagen com Some of these additional protocols have been provided by QIAGEN users for QIAGEN users These protocols have not been thoroughly tested or optimized by QIAGEN QIAGEN neither guarantees them nor warrants that they do not infringe the rights of third parties Other than expressly stated licenses QIAGEN makes no warranty that this kit and or its use s do not infringe the rights of third parties This kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other license
3. RNeasy Protect Animal Blood Handbook For purification of total RNA or total RNA including miRNA from animal blood stabilized in RNAprotect Animal Blood Tubes QIAGEN QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Kit Contents 4 Shipping and Storage 5 Intended Use 5 Safety Information 5 Quality Control 6 Introduction 7 Principle and procedure 7 Automated purification 8 Equipment and Reagents to Be Supplied by User 10 Important Notes 11 Collection of starting material 11 Protocols H Purification of Total RNA gt 200 Nucleotides Excluding miRNA from RNAprotect Stabilized Animal Blood 12 E Purification of Total RNA Including miRNA from RNAprotect Stabilized Animal Blood 16 Troubleshooting Guide 20 Appendix A General Remarks on Handling RNA 22 Appendix B Storage Quantification and Determination of Quality of RNA 24 Appendix C Purification of Small RNAs lt 200 nt from RNAprotec
4. Including miRNA from RNAprotect Stabilized Animal Blood on page 16 The system has been developed for and tested with blood from rats and mice Performance with blood from other species may vary The purified total RNA is ready to use and is highly suited for any downstream application including RT PCR and real time RT PCR Differential display cDNA synthesis Northern dot and slot blot analyses Primer extension Poly A RNA selection RNase S1 nuclease protection Microarrays The RNeasy Protect Animal Blood Kit allow parallel processing of multiple samples in less than 80 minutes including hands on time of 30 minutes Methods involving the use of toxic substances such as phenol and or chloroform or time consuming and tedious methods such as alcohol precipitation are replaced by the RNeasy miRNeasy procedure Important To ensure optimal performance of theRNeasy Protect Animal Blood Kit blood samples must be collected in RNAprotect Animal Blood Tubes The tubes are not included in the kits and are supplied separately in 2 sizes RNAprotect Animal Blood Tubes 100 pl for 100 pl blood and RNAprotect Animal Blood Tubes 500 pl for 500 pl blood For ordering information see page 30 Important Buffer RWT must be ordered separately see ordering information page 30 for use when following the protocol Purification of Total RNA Including miRNA from RNAprotect Stabilized Animal Blood page 16 Principle and procedu
5. page 24 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 vast majority of cellular DNA will by removed by the DNase digestion step trace amounts may still remain in the purified RNA 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 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 T Values up to 2 3 are routinely obtained for pure RNA in 10 mM Tris Cl pH 7 5 with some spectrophotometers zz gt m gt m mm s6sv _zojzpb zz zpx z z ztzvjzizxjxz yzz vzzzkzzz m FT RNeasy Protect Animal Blood Handbook 06 2012 25 To prevent any interference by DNA in real time RT PCR applications such as with Rotor Gene and Applied Biosystems instruments we recommend designing primers that anneal at intron splice junctions so that genomic DNA will not be amplified QuantiTect Primer Assays from QIAGEN www giagen com GeneGlobe are designed for SYBR Green based real time RT PCR analysis of RNA sequences without detection of genomic DNA where possible For realtime RT PCR assays where amplification of genomic D
6. redissolve by warming at 37 C and then place at room temperature E Ifusing the RNase Free DNase Set for the first time prepare DNase stock solution Dissolve the lyophilized DNase 1500 Kunitz units in 550 pl of the RNase free water provided with the set 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 16 RNeasy Protect Animal Blood Handbook 06 2012 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 Procedure 1 Centrifuge the RNAprotect Animal Blood Tube for 3 min at 5000 x g Note Be sure to incubate the tube for at least 2 h at room temperature 15 25 C after blood collection to achieve complete lysis of blood cells Note If using an RNAprotect Animal Blood Tube 100 pl transfer the blood sample to a new 1 5 ml collection tube supplied the same tube can be used in step 5 Alternatively use only 450 pl RNase free water in step 2 below Remove the supernatant by decanting or pipetting Add 1 ml RNase free water to the pellet and close the tube If decanting the supernatant take care not to disturb the pellet and dry the rim of the tube with a clean paper towel Vortex until the
7. free RNases can be introduced during use Be certain not to introduce any RNases during the RNeasy procedure or later handling See Appendix A page 22 for general remarks on handling RNA Do not put RNA samples into a vacuum dryer or microcentrifuge that has been used in DNA preparation where RNases may have been used RNA does not perform well in downstream experiments High levels of globin High levels of globin mRNA in purified RNA from mRNA affect microarray whole blood reduce the sensitivity of gene analysis expression analysis with Affymetrix GeneChip arrays To avoid this problem we recommend preparing targets for GeneChip arrays using the Ovation Whole Blood Solution from NuGEN www nugeninc com RNeasy Protect Animal Blood Handbook 06 2012 21 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 Care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure 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 RN
8. is for purification of small RNAs The RNA eluate is enriched in miRNA and other small RNA species of lt 200 nucleotides e g tRNAs The protocol requires the RNeasy MinElute Cleanup Kit and the RNeasy Protect Animal Blood Kit The protocol is for use with blood samples collected in RNAprotect Animal Blood Tubes It is important to collect blood as described in the product sheet supplied with RNAprotect Animal Blood Tubes If transporting the tubes be sure to handle the tubes according to the recommendations in the product sheet Quantification of miRNA Since the RNA eluate obtained using this procedure is enriched in various small RNA species the yield of specific small RNA species e g miRNA cannot be quantified by OD measurement or fluorogenic assays Instead we recommend using quantitative real time RT PCR assays specific for the type of small RNA under study For example to estimate miRNA recovery an assay directed against any miRNA known to be adequately expressed in the samples being processed may be used Reagents to be supplied by user E Ethanol 100 and 80 M RNeasy MinElute Cleanup Kit cat no 74204 Important points before starting E Perform all steps of the procedure at room temperature 15 25 C During the procedure work quickly E Ifusing a refrigerated microcentrifuge make sure cooling is switched off i e set the temperature to just above ambient temperature Make sure the microcentrifuge is at room
9. pellet is visibly dissolved and centrifuge for 3 min at 5000 x g Remove the entire supernatant by decanting or pipetting and discard Small debris remaining in the sample after vortexing does not affect subsequent RNA purification Note Incomplete removal of the supernatant will inhibit proteinase K digestion and dilute the lysate affecting the conditions for binding RNA to the RNeasy MinElute membrane Add 240 pl Buffer RSB and vortex until the pellet is visibly dissolved Pipet the sample into a 1 5 ml collection tube supplied Add 200 pl Buffer RBT and 20 pl proteinase K Mix by vortexing for 5 s and incubate for 10 min at 55 C in a shaker incubator at 400 1400 rpm After incubation set the temperature of the shaker incubator to 65 C for step 18 Note Do not mix Buffer RBT and proteinase K together before adding them to the sample Pipet the sample into a QlAshredder spin column lilac placed in a 2 ml collection tube and centrifuge for 3 min at full speed do not exceed 20 000 x g Carefully transfer the entire supernatant of the flow through from the QlAshredder spin column to a new 1 5 ml collection tube supplied without disturbing the pellet Add 1 5 volumes usually 690 pl of 100 ethanol and mix by vortexing RNeasy Protect Animal Blood Handbook 06 2012 17 2 ao at 32 gt Z gt 9 Pipet 700 pl sample into an RNeasy MinElute spin column pink placed in a 2 ml collection tube Close the lid gent
10. temperature Things to do before starting Buffer RPE is supplied as a concentrate Before using for the first time add 4 volumes of ethanol 96 100 as indicated on the bottle to obtain a working solution When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier RNeasy Protect Animal Blood Handbook 06 2012 27 Procedure Carry out the protocol starting on page 16 up to and including step 6 Instead of performing steps 8 19 purification of total RNA including miRNA follow steps C1 C8 below purification of small RNAs C1 Add 240 pl ethanol 96 100 to the flow through from the QlAshredder spin column and mix by vortexing C2 Pipet the sample into an RNeasy MinElute spin column pink placed in a 2 ml collection tube Close the lid gently and centrifuge for 1 min at 8000 20 000 x g Keep the flow through for purification of small RNAs in steps C3 C8 Optional If purification of total RNA gt 200 nt is desired save the RNeasy MinElute spin column and follow steps 10 19 of the protocol on page 16 Otherwise discard the RNeasy MinElute spin column C3 Add 455 pl of 100 ethanol 0 65 volumes to the flow through from step C2 and mix thoroughly by vortexing Do not centrifuge Proceed immediately to step C4 C4 Pipet 700 pl of the sample into an RNeasy MinElute sp
11. 0 reactions DNA Wipeout Buffer Quantiscript Reverse Transcriptase Quantiscript RT Buffer RT Primer Mix RNase Free Water Other kit sizes are available see www giagen com 30 RNeasy Protect Animal Blood Handbook 06 2012 Ordering Information Product Contents Cat no Quantilect Primer For 200 x 50 pl reactions or Varies Assay 200 400 x 25 pl reactions 10x QuantiTect Primer Assay lyophilized RNeasy MinElute For 50 preps RNeasy MinElute Spin 74204 Cleanup Kit 50 Columns Collection Tubes 1 5 ml and 2 ml RNase Free Reagents and Buffers For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor Visit www giagen com geneXpression to find out more about standardized solutions for gene expression analysis from RNA preparation to real time RT PCR Assay also available in a 96 or 384 well format To search for and order assays visit www giagen com GeneGlobe RNeasy Protect Animal Blood Handbook 06 2012 31 Notes 32 RNeasy Protect Animal Blood Handbook 06 2012 Notes RNeasy Protect Animal Blood Handbook 06 2012 33 Notes 34 RNeasy Protect Animal Blood Handbook 06 2012 Trademarks QIAGEN QlAcube QlAxcel MinElute GeneGlobe Quantiscript QuantiTect RNAprotect
12. A 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 To remove RNase contamination from bench surfaces nondisposable plasticware and laboratory equipment e g pipets and electrophoresis tanks use of RNaseKiller cat no 2500080 from 5 PRIME www 5prime com is recommended RNase contamination can alternatively be removed using general laboratory reagents To decontaminate plasticware rinse with 0 1 M NaOH 1 mM EDTA followed by RNase free water see Solutions page 23 or rinse with chloroform if the plasticware is chloroform resistant To decontaminate electrophoresis tanks clean with detergent e g 0 5 SDS rinse with RNase free water rinse with ethanol if the tanks are ethanol resistant and allow to dry 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 When working with chemicals alwa
13. NA cannot be avoided the QuantiTect Reverse Transcription Kit provides fast cDNA synthesis with integrated removal of genomic DNA contamination see ordering information page 30 Integrity of RNA The integrity and size distribution of total RNA purified with RNeasy Protect Animal Blood Kits can be checked by denaturing agarose gel electrophoresis and ethidium bromide staining or by using the QlAxcel Advanced system or Agilent 2100 bioanalyzer The respective ribosomal RNAs should appear as sharp bands or peaks The apparent ratio of 28S rRNA to 18S rRNA 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 The Agilent 2100 bioanalyzer also provides an RNA Integrity Number RIN as a useful measure of RNA integrity Ideally the RIN should be close to 10 but in many cases particularly with tissue samples RNA quality is greatly influenced by how well the original sample was preserved When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier 26 RNeasy Protect Animal Blood Handbook 06 2012 Appendix C Purification of Small RNAs lt 200 nt from RNAprotect Stabilized Animal Blood This protocol
14. Protect Animal Blood Handbook 06 2012 11 Protocol Purification of Total RNA gt 200 Nucleotides Excluding miRNA from RNAprotect Stabilized Animal Blood This protocol is for purification of total RNA that excludes small RNAs such as miRNA and requires the RNeasy Protect Animal Blood Kit The protocol is for use with blood samples collected in RNAprotect Animal Blood Tubes It is important to collect blood as described in the product sheet supplied with RNAprotect Animal Blood Tubes If transporting the tubes be sure to handle the tubes according to the recommendations in the product sheet lt Z A 3 Ale Important points before starting E Ifusing the RNeasy Protect Animal Blood Kit for the first time read Safety Information page 5 and Important Notes page 11 E If preparing RNA for the first time read Appendix A page 22 EB Buffer RBT and Buffer RW 1 contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach See page 5 for safety information MH Unless otherwise indicated perform all steps of the procedure at room temperature 15 25 C During the procedure work quickly E fusing a refrigerated microcentrifuge make sure cooling is switched off i e set the temperature to just above ambient temperature Make sure the microcentrifuge is at room temperature Things to do before starting E Ensure that RNAprotect Animal Blood Tubes are incubated for at
15. RBT or Buffer RW1 and is therefore not compatible with bleach See page 5 for safety information 14 RNeasy Protect Animal Blood Handbook 06 2012 16 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 VN P101 lt 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 17 Place the RNeasy MinElute spin column in a new 1 5 ml collection tube and pipet 14 30 pl Buffer REB directly onto the spin column membrane Close the lid gently and centrifuge for 1 min at 8000 x g 10 000 rpm to elute the RNA Be sure to add Buffer REB directly to the spin column membrane This wets the entire membrane ensuring maximum elution efficiency 18 Incubate the RNA eluate for 5 min at 65 C in the shaker incubator without shaking After incubation chill immediately on ice This incubation at 65 C denatures the RNA for downstream applications Do not exceed the incubation ti
16. RNAprotect Animal Blood Tubes 100 pl 50 RNAprotect Animal Blood Tubes 500 pl 50 4 RNeasy Protect Animal Blood Handbook 06 2012 Shipping and Storage The RNeasy Protect Animal Blood Kit is shipped at ambient temperature The RNeasy MinElute spin columns and RNase Free DNase Set box containing RNase free DNase Buffer RDD and RNase free water should be stored immediately upon receipt at 2 8 C The remaining components of the kit should be stored dry at room temperature 15 25 C All kit components are stable for at least 9 months under these conditions Intended Use The RNeasy Protect Animal Blood Kit is intended for molecular biology applications This product is not intended for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate safety data sheets SDSs These are available online in convenient and compact PDF format at www giagen com safety where you can find view and print the SDS for each QIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the
17. atant not completely removed after centrifuging RNAprotect Animal Blood Tube fl Blood incubated for less than 2 h in RNAprotect Animal Blood Tubes g RNeasy MinElute spin columns incorrectly stored Low Az40 Azs value Water used to dilute RNA for Asl Argo measurement 20 If using RNAprotect Animal Blood Tubes 100 yl do not collect less than the 100 pl blood If using RNAprotect Animal Blood Tubes 500 pl do not collect less than the 500 pl blood For accurate quantification RNA must be diluted in 10 mM Tris Cl pH 7 0 see Appendix B page 24 Repeat RNA elution but incubate the RNeasy MinElute spin column on the benchtop for 10 min with Buffer REB before centrifuging Be sure to vortex the pellet at step 3 of the protocol however the presence of small debris does not affect the procedure Be sure to remove the entire supernatant If decanting the supernatant remove drops from the rim of the tube by dabbing onto a paper towel After collecting blood in RNAprotect Animal Blood Tubes be sure to incubate for at least 2 h at room temperature 15 25 C Store RNeasy MinElute spin columns at 2 8 C Use 10 mM Tris Cl pH 7 5 not RNase free water to dilute the sample before measuring purity see Appendix B page 24 RNeasy Protect Animal Blood Handbook 06 2012 Comments and suggestions RNA degraded RNase contamination Although all RNeasy buffers have been tested and are guaranteed RNase
18. ation consult the appropriate safety data sheets SDSs available from the product supplier t In addition dilute 1 pl Buffer REB with 499 pl of 10 mM Tris Cl pH 7 0 and use this to zero the spectrophotometer 24 RNeasy Protect Animal Blood Handbook 06 2012 Concentration of RNA sample 44 pg ml x Aygo x dilution factor 44 pg ml x 0 2 x 500 4400 pg ml Total amount concentration x volume in milliliters 4400 pg ml x 0 01 ml 44 pg of RNA Purity of RNA The ratio of the readings at 260 nm and 280 nm Ao60 Aogo provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV spectrum such as protein However the A260 A2s0 ratio is influenced considerably by pH Since water is not buffered the pH and the resulting A260 A2s0 ratio can vary greatly Lower pH results in a lower Asso Acgo 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 Ayeo Aogo 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
19. be for at least 2 h at room temperature 15 25 C after blood collection to achieve complete lysis of blood cells Note If using an RNAprotect Animal Blood Tube 100 pl transfer the blood sample to a new 1 5 ml collection tube supplied the same tube can be used in step 5 Alternatively use only 450 pl RNase free water in step 2 below Remove the supernatant by decanting or pipetting Add 1 ml RNase free water to the pellet and close the tube If decanting the supernatant take care not to disturb the pellet and dry the rim of the tube with a clean paper towel Vortex until the pellet is visibly dissolved and centrifuge for 3 min at 5000 x g Remove the entire supernatant by decanting or pipetting and discard Small debris remaining in the sample after vortexing does not affect subsequent RNA purification Note Incomplete removal of the supernatant will inhibit proteinase K digestion and dilute the lysate affecting the conditions for binding RNA to the RNeasy MinElute membrane Add 240 pl Buffer RSB and vortex until the pellet is visibly dissolved Pipet the sample into a 1 5 ml collection tube supplied Add 200 pl Buffer RBT and 20 pl proteinase K Mix by vortexing for 5 s and incubate for 10 min at 55 C in a shaker incubator at 400 1400 rpm After incubation set the temperature of the shaker incubator to 65 C for step 18 Note Do not mix Buffer RBT and proteinase K together before adding them to the sample Pipet the sampl
20. e into a QlAshredder spin column lilac placed in a 2 ml collection tube and centrifuge for 3 min at full speed do not exceed 20 000 x g Carefully transfer the entire supernatant of the flow through from the QlAshredder spin column to a new 1 5 ml collection tube supplied without disturbing the pellet Add 240 pl ethanol 96 100 and mix by vortexing RNeasy Protect Animal Blood Handbook 06 2012 13 VNU P101 lt Z A 3 Ae Pipet the sample into an RNeasy MinElute spin column pink placed in a 2 ml collection tube Close the lid gently and centrifuge for 1 min at 8000 x g 210 000 rpm Discard the flow through Reuse the collection tube in step 10 Add 350 pl Buffer RW1 to the RNeasy MinElute spin column Close the lid gently and centrifuge for 15 s at 8000 x g 10 000 rpm Discard the flow through Reuse the collection tube in step 13 Note After centrifugation carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow through Be sure to empty the collection tube completely Add 10 pl DNase I stock solution to 70 pl Buffer RDD in a 1 5 ml microcentrifuge tube not supplied Mix by gently inverting the tube and centrifuge briefly to collect residual liquid from the sides of the tube Note DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube Do not vortex Pipe
21. ed system www giagen com QlAxcel or Agilent 2100 bioanalyzer fluorometric quantification or quantitative real time RT PCR 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 Aggo 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 25 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 23 When zeroing the spectrophotometer be sure fo use a solution that contains Buffer REB at the same dilution as the RNA solution to be quantified An example of the calculation involved in RNA quantification is shown below Volume of RNA sample 10 pl Dilution 1 pl of RNA sample 499 pl of 10 mM Tris Cl pH 7 0 1 500 dilution Measure absorbance of diluted sample in a 1 ml cuvette RNase free Azs 0 2 When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more inform
22. fety data sheets SDSs available from the product supplier E For 100 pl blood samples RNAprotect Animal Blood Tubes 50 x 100 pl cat no 76544 E For 500 pl blood samples RNAprotect Animal Blood Tubes 50 x 500 yl cat no 76554 E Ethanol 96 100 and 80 E Ethanol 100 E Sterile RNase free pipet tips Microcentrifuge tubes 1 5 ml E ice MH Microcentrifuge with rotor for 2 ml tubes BE Shaker incubator capable of incubating at 55 C and 65 C and shaking at 400 rpm but not exceeding 1400 rpm e g Eppendorf Thermomixer Compact or equivalent E Vortexer E If using the protocol Purification of Total RNA Including miRNA from RNAprotect Stabilized Animal Blood Buffer RWT concentrate 80 ml cat no 1067933 Do not use denatured alcohol which contains other substances such as methanol or methylethylketone t If using the protocol Purification of Total RNA Including miRNA from RNAprotect Stabilized Animal Blood This is not a complete list of suppliers and does not include many important vendors of biological supplies p pp y Imp g pp 10 RNeasy Protect Animal Blood Handbook 06 2012 Important Notes Collection of starting material Before using the RNeasy Protect Animal Blood Kit it is important to collect blood samples as described in the product sheet supplied with RNAprotect Animal Blood Tubes see ordering information page 30 The kits are designed specifically for use w
23. gh Otherwise carryover of ethanol will occur 16 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 VNA P101 2 Qa S a E A Z gt 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 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 17 Place the RNeasy MinElute spin column in a new 1 5 ml collection tube and pipet 14 30 pl Buffer REB directly onto the spin column membrane Close the lid gently and centrifuge for 1 min at 8000 x g 10 000 rpm to elute the RNA Be sure to add Buffer REB directly to the spin column membrane This wets the entire membrane ensuring maximum elution efficiency 18 Incubate the RNA eluate for 5 min at 65 C in the shaker incubator without shaking After incubation chill immediately on ice This incubation at 65 C denatures the RNA for downstream applications Do not exceed the incubation time or temperature 19 If the RNA eluate will not be used immediate
24. in column placed in a 2 ml collection tube Close the lid gently and centrifuge for 15 s at 8000 x g 210 000 rpm Discard the flow through Repeat step C4 until the whole sample has passed through the membrane Discard the flow through each time C5 Add 500 pl Buffer RPE to the RNeasy MinElute spin column Close the lid gently and centrifuge for 15 s at 8000 x g 10 000 rpm Discard the flow through C6 Add 500 pl of 80 ethanol to the RNeasy MinElute spin column Close the lid gently and centrifuge for 2 min at 8000 x g 210 000 rpm to dry the spin column membrane Discard the flow through and collection tube Note After centrifugation remove the RNeasy MinElute spin column from the collection tube carefully so that the column does not contact the flow through Otherwise carryover of ethanol will occur C7 Place the RNeasy MinElute spin column in a new 2 ml collection tube Open the lid and centrifuge for 5 min at 8000 x g 210 000 rpm 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 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
25. ith animal rat and mouse blood collected in RNAprotect Animal Blood Tubes The kits should not be used with unstabilized blood or with blood stabilized by other methods When col lecting blood with RNAprotect Animal Blood Tubes be sure to collect the specified vol ume of blood i e 100 pl or 500 pl Collection of larger or smaller volumes of blood may lead to incomplete RNA stabilization and RNA yields may be significantly lower than expected The specifications of the RNeasy MinElute spin column and the typical yields of total RNA that can be achieved with it are shown in Tables 1 and 2 Table 1 RNeasy MinElute spin column specifications Maximum binding capacity A5 pg RNA Maximum loading volume 700 pl Minimum elution volume 14 pl Table 2 Typical yields of total RNA with RNeasy Protect Animal Blood Kits Sample type Yield of total RNA yg Rat blood 500 yl 10 30 Mouse blood 100 pl 4 8 Amounts can vary due to factors such as species age and health of the animal Since the RNeasy Protect Animal Blood Kit enriches for mRNA and other RNA species gt 200 nucleotides the total RNA yield obtained with this kit does not include miRNA 5S rRNA tRNA and other low molecular weight RNAs which make up 15 20 of total cellular RNA To purify RNA species smaller than 200 nt follow the protocol Purification of Total RNA Including miRNA from RNAprotect Stabilized Animal Blood on page 16 of the handbook RNeasy
26. ity RNA For more information about the automated procedure see the relevant protocol sheet available at www giagen com MyQlAcube 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 at www giagen com MyQlAcube 8 RNeasy Protect Animal Blood Handbook 06 2012 RNeasy Protect Animal Blood Procedure Animal blood collected in RNAprotect Animal Blood Tubes Wash blood pellet with water and then resuspend in Buffer RSB Digest in Buffer RBT with proteinase K Homogenize by centrifugation in QlAshredder column and then add ethanol ale Bind RNA to RNeasy MinElute column and then digest DNA Total RNA Wash with Buffer Wash with Buffer RW1 and Buffer RPE RWT and Buffer RPE Elute with Elute with Buffer REB Buffer REB Total RNA gt 200 nt Total RNA including miRNA Included in RNeasy Protect Animal Blood Kit t Ordered separately Refer to ordering information on page 30 ZZ a ss srjyij w m gt gt gt gt m m mmzz zz rjzsz z zss GG RNeasy Protect Animal Blood Handbook 06 2012 9 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 sa
27. least 2 h at room temperature 15 25 C after blood collection to ensure complete lysis of blood cells Incubating the tubes overnight may increase RNA yields If the tubes were stored at 2 8 C or below directly after blood collection first equilibrate the tubes to room temperature and then store at room temperature for 2 h before starting the procedure M Buffer RPE is supplied as a concentrate Before using for the first time add 4 volumes of ethanol 96 100 as indicated on the bottle to obtain a working solution HE Buffer RBT may form a precipitate during storage If necessary redissolve by warming at 37 C and then place at room temperature E Ifusing the RNase Free DNase Set for the first time prepare DNase stock solution Dissolve the lyophilized DNase 1500 Kunitz units in 550 pl of the RNase free water provided with the set To avoid loss of DNase I 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 12 RNeasy Protect Animal Blood Handbook 06 2012 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 Procedure 1 Centrifuge the RNAprotect Animal Blood Tube for 3 min at 5000 x g Note Be sure to incubate the tu
28. ly store at 20 C or 70 C Since the RNA remains denatured after freezing and thawing it is not necessary to repeat the incubation at 65 C Note If quantifying the RNA using a spectrophotometer refer to the guidelines in Appendix B page 24 Buffer REB shows high absorbance at 220 nm which can lead to high background absorbance levels if the spectrophotometer is not properly zeroed 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 and www qiagen com miRNA RNeasy Protect Animal Blood Handbook 06 2012 19 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www giagen com FAQ FAQList aspx 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 giagen com Comments and suggestions Low RNA yield a Insufficient blood collected in RNAprotect Animal Blood Tube b RNA diluted in water for A measurement c RNA still bound to RNeasy MinElute spin column membrane d Cell debris transferred to RNeasy MinElute spin column e Supern
29. ly and centrifuge for 1 min at 8000 x g 210 000 rpm Discard the flow through Transfer the remaining sample repeat the centrifugation and discard the flow through Reuse the collection tube in step 10 10 Add 350 pl Buffer RWT to the RNeasy MinElute spin column Close the lid gently and centrifuge for 15 s at 8000 x g 10 000 rpm Discard the flow through Reuse the collection tube in step 13 Note Buffer RWT is supplied as a concentrate Ensure that ethanol is added to Buffer RWT before use see Things to do before starting Note It is essential to use Buffer RWT Do not use Buffer RW 1 and do not omit this washing step lt Z 2 O Including miRNA Note After centrifugation carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow through Be sure to empty the collection tube completely 11 Add 10 pl DNase I stock solution to 70 pl Buffer RDD in a 1 5 ml microcentrifuge tube not supplied Mix by gently inverting the tube and centrifuge briefly to collect residual liquid from the sides of the tube Note DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube Do not vortex 12 Pipet the DNase I incubation mix 80 pl directly onto 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 mi
30. me or temperature 19 If the RNA eluate will not be used immediately store at 20 C or 70 C Since the RNA remains denatured after freezing and thawing it is not necessary to repeat the incubation at 65 C Note If quantifying the RNA using a spectrophotometer refer to the guidelines in Appendix B page 24 Buffer REB shows high absorbance at 220 nm which can lead to high background absorbance levels if the spectrophotometer is not properly zeroed 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 RNeasy Protect Animal Blood Handbook 06 2012 15 Protocol Purification of Total RNA Including miRNA from RNAprotect Stabilized Animal Blood This protocol is for purification of total RNA that includes miRNA and other small RNAs e g tRNA and requires the RNeasy Protect Animal Blood Kit and Buffer RWT ordered separately The protocol is for use with blood samples collected in RNAprotect Animal Blood Tubes It is important to collect blood as described in the product sheet supplied with RNAprotect Animal Blood Tubes If transporting the tubes be sure to handle the tubes according to the recommendations in the product sheet Important points before starting MH If using the RNeasy Protect Animal Blood Kit for the first time read Safety Information page 5 and Important Note
31. 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 When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier RNeasy Protect Animal Blood Handbook 06 2012 23 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 Buffer REB Under these conditions no degradation of RNA is detectable after 1 year Quantification of RNA The concentration of RNA can be determined by measuring the absorbance at 260 nm Asso in a spectrophotometer see Spectrophotometric quantification of RNA below For small amounts of RNA however it may not be possible to accurately determine amounts photometrically Small amounts of RNA can be quantified using the QlAxcel Advanc
32. re Blood samples are collected in RNAprotect Animal Blood Tubes which contain a reagent that lyses blood cells and immediately stabilizes intracellular RNA fo preserve the gene expression profile RNA stabilization is critical for reliable downstream gene expression analysis as degradation of RNA and upregulation or downregulation of transcripts RNeasy Protect Animal Blood Handbook 06 2012 7 occur straightaway after blood is drawn from the animal Blood samples collected in RNAprotect Animal Blood Tubes can be safely stored or transported at 15 25 C for up to 48 hours at 2 8 C for up to 2 weeks or at 20 C for at least 3 months without showing any significant RNA degradation or changes in transcript levels Total RNA is purified from the stabilized blood samples using well established RNeasy silica membrane technology RNAprotect Animal Blood Tubes are first centrifuged to pellet the samples which are then washed with water and resuspended in Buffer RSB After digestion in Buffer RBT with proteinase K the samples are homogenized by centrifugation in QlAshredder spin columns Ethanol is added to the samples to optimize binding conditions and the samples are then centrifuged in RNeasy MinElute spin columns where total RNA binds to the RNeasy MinElute silica membrane The total RNA that is bound to the membrane is subjected to DNase digestion to remove genomic DNA contamination and washed with Buffer RW1 RNeasy Kit or Buffer RWT ordered
33. s page 11 lt Z 2 O Including miRNA Itis essential to use Buffer RWT ordered separately for steps 10 and 13 of the protocol Do not use Buffer RW1 for these steps E If preparing RNA for the first time read Appendix A page 22 Buffer RBT and Buffer RWT contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach See page 5 for safety information M Unless otherwise indicated perform all steps of the procedure at room temperature 15 25 C During the procedure work quickly MH If using a refrigerated microcentrifuge make sure cooling is switched off i e set the temperature to just above ambient temperature Make sure the microcentrifuge is at room temperature Things to do before starting E Ensure that RNAprotect Animal Blood Tubes are incubated for at least 2 h at room temperature 15 25 C after blood collection to ensure complete lysis of blood cells Incubating the tubes overnight may increase RNA yields If the tubes were stored at 2 8 C or below directly after blood collection first equilibrate the tubes to room temperature and then store at room temperature for 2 h before starting the procedure BE Buffers RWT and RPE are supplied as concentrates Before using for the first time add the required volume of ethanol 96 100 as indicated on the bottle to obtain a working solution E Buffer RBT may form a precipitate during storage If necessary
34. s expressed or implied other than those expressly stated The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and or its components For updated license terms see www giagen com 2009 2012 QIAGEN all rights reserved www giagen com Australia techservice au giagen com Austria techservice at qiagen com Belgium techservice bni qiagen com Brazil suportetecnico brasil giagen com Canada techservice ca qiagen com China techservice cn qiagen com Denmark techservice nordic giagen com Finland techservice nordic qiagen com France techservice fr qiagen com Germany techservice de qiagen com Hong Kong techservice hk qiagen com India techservice india qiagen com Ireland techservice uk qiagen com Italy techservice it giagen com Japan techservice jp giagen com Korea South techservice kr qiagen com Luxembourg techservice bnl giagen com Mexico techservice mx qiagen com The Netherlands techservice bnl giagen com Norway techservice nordic qiagen com Singapore techservice sg qiagen com Sweden techservice nordic qiagen com S
35. sample preparation waste Buffer RBT contains guanidine thiocyanate and Buffer RW1 contains a small amount of guanidine thiocyanate Guanidine salts can form highly reactive compounds when combined with bleach If liquid containing these buffers is 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 1 v v sodium hypochlorite 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 613 1 19240 RNeasy Protect Animal Blood Handbook 06 2012 5 Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of the RNeasy Protect Animal Blood Kit is tested against predetermined specifications to ensure consistent product quality 6 RNeasy Protect Animal Blood Handbook 06 2012 Introduction The RNeasy Protect Animal Blood System provides a complete solution for stabilization and purification of high quality RNA from animal blood Blood from small animals is conveniently collected and stabilized in RNAprotect Animal Blood Tubes Total RNA is then purified using the RNeasy Protect Animal Blood Kit To obtain microRNA miRNA either in a total RNA fraction or in a small RNA fraction please refer to the protocol Purification of Total RNA
36. separately followed by Buffer RPE Pure RNA is then eluted in Buffer REB When following the protocol Purification of Total RNA Including miRNA from RNAprotect Stabilized Animal Blood all RNA molecules longer than 200 nucleotides are purified see protocol on page 16 This is an enrichment procedure for mRNA since most RNAs lt 200 nucleotides such as miRNA 5 85 rRNA 5S rRNA and tRNAs which together make up 15 20 of total RNA are selectively excluded When following the protocol Purification of Total RNA Including miRNA from RNAprotect Stabilized Animal Blood all RNA molecules longer than approximately 18 nucleotides are purified see protocol on page 16 The purified RNA includes both mRNA and small RNAs such as miRNA Alternatively the RNeasy Protect Animal Blood Kit can be used in combination with the RNeasy MinElute Cleanup Kit to purify a small RNA fraction containing miRNA and other RNAs shorter than 200 nucleotides see Appendix C page 27 Automated purification Purification of RNA can be fully automated on the QlAcube 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 RNeasy Protect Animal Blood Kits for purification of high qual
37. t Stabilized Animal Blood 27 Ordering Information 30 RNeasy Protect Animal Blood Handbook 06 2012 3 Kit Contents RNeasy Protect Animal Blood Kit 50 Catalog no 73224 Number of preps 50 RNeasy MinElute Spin Columns pink in 2 ml Collection Tubes 50 QlAshredder Spin Columns lilac in 2 ml Collection Tubes 50 Collection Tubes 1 5 ml 150 Collection Tubes 2 ml 50 Buffer RSB resuspension buffer 20 ml Buffer RBT binding buffer 18 ml Buffer RW 1 wash buffer A5 ml Buffer RPE wash buffer concentrate 11 ml Buffer REB elution buffer 5 ml RNase Free Water 100 ml Proteinase K 1 25 ml RNase Free DNase Set E RNase Free DNase lyophilized 1500 units E Buffer RDD DNA digestion buffer 2x2 ml E RNase Free Water 1 5 ml Quick Start Protocol 1 Contains a guanidine salt Not compatible with disinfectants containing bleach See page 5 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 Note RNAprotect Animal Blood Tubes are supplied separately Blood samples must be collected in RNAprotect Animal Blood Tubes For details see Introduction page 7 Note The protocol Purification of Total RNA Including miRNA from RNAprotect Stabilized Animal Blood requires Buffer RWT ordered separately cat no 1067933 RNAprotect Animal Blood Tubes 50 x 100 pl 50 x 500 pl Catalog no 76544 76554
38. t the DNase I incubation mix 80 pl directly onto 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 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 13 Add 350 pl Buffer RW1 to the RNeasy MinElute spin column Close the lid gently and centrifuge for 15 s at 8000 x g 10 000 rpm Discard the flow through Reuse the collection tube in step 14 Note After centrifugation carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow through Be sure to empty the collection tube completely Add 500 pl Buffer RPE to the RNeasy MinElute spin column Close the lid gently and centrifuge for 15 s at 8000 x g 10 000 rpm Discard the flow through Reuse the collection tube in step 15 Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use see Things to do before starting Add 500 pl of 80 ethanol to the RNeasy MinElute spin column Close the lid gently and centrifuge for 2 min at 8000 x g 210 000 rpm 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 Flow through contains Buffer
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40. x directly to the 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 13 Add 350 pl Buffer RWT to the RNeasy MinElute spin column Close the lid gently and centrifuge for 15 s at 8000 x g 10 000 rpm Discard the flow through Reuse the collection tube in step 14 Note It is essential to use Buffer RWT Do not use Buffer RW1 and do not omit this washing step Note After centrifugation carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow through Be sure to empty the collection tube completely Flow through contains Buffer RBT or Buffer RWT and is therefore not compatible with bleach See page 5 for safety information 18 RNeasy Protect Animal Blood Handbook 06 2012 14 Add 500 pl Buffer RPE to the RNeasy MinElute spin column Close the lid gently and centrifuge for 15 s at 8000 x g 10 000 rpm Discard the flow through Reuse the collection tube in step 15 Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use see Things to do before starting 15 Add 500 pl of 80 ethanol to the RNeasy MinElute spin column Close the lid gently and centrifuge for 2 min at 8000 x g 10 000 rpm Note After centrifugation carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow throu
41. ys wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier 22 RNeasy Protect Animal Blood Handbook 06 2012 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 as described in Solutions below Solutions Note QIAGEN solutions such as RNeasy buffers are guaranteed RNase free without using DEPC treatment and are therefore free of any DEPC contamination 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
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