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Sample & Assay Technologies AllPrep® DNA/RNA Micro

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1. 58 AllPrep DNA RNA Micro Handbook 07 2007 Product Contents Cat no QuantiTect Reverse Transcription Kit for fast cDNA synthesis for sensitive real time two step RT PCR QuantiTect Reverse For 50 x 20 ul reverse transcription 205311 Transcription Kit 50 reactions gDNA Wipeout Buffer Quantiscript Reverse Transcriptase Quantiscript RT Buffer RT Primer Mix RNase Free Water QuantiTect Primer Assays for use in real time RT PCR with SYBR Green detection search for and order assays at www giagen com GeneGlobe QuantiTect Primer For 200 x 50 ul reactions or 400 x Varies Assay 200 25 ul reactions 10x QuantiTect Primer Assay lyophilized QuantiFast SYBR Green PCR Kit for fast SYBR Green based real time PCR and two step RT PCR QuantiFast SYBR Green For 400 x 25 ul reactions 3x 1 7 ml 2x 204054 PCR Kit 400 Master Mix contains ROX dye 2 x 2 ml RNase Free Water QuantiFast SYBR Green RT PCR Kit for fast SYBR Green based real time one step RT PCR QuantiFast SYBR Green For 400 x 25 ul reactions 3x 1 7 ml 2x 204154 RT PCR Kit 400 Master Mix contains ROX dye 100 ul RT Mix 2 x 2 ml RNase Free Water RNAprotect Cell Reagent RNAlater RNA Stabilization Reagent RNAlater TissueProtect Tubes Allprotect Tissue Reagent AllPrep Kits the DNeasy Blood amp Tissue Kit the RNeasy Fibrous Tissue Mini Kit the RNeasy FFPE Kit REPLI g Kits the QIAGEN Fast Cycling PCR Kit the QuantiTect Whole T
2. 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 D6 Place the RNeasy MinElute spin column in a new 1 5 ml collection tube supplied 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 full speed to elute the RNA total RNA containing small RNAs As little as 10 ul RNase free water can be used for elution if a higher RNA concentration is required but the yield will be reduced by approximately 20 Do not elute with less than 10 ul RNase free water as the spin column membrane will not be sufficiently hydrated The dead volume of the RNeasy MinElute spin column is 2 ul elution with 14 ul RNase free water results in a 12 pl eluate For real time RT PCR with the purified RNA QIAGEN offers the miScript System which allows detection of hundreds of miRNAs from a single cDNA synthesis reaction For details visit www qiagen com miRNA 52 AllPrep DNA RNA Micro Handbook 07 2007 Appendix E Acetone Precipitation of Protein from Lysates The following procedure describes how to recover denatured protein by acetone precipitation from lysates of cells and easy to lyse tissues Reagents to be supplied by user E Ice E Acetone E Optional Ethanol E Buffer for downstream application e g loading buf
3. Total RNA purification 5 Add 1 volume usually 350 pl of 70 ethanol to the flow through from step 4 and mix well by pipetting Do not centrifuge Proceed immediately to step 6 Note The volume of 70 ethanol to add may be less than 350 ul if some lysate was lost during homogenization and DNA isolation Note Precipitates may be visible after addition of ethanol but this does not affect the procedure Note For maximum RNA yields from liver use 5096 ethanol instead of 7096 ethanol 6 Transfer the sample including any precipitate that may have formed to an RNeasy MinElute spin column placed in a 2 ml collection tube supplied Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm Discard the flowthrough Optional If recovery of protein is desired keep the flow through on ice and follow steps E1 E5 in Appendix E on page 53 Reuse the collection tube in step 7 7 Add 700 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 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 Optional If purifying RNA from tissues with high DNA content and if the RNA will be used in sensitive downstream a
4. Before using the kit for the first time prepare 8096 ethanol by mixing 24 ml ethanol 96 100 and 6 ml RNase free water supplied The procedure also requires 7096 ethanol which can be prepared by diluting ethanol 96 100 with distilled water not supplied Buffer RLT Plus may form a precipitate upon storage If necessary redissolve by warming and then place at room temperature Preheat Buffer EB to 70 C to ensure optimal DNA elution Procedure Sample disruption and homogenization l 36 Collect the sample directly into an appropriate volume of Buffer RLT Plus the volume depends on the collection vessel used for the microdissection but should not be greater than 65 pl Leica instruments or 300 pl other instruments Note Ensure that B ME or DTT is added to Buffer RLT Plus before use see Things to do before starting If necessary transfer the sample and buffer to a larger vessel e g 1 5 or 2 ml tube Adjust the volume to 350 ul with Buffer RLT Plus Note If processing 500 cells 20 ng carrier RNA 5 ul of a 4 ng ul solution may be added to the lysate before homogenization Prepare the carrier RNA as described in Things to do before starting AllPrep DNA RNA Micro Handbook 07 2007 3 Vortex the sample for 30 s No further homogenization is necessary 4 Transfer the sample to an AllPrep DNA spin column placed in a 2 ml collection tube supplied Close the lid gently and centrifuge fo
5. Spectrophotometric quantification of DNA below For small amounts of DNA however it may not be possible to accurately determine amounts photometrically Small amounts of DNA can be quantified using quantitative real time PCR or fluorometric quantification When purifying DNA from particularly small samples e g laser microdissected samples quantitative real time PCR should be used for quantification Spectrophotometric quantification of DNA DNA concentration can be determined by measuring the absorbance at 260 nm A240 in a spectrophotometer using a quartz cuvette For greatest accuracy readings should be between 0 1 and 1 0 Using a standard 1 cm path length an absorbance of 1 unit at 260 nm corresponds to 50 ug genomic DNA per ml Aygo 1 50 ug ml This relation is valid only for measurements made at neutral pH Therefore samples should be diluted in a low salt buffer with neutral pH e g Tris Cl pH 7 0 Use the buffer in which the DNA is diluted to zero the spectrophotometer An example of the calculation involved in DNA quantification is shown below Volume of DNA sample 100 ul Dilution 20 ul of DNA sample 180 pl of buffer 1 10 dilution Measure absorbance of diluted sample in a 0 2 ml cuvette Axo 0 2 Concentration of DNA sample 50 ug ml x A s x dilution factor 50 ug ml x 0 2 x 10 100 ug ml When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For
6. Table 3 gives an overview of various disruption and homogenization methods and is followed by a detailed description of each method AllPrep DNA RNA Micro Handbook 07 2007 15 Table 3 Disruption and homogenization methods Sample Disruption method Homogenization method Microdissected Addition of lysis buffer Vortexing samples Cells and fine Addition of lysis buffer TissueRuptor needle aspirates or QlAshredder homogenizer FNA or syringe and needle Tissues TissueRuptor TissueRuptor TissueLysert TissueLysert Mortar and pestle QlAshredder homogenizer or syringe and needle Simultaneously disrupts and homogenizes individual samples t Simultaneously disrupts and homogenizes up to 192 samples in parallel Results are comparable to those obtained using the TissueRuptor or other rotor stator homogenizer Disruption and homogenization using the TissueRuptor The TissueRuptor is a rotor stator homogenizer that thoroughly disrupts and simultaneously homogenizes single tissue samples in the presence of lysis buffer in 15 90 seconds depending on the toughness and size of the sample The TissueRuptor can also be used to homogenize cell lysates The blade of the TissueRuptor disposable probe rotates at a very high speed causing the sample to be disrupted and homogenized by a combination of turbulence and mechanical shearing For guidelines on using the TissueRuptor refer to the TissueRuptor Handbook For other rotor stator h
7. Flow through contains Buffer RLT Plus or Buffer RW1 and is therefore not compatible with bleach See page 7 for safety information 24 AllPrep DNA RNA Micro Handbook 07 2007 9 10 11 12 Add 500 pl Buffer RPE 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 10 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 28000 x g 210 000 rpm to wash the spin column membrane Discard the collection tube with the flow through Prepare the 80 ethanol with 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 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 collection tube with the flow through 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 dir
8. cells and tissues we recommend using the miRNeasy Mini Kit For details visit www giagen com miRNA Visit www qiagen com geneXpression for information on standardized solutions for gene expression analysis from QIAGEN 8 AllPrep DNA RNA Micro Handbook 07 2007 Northern dot and slot blot analyses Primer extension Poly A RNA selection RNase S1 nuclease protection S Microarrays Optionally the AllPrep DNA RNA procedure for cells can be modified to allow the purification of total RNA containing small RNAs such as miRNA see Appendix D page 51 In addition if processing cells and easy to lyse tissues optional steps for the AllPrep DNA RNA procedure allow the isolation of protein by acetone precipitation see Appendix E page 53 The protein is denatured and suitable for applications such as SDS PAGE and western blotting Principle and procedure The AllPrep DNA RNA procedure integrates QIAGEN s patented technology for selective binding of double stranded DNA with well established RNeasy technology Efficient purification of high quality DNA and RNA is guaranteed without the need for additional RNase and DNase digestions Biological samples are first lysed and homogenized in a highly denaturing guanidine isothiocyanate containing buffer which immediately inactivates DNases and RNases to ensure isolation of intact DNA and RNA The lysate is then passed through an AllPrep DNA spin column This column in combination with the h
9. elution but incubate the RNeasy RNeasy MinElute spin MinElute spin column on the benchtop for column membrane 10 min with RNase free water before centrifuging d DNA still bound to Repeat DNA elution but incubate the AllPrep AllPrep DNA spin DNA spin column on the benchtop for 10 min column membrane with Buffer EB before centrifuging e Ethanol carryover After the wash with 80 ethanol be sure to centrifuge at full speed for 5 min to dry the RNeasy MinElute spin column membrane 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 f Incomplete removal of When processing cultured cells ensure complete cell culture medium removal of cell culture medium after harvesting cell samples cells see protocol page 19 DNA contaminated with RNA a Lysate applied to the Add ethanol to the lysate after passing the lysate AllPrep DNA spin through the AllPrep DNA spin column column contains ethanol b Sample is affecting pH The final homogenate should have a pH of 7 of homogenate Make sure that the sample is not highly acidic or basic Contamination of RNA with DNA affects downstream applications a Cell number too high For some cell types the efficiency of DNA binding to the AllPrep DNA spin column may be reduced when processing very high cell numbers If the eluted RNA contains subs
10. meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www giagen com Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology and the use of QIAGEN products If you have any questions or experience any difficulties regarding the AllPrep DNA RNA Micro Kit or QIAGEN products in general please do not hesitate to contact us QIAGEN 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 Ser
11. more information consult the appropriate material safety data sheets MSDSs available from the product supplier AllPrep DNA RNA Micro Handbook 07 2007 49 Total amount concentration x volume of sample in milliliters 100 ug ml x 0 1 ml 10 ug of DNA RNA concentration can also be determined by measuring the absorbance at 260 nm If the eluate contains both DNA and RNA a fluorometer must be used to quantify the DNA Determination of DNA purity The ratio of the readings at 260 nm and 280 nm As o A280 provides an estimate of DNA purity with respect to contaminants that absorb UV light such as protein The A26o Argo ratio is influenced considerably by pH Since water is not buffered the pH and the resulting A A280 ratio can vary greatly Lower pH results in a lower A 4 A2g0 ratio and reduced sensitivity to protein contamination For accurate A o Argo values we recommend measuring absorbance in a slightly alkaline buffer e g 10 mM Tris Cl pH 7 5 Make sure to zero the spectrophotometer with the appropriate buffer Pure DNA has an A so Aggo ratio of 1 7 1 9 Scanning the absorbance from 220 320 nm will show whether there are contaminants affecting absorbance at 260 nm Absorbance scans should show a peak at 260 nm and an overall smooth shape Determination of DNA length The precise length of genomic DNA can be determined by pulsed field gel electrophoresis PFGE through an agarose gel The DNA should be concentr
12. steps 6 12 in this protocol Note Do not store the AllPrep DNA spin column at room temperature or at 4 C for long periods Do not freeze the column Total RNA purification 6 Add 1 volume usually 350 pl of 70 ethanol to the flow through from step 5 and mix well by pipetting Do not centrifuge Proceed immediately to step 7 Note The volume of 70 ethanol to add may be less than 350 ul if some lysate was lost during homogenization and DNA isolation Note When purifying RNA from certain cell lines precipitates may be visible after addition of ethanol This does not affect the procedure 7 Transfer the sample including any precipitate that may have formed to an RNeasy MinElute spin column placed in a 2 ml collection tube supplied Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm Discard the flow through Optional If recovery of protein is desired keep the flow through on ice and follow steps E1 E5 in Appendix E on page 53 Reuse the collection tube in step 8 8 Add 700 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 9 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
13. then centrifuge for 1 min at 28000 x g 10 000 rpm to elute the DNA 16 Repeat step 15 to elute further DNA To prevent dilution of the first DNA eluate use a new 1 5 ml collection tube not supplied to collect the second DNA eluate To combine the first and second DNA eluates reuse the collection tube from step 15 Note To achieve a higher DNA concentration elute with 2 x 30 ul Buffer EB The final DNA yield however may be reduced For PCR and real time PCR with the purified DNA QIAGEN offers a range of optimized ready to use kits that provide highly specific and sensitive results For details visit www qiagen com PCR QIAGEN kits and services for whole genome amplification WGA of limited amounts of DNA are also available For details visit www giagen com WGA Flow through contains Buffer AW1 and is therefore not compatible with bleach See page 7 for safety information AllPrep DNA RNA Micro Handbook 07 2007 39 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 for contact information see back cover or visit www qiagen com Comments and suggestions Clogged AllPrep DNA or RNeasy MinElute spin column a Inefficient disruption See Disrupting and homogenizing start
14. 1 ml Buffer RLT Plus Dispense in a fume hood and wear appropriate protective clothing Buffer RLT Plus containing B ME can be stored at room temperature 15 25 C for up to 1 month Alternatively AllPrep DNA RNA Micro Handbook 07 2007 35 add 20 ul of 2 M dithiothreitol DTT per 1 ml Buffer RLT Plus The stock solution of 2 M DTT in water should be prepared fresh or frozen in single use aliquots Buffer RLT Plus containing DTT can be stored at room temperature for up to 1 month When processing lt 500 cells carrier RNA may be added to the lysate before homogenization see Carrier RNA page 17 Before using for the first time dissolve the carrier RNA 310 ug in 1 ml RNase free water Store this stock solution at 20 C and use it to make fresh dilutions for each set of preps The concentration of this stock solution is 310 ug ml i e 310 ng ul To make a working solution 4 ng Ll for 10 preps add 5 ul stock solution to 34 ul Buffer RLT Plus and mix by pipetting Add 6 ul of this diluted solution to 54 ul Buffer RLT Plus to give a working solution of 4 ng ul Add 5 ul of this solution to the lysate in step 2 Do not add the carrier RNA to the lysate if purifying RNA for use in oligo dT based amplification Buffer RPE Buffer AW1 and Buffer AW2 are each supplied as a concentrate Before using for the first time add the appropriate volume of ethanol 96 10096 as indicated on the bottle to obtain a working solution
15. A RNA procedure enriches for mRNA and other RNA species gt 200 nucleotides the total RNA yield does not include 5S rRNA tRNA and other low molecular weight RNAs which make up 15 2096 of total cellular RNA Handling and storing starting material Cells After harvesting cells should be immediately lysed in Buffer RLT Plus to prevent unwanted changes in the gene expression profile This highly denaturing lysis buffer inactivates RNases and other proteins to prevent RNA degradation as well as downregulation or upregulation of transcripts If the cells are to be shipped to another lab for nucleic acid purification they should be pelleted frozen in liquid nitrogen and transported on dry ice Alternatively the cells can be mixed with RNAprotect Cell Reagent at room temperature and then shipped at ambient temperature ENR 14 AllPrep DNA RNA Micro Handbook 07 2007 Tissues RNA in harvested tissue is not protected until the sample is treated with RNAlater RNA Stabilization Reagent flash frozen or disrupted and homogenized in the presence of RNase inhibiting or denaturing reagents Otherwise unwanted changes in the gene expression profile will occur It is therefore important that tissue samples are immediately frozen in liquid nitrogen and stored at 70 C or immediately immersed in RNAlater RNA Stabilization Reagent An alternative to RNAlater RNA Stabilization Reagent is Allprotect Tissue Reagent which provides immediate stabilizat
16. July 2007 AllPrep DNA RNA Micro Handbook For simultaneous purification of genomic DNA and total RNA from the same small sample including animal and human cells lt 5 x 10 animal and human tissues lt 5 mg microdissected cryosections QIAGEN Sample amp Assay Technologies Trademarks QIAGEN AllPrep Allprotect DNeasy GeneGlobe MinElute QuantiFast Quantiscript QuantiTect REPLI g RNAprotect RNeasy QIAGEN Group Agilent Agilent Technologies Inc Applied Biosystems Applera Corporation or its subsidiaries Leica Leica Microsystems GmbH LightCycler Roche Group SYBR Molecular Probes Inc RNAlater is a trademark of AMBION Inc Austin Texas and is covered by various U S and foreign patents 2007 QIAGEN all rights reserved Contents Kit Contents 5 Storage 5 Quality Control 5 Product Use Limitations 6 Product Warranty and Satisfaction Guarantee 6 Technical Assistance 6 Safety Information 7 Introduction 8 Principle and procedure 9 Equipment and Reagents to Be Supplied by User 11 Important Notes 12 Determining the amount of starting material 12 Handling and storing starting material 14 Disrupting and homogenizing starting material 15 Carrier RNA 17 Limitations of small samples 18 Protocols B Simultaneous Purification of Genomic DNA and Total RNA from Animal and Human Cells 19 B Simultaneous Purification of Genomic DNA and Tot
17. NA Micro Handbook 07 2007 51 Repeat step D2 until the whole sample has passed through the membrane Discard the flow through each time Reuse the collection tube in step D3 D3 Add 500 ul Buffer RPE 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 D4 Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use see Things to do before starting D4 Add 500 pl Buffer RPE to the RNeasy MinElute spin column Close the lid gently and centrifuge for 2 min at 28000 x g 10 000 rpm to wash the spin column membrane Discard the collection tube with the flow through 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 D5 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 collection tube with the flow through 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
18. RNase free 0 0 2 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 46 AllPrep DNA RNA Micro Handbook 07 2007 Concentration of RNA sample 44 ug ml x Aso x dilution factor 44 ug ml x 0 2 x 50 440 ug ml concentration x volume in milliliters 440 ug ml x 0 1 ml 44 ug of RNA Total amount Purity of RNA The ratio of the readings at 260 nm and 280 nm A6o Az90 provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV spectrum such as protein However the Aj49 Az 9 ratio is influenced considerably by pH Since water is not buffered the pH and the resulting Ax60 A280 ratio can vary greatly Lower pH results in a lower A260 A2g0 ratio and reduced sensitivity to protein contamination For accurate values we recommend measuring absorbance in 10 mM Tris Cl pH 7 5 Pure RNA has an A so Aago 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 Asso reading of 1 44 ug ml RNA is based on an extinction coefficient calculated for RNA at neutral pH see Spectrophot
19. Reuse the collection tube in step 9 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 RLT Plus or Buffer RW1 and is therefore not compatible with bleach See page 7 for safety information AllPrep DNA RNA Micro Handbook 07 2007 37 9 10 11 12 38 Add 500 pl Buffer RPE 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 10 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 28000 x g 210 000 rpm to wash the spin column membrane Discard the collection tube with the flow through Prepare the 80 ethanol with 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 Place the RNeasy MinElute spin column in a new 2 ml collection tube supplied Open the lid of the spin column and cen
20. al RNA from Animal and Human Tissues 27 B Simultaneous Purification of Genomic DNA and Total RNA from Microdissected Cryosections 35 Troubleshooting Guide 40 Appendix A General Remarks on Handling RNA 44 Appendix B Storage Quantification and Determination of Quality of RNA 46 Appendix C Storage Quantification and Determination of Quality of Genomic DNA 49 Appendix D Purification of Total RNA Containing Small RNAs from Cells 51 Appendix E Acetone Precipitation of Protein from Lysates 53 AllPrep DNA RNA Micro Handbook 07 2007 3 Appendix F Optional On Column DNase Digestion Using the RNase Free DNase Set 54 Ordering Information 56 4 AllPrep DNA RNA Micro Handbook 07 2007 Kit Contents AllPrep DNA RNA Micro Kit 50 Catalog no 80284 Number of preps 50 AllPrep DNA Mini Spin Columns uncolored 50 each in a 2 ml Collection Tube RNeasy MinElute Spin Columns pink 50 each in a 2 ml Collection Tube Collection Tubes 1 5 ml 100 Collection Tubes 2 ml 100 Buffer RLT Plus 45 ml Buffer RW1 45 ml Buffer RPEt concentrate 11 ml RNase Free Water 10 ml Buffer AW1 t concentrate 19 ml Buffer AW2 concentrate 13 ml Buffer EB 15 ml Carrier RNA poly A 310 ug Handbook 1 Contains a guanidine salt Not compatible with disinfectants containing bleach See page 7 for safety information t Before using for the first time add the appropriate volume of ethanol 96 10096 as indicated on the bottle to ob
21. am experiments a Salt carryover during Ensure that buffers are at 20 30 C elution Ensure that the correct buffer is used for each step of the procedure When reusing collection tubes between washing steps remove residual flow through from the rim by blotting on clean paper towels b Ethanol carryover After the wash with 8096 ethanol be sure to centrifuge at full speed for 5 min to dry the RNeasy MinElute spin column membrane 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 EEE SSSSSS__ee_x_ AllPrep DNA RNA Micro Handbook 07 2007 43 Appendix A General Remarks on Handling RNA Handling RNA Ribonucleases RNases are very stable and active enzymes that generally do not require cofactors to function Since RNases are difficult to inactivate and even minute amounts are sufficient to destroy RNA do not use any plasticware or glassware without first eliminating possible RNase contamination Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure In order to create and maintain an RNase free environment the following precautions must be taken during pretreatment and use of disposable and nondisposable vessels and solutions wh
22. amounts of RNA we recommend the QuantiTect Whole Transcriptome Kit For details visit www gqiagen com goto WTA AllPrep DNA RNA Micro Handbook 07 2007 Genomic DNA purification 13 Add 500 pl Buffer AW1 to the AllPrep DNA spin column from step 5 Close the lid gently and centrifuge for 15 s at 28000 x g 10 000 rpm Discard the flow through Reuse the spin column in step 14 Note Buffer AW is supplied as a concentrate Ensure that ethanol is added to Buffer AW1 before use see Things to do before starting 14 Add 500 pl Buffer AW2 to the AllPrep DNA spin column Close the lid gently and centrifuge for 2 min at full speed to wash the spin column membrane Note Buffer AW2 is supplied as a concentrate Ensure that ethanol is added to Buffer AW2 before use see Things to do before starting The long centrifugation dries the spin column membrane ensuring that no ethanol is carried over during DNA elution Residual ethanol may interfere with downstream reactions Note After centrifugation carefully remove the AllPrep DNA spin column from the collection tube If the column contacts the flow through empty the collection tube and centrifuge the spin column again for 1 min at full speed 15 Place the AllPrep DNA spin column in a new 1 5 ml collection tube supplied Add 50 pl Buffer EB preheated to 70 C directly to the spin column membrane and close the lid Incubate at room temperature 15 25 C for 2 min and
23. and is therefore not compatible with bleach See page 7 for safety information AllPrep DNA RNA Micro Handbook 07 2007 53 Appendix F Optional On Column DNase Digestion Using the RNase Free DNase Set Although DNA binds very efficiently to the AllPrep DNA spin column some tissues contain very high amounts of DNA e g spleen and thymus that may result in trace amounts of DNA passing through the membrane For these tissues DNase digestion can be performed on the RNeasy MinElute spin column membrane if the eluted RNA will be used in downstream applications sensitive to very small amounts of DNA Tissues containing moderate amounts of DNA and cultured cells do not require DNase digestion The QIAGEN RNase Free DNase Set see page 56 for ordering information provides efficient on column digestion of DNA during RNA purification The DNase is efficiently removed in subsequent wash steps Note Buffer RDD supplied with the QIAGEN RNase Free DNase Set is specially optimized for on column DNase digestion Use of other DNase buffers may affect the binding of the RNA to the RNeasy MinElute spin column membrane reducing RNA yield and integrity Preparation of tissue homogenates and binding of RNA to the RNeasy MinElute spin column membrane are performed according to the protocol starting on page 27 After washing with a reduced volume of Buffer RW1 RNA is treated with DNase while bound to the spin column membrane DNase is removed by a second
24. andbook 07 2007 Table 1 Specifications of the spin columns in the AllPrep DNA RNA Micro Kit AllPrep DNA spin RNeasy MinElute spin Specification column column Maximum binding capacity 100 ug DNA 45 ug RNA Maximum loading volume 700 ul 700 ul Nucleic acid size DNA of 15 30 kb RNA gt 200 nucleotides distribution Minimum elution volume 30 ul 10 ul Maximum amount of starting material E Animal and human 5 x 10 cells Entire flow through from cells AllPrep DNA spin column E Animal and human 5 mg Entire flow through from tissues AllPrep DNA spin column Loading more than 20 ug DNA may lead to DNA contamination of the RNA eluate t Depending on homogenization conditions Purification of total RNA containing small RNAs from cells is possible through a modification of the AllPrep DNA RNA procedure For details see Appendix D page 51 AllPrep DNA RNA Micro Handbook 07 2007 13 Table 2 Typical yields of genomic DNA and total RNA with the AllPrep DNA RNA Micro Kit Typical yield of Typical yield of Sample type genomic DNA yg total RNA ug Cell cultures 5 x 10 cells SW NIH 3T3 4 5 E HeLa Jurkat 3 8 E COS 7 4 15 Mouse rat tissues 5 mg E Brain 2 5 2 5 m Heart 2 5 2 4 E Kidney 15 10 15 Liver 7 15 20 30 E Spleen 25 35 15 40 E Thymus 25 50 20 40 E Lung 5 10 5 10 Amounts can vary due to factors such as species developmental stage and growth conditions Since the AllPrep DN
25. anscription Kit provides fast cDNA synthesis with integrated removal of genomic DNA contamination see ordering information page 56 Integrity of RNA The integrity and size distribution of total RNA purified with the AllPrep DNA RNA Micro Kit can be checked by denaturing agarose gel electrophoresis and ethidium bromide staining or by using an Agilent 2100 bioanalyzer The respective ribosomal RNAs should appear as sharp bands or peaks The apparent ratio of 28S rRNA to 18S RNA should be approximately 2 1 If the ribosomal bands or peaks of a specific sample are not sharp but appear as a smear towards smaller sized RNAs it is likely that the sample suffered major degradation either before or during RNA purification When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 48 AllPrep DNA RNA Micro Handbook 07 2007 Appendix C Storage Quantification and Determination of Quality of Genomic DNA Storage of DNA For long term storage purified DNA in Buffer EB can be stored at 20 C Avoid any contamination as this may lead to DNA degradation We recommend storing samples in aliquots in order to avoid repeated freezing and thawing Quantification of DNA The concentration of DNA can be determined by measuring the absorbance at 260 nm A350 in a spectrophotometer see
26. ated by alcohol precipitation and reconstituted by gentle agitation in approximately 30 ul TE buffer pH 8 0 for at least 30 minutes at 60 C Avoid drying the DNA pellet for more than 10 minutes at room temperature 15 25 C since over dried genomic DNA is very difficult to redissolve Load 3 5 ug of DNA per well Standard PFGE conditions are as follows 196 agarose gel in 0 5x TBE electrophoresis buffer Switch intervals 5 40 s Run time 17h Voltage 170 V 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 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 50 AllPrep DNA RNA Micro Handbook 07 2007 Appendix D Purification of Total RNA Containing Small RNAs from Cells The following procedure allows the purification of total RNA containing small RNAs such as miRNA from animal and human cells Reagents to be supplied by user M Ethanol 100 Important points before starting E Perform all steps of the procedure at room temperature 15 25 C During the procedure work quickly E Perform all centrifugation steps at 20 25 C in a standard microcentrifuge Ensure that the centrifuge does not cool below 20 C Things to do befor
27. binding capacity of the RNeasy MinElute spin column and the lysing capacity of Buffer RLT Plus will not be exceeded by these amounts Typical DNA and RNA yields from various tissues are given in Table 2 page 14 For maximum RNA yields from liver 50 ethanol instead of 70 ethanol should be used in step 5 of the procedure Some tissues such as spleen and thymus contain very high amounts of DNA which may lead to copurification of RNA with trace amounts of DNA For these tissues we recommend performing DNase digestion on the RNeasy MinElute spin column membrane if the eluted RNA will be used in downstream applications sensitive to very small amounts of DNA for further details see Appendix F page 54 RNA yields from skeletal muscle heart and skin tissue may be low due to the abundance of contractile proteins connective tissue and collagen For purification of genomic DNA and total RNA from these tissues we recommend using the DNeasy Blood amp Tissue Kit and the RNeasy Fibrous Tissue Mini Kit respectively see page 56 for ordering information Do not overload the AllPrep DNA spin column as this will lead to copurification of DNA with RNA Do not overload the RNeasy MinElute spin column as this will significantly reduce RNA yield and purity Weighing tissue is the most accurate way to quantitate the amount of starting material As a guide a 1 5 mm cube 3 4 mm of most animal tissues weighs 3 5 4 5 mg Important points b
28. book 07 2007 33 Genomic DNA purification 12 Add 500 pl Buffer AW1 to the AllPrep DNA spin column from step 4 Close the lid gently and centrifuge for 15 s at 28000 x g 10 000 rpm Discard the flow through Reuse the spin column in step 13 Note Buffer AW is supplied as a concentrate Ensure that ethanol is added to Buffer AW1 before use see Things to do before starting 13 Add 500 pl Buffer AW2 to the AllPrep DNA spin column Close the lid gently and centrifuge for 2 min at full speed to wash the spin column membrane Note Buffer AW2 is supplied as a concentrate Ensure that ethanol is added to Buffer AW2 before use see Things to do before starting The long centrifugation dries the spin column membrane ensuring that no ethanol is carried over during DNA elution Residual ethanol may interfere with downstream reactions Note After centrifugation carefully remove the AllPrep DNA spin column from the collection tube If the column contacts the flow through empty the collection tube and centrifuge the spin column again for 1 min at full speed 14 Place the AllPrep DNA spin column in a new 1 5 ml collection tube supplied Add 50 pl Buffer EB preheated to 70 C directly to the spin column membrane and close the lid Incubate at room temperature 15 25 C for 2 min and then centrifuge for 1 min at 28000 x g 10 000 rpm to elute the DNA 15 Repeat step 14 to elute further DNA To prevent dilution of the firs
29. bottle to obtain a working solution W Before using the kit for the first time prepare 80 ethanol by mixing 24 ml ethanol 96 100 and 6 ml RNase free water supplied The procedure also requires 7096 ethanol which can be prepared by diluting ethanol 96 10096 with distilled water not supplied W Buffer RLT Plus may form a precipitate during storage If necessary redissolve by warming and then place at room temperature E Preheat Buffer EB to 70 C to ensure optimal DNA elution AllPrep DNA RNA Micro Handbook 07 2007 21 Procedure Sample disruption and homogenization 1 la Tb 22 Harvest cells according to step 1a or 1b Cells grown in suspension do not use more than 5 x 10 cells Determine the number of cells Pellet the appropriate number of cells by centrifuging for 5 min at 300 x g in a centrifuge tube not supplied Carefully remove all supernatant by aspiration and proceed to step 2 Note Incomplete removal of cell culture medium will inhibit lysis and dilute the lysate affecting the conditions for nucleic acid purification Both effects may reduce nucleic acid yields and purity Cells grown in a monolayer do not use more than 5 x 10 cells Cells grown in a monolayer in cell culture vessels can be either lysed directly in the vessel up to 10 cm diameter or trypsinized and collected as a cell pellet prior to lysis Cells grown in a monolayer in cell culture flasks should always be trypsiniz
30. 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 11 Place the RNeasy MinElute spin column in a new 1 5 ml collection tube supplied Add 14 ul 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 As little as 10 ul RNase free water can be used for elution if a higher RNA concentration is required but the yield will be reduced by approximately 20 Do not elute with less than 10 ul RNase free water as the spin column membrane will not be sufficiently hydrated The dead volume of the RNeasy MinElute spin column is 2 ul elution with 14 ul RNase free water results in a 12 pl eluate 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 qiagen com PCR For whole transcriptome amplification WTA of limited amounts of RNA we recommend the QuantiTect Whole Transcriptome Kit For details visit www qiagen com goto WTA AllPrep DNA RNA Micro Hand
31. cially the first few steps See Appendix A page 44 and Handling and storing starting material page 14 b RNase contamination Although all AllPrep buffers have been tested and are guaranteed RNase free RNases can be introduced during use Be certain not to introduce any RNases during the AllPrep DNA RNA procedure or later handling See Appendix A page 44 for general remarks on handling RNA 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 42 AllPrep DNA RNA Micro Handbook 07 2007 Comments and suggestions DNA fragmented Homogenization too The length of the purified DNA usually vigorous 15 30 kb depends strongly on the homogenization conditions If longer DNA fragments are required keep the homogenization time to a minimum or use a gentler homogenization method if possible e g use a QlAshredder homogenizer instead of a rotor stator homogenizer Nucleic acid concentration too low Elution volume too high Elute nucleic acids in a smaller volume Do not use less than 30 pl Buffer EB for the AllPrep DNA spin column or less than 12 ul RNase free water for the RNeasy MinElute spin column Although eluting in smaller volumes results in increased nucleic acid concentrations yields may be reduced Nucleic acids do not perform well in downstre
32. ct stabilized tissues Remove the tissue from the stabilization reagent using forceps and be sure to remove any crystals that may have formed RNA in RNAlater or Allprotect stabilized tissues is protected during cutting and weighing of tissues at ambient temperature 15 259C It is not necessary to cut the tissues on ice or dry ice or in a refrigerated room Remaining tissues can be stored in RNAlater or Allprotect Reagent Previously stabilized tissues can be stored at 80 C without the reagent For unstabilized fresh or frozen tissues RNA in harvested tissues is not protected until the tissues are treated with RNAlater or Allprotect Reagent flash frozen or disrupted and homogenized in step 2 Frozen tissues should not be allowed to thaw during handling The relevant procedures should be carried out as quickly as possible Remaining fresh tissues can be placed into RNAlater Reagent to stabilize RNA or in Allprotect Tissue Reagent to stabilize DNA RNA and protein However previously frozen tissues thaw too slowly in the reagent preventing the reagent from diffusing into the tissues quickly enough to prevent RNA degradation AllPrep DNA RNA Micro Handbook 07 2007 29 2 Disrupt the tissue and homogenize the lysate in Buffer RLT Plus do not use more than 5 mg tissue according to step 2a 2b or 2c See Disrupting and homogenizing starting material page 15 for more details on disruption and homogenization Note Ensure that B ME o
33. e vessels after confluent growth is given in Table 4 Important points before starting E If using the AllPrep DNA RNA Micro Kit for the first time read Important Notes page 12 E If preparing RNA for the first time read Appendix A page 44 M f using the TissueRuptor ensure that you are familiar with operating it by referring to the TissueRuptor User Manual and TissueRuptor Handbook E Cell pellets can be stored at 70 C for later use or used directly in the procedure Determine the number of cells before freezing Frozen cell pellets should be thawed slightly so that they can be dislodged by flicking the tube in step 2 Homogenized cell lysates from step 3 can be stored at 70 C for several months Frozen lysates should be incubated at 37 C in a water bath until completely thawed and salts are dissolved Avoid prolonged incubation which may compromise RNA integrity If any insoluble material is visible centrifuge for 5 min at 3000 5000 x g Transfer the supernatant to a new RNase free glass or polypropylene tube and continue with step 4 AllPrep DNA RNA Micro Handbook 07 2007 19 Table 4 Growth area and number of HeLa cells in various culture vessels Cell culture vessel Growth area cm Number of cells Multiwell plates mM 96 well 0 32 0 6 4 5 x 104 5 48 well 1 x 10 mM 24 well 2 2 5 x 10 E 12 well 4 5x10 E 6 well 9 5 1x104 Dishes E 35mm 8 Tx Flasks E 40 50 ml 25 3 x 10 Per well if mult
34. e 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 Procedure Carry out the protocol starting on page 19 up to and including step 5 Instead of performing steps 6 12 purification of total RNA gt 200 nucleotides follow steps D1 D6 below purification of total RNA containing small RNAs D1 Add 1 5 volumes usually 525 pl of 100 ethanol to the flow through from the AllPrep DNA spin column and mix well by pipetting Do not centrifuge Proceed immediately to step D2 Note The volume of 100 ethanol to add may be less than 525 ul if some lysate was lost during homogenization and DNA isolation Note Precipitates may be visible after addition of ethanol but this does not affect the procedure D2 Transfer 700 ul of the sample including any precipitate that may have formed to an RNeasy MinElute spin column placed in a 2 ml collection tube supplied Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm Discard the flow through 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 Flow through contains Buffer RLT Plus and is therefore not compatible with bleach See page 7 for safety information AllPrep DNA R
35. ection 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 Place the RNeasy MinElute spin column in a new 1 5 ml collection tube supplied 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 full speed to elute the RNA As little as 10 ul RNase free water can be used for elution if a higher RNA concentration is required but the yield will be reduced by approximately 20 Do not elute with less than 10 ul RNase free water as the spin column membrane will not be sufficiently hydrated The dead volume of the RNeasy MinElute spin column is 2 ul elution with 14 ul RNase free water results in a 12 pl eluate 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 qiagen com PCR For whole transcriptome amplification WTA of limited amounts of RNA we recommend the QuantiTect Whole Transcriptome Kit For details visit www gqiagen com goto WTA AllPrep DNA RNA Micro Handbook 07 2007 25 Genomic DNA purification 13 14 15 16 Add 500 pl Buffer AW1 to the Al
36. ed To lyse cells directly Determine the number of cells Completely aspirate the cell culture medium and proceed immediately to step 2 Note Incomplete removal of cell culture medium will inhibit lysis and dilute the lysate affecting the conditions for nucleic acid purification Both effects may reduce nucleic acid yields and purity To trypsinize and collect cells Determine the number of cells Aspirate the medium and wash the cells with PBS Aspirate the PBS and add 0 10 0 25 trypsin in PBS After the cells detach from the dish or flask add medium containing serum to inactivate the trypsin transfer the cells to an RNase free glass or polypropylene centrifuge tube not supplied and centrifuge at 300 x g for 5 min Completely aspirate the supernatant and proceed to step 2 Note Incomplete removal of cell culture medium will inhibit lysis and dilute the lysate affecting the conditions for nucleic acid purification Both effects may reduce nucleic acid yields and purity Disrupt the cells by adding Buffer RLT Plus For pelleted cells loosen the cell pellet thoroughly by flicking the tube Add 350 ul Buffer RLT Plus Vortex or pipet to mix and proceed to step 3 If processing lt 1 x 10 cells 75 ul Buffer RLT Plus can be added instead This allows cell pelleting in smaller tubes Pipet up and down to lyse the cells AllPrep DNA RNA Micro Handbook 07 2007 Note Incomplete loosening of the cell pellet may lead to
37. efore starting E If using the AllPrep DNA RNA Micro Kit for the first time read Important Notes page 12 E If preparing RNA for the first time read Appendix A page 44 AllPrep DNA RNA Micro Handbook 07 2007 27 E f using the TissueRuptor ensure that you are familiar with operating it by referring to the TissueRuptor User Manual and TissueRuptor Handbook M f using the TissueLyser ensure that you are familiar with operating it by referring to the operating instructions and TissueLyser Handbook M For optimal results stabilize harvested tissues immediately in RNAlater RNA Stabilization Reagent see the RNAlater Handbook or Allprotect Tissue Reagent see the Allprotect Tissue Reagent Handbook Tissues can be stored in the reagent at 37 C for up to 1 day at 15 25 C for up to 7 days or at 2 8 C for up to 4 weeks RNAlater or 6 months Allprotect Alternatively tissues can be archived at 20 C or 80 C E Fresh frozen or RNAlater Allprotect stabilized tissues can be used Tissues can be stored at 70 C for several months Flash freeze tissues in liquid nitrogen and immediately transfer to 70 C Do not allow tissues to thaw during weighing or handling prior to disruption in Buffer RLT Plus Homogenized tissue lysates from step 2 can also be stored at 70 C for several months Incubate frozen lysates at 37 C in a water bath until completely thawed and salts are dissolved before continuing with step 3 Avoid prolon
38. er M 14 3 M B mercaptoethanol B ME commercially available solutions are usually 14 3 M or alternatively 2 M dithiothreitol DTT in water Sterile RNase free pipet tips Microcentrifuge with rotor for 2 ml tubes 96 100 ethanol 70 ethanol in water Disposable gloves Reagent for RNA stabilization see pages 14 15 W For cell samples RNAprotect Cell Reagent or liquid nitrogen For tissue samples RNAlater RNA Stabilization Reagent stabilizes RNA only Allprotect Tissue Reagent stabilizes DNA RNA and protein or liquid nitrogen M Equipment for sample disruption and homogenization see pages 15 17 Depending on the method chosen one or more of the following are required E Trypsin and PBS QlAshredder homogenizer Blunt ended needle and syringe Mortar and pestle TissueRuptor with TissueRuptor Disposable Probes TissueLysert Do not use denatured alcohol which contains other substances such as methanol or methylethylketone t For ordering information see page 56 AllPrep DNA RNA Micro Handbook 07 2007 11 Important Notes Determining the amount of starting material It is essential to use the correct amount of starting material in order to obtain optimal nucleic acid yield and purity The maximum amount that can be used is determined by E The type of sample and its DNA and RNA content M The volume of Buffer RLT Plus required for efficient lysis M The DNA binding capacity of the A
39. fer for SDS PAGE gel Important points before starting E Do not use trichloroacetic acid TCA to precipitate protein from Buffer RLT Plus lysates This buffer contain guanidine thiocyanate which can form highly reactive compounds when combined with acidic solutions Procedure Bind total RNA to the RNeasy MinElute spin column as described in the cell protocol from page 19 steps 1 7 or the tissue protocol from page 27 steps 1 6 Then follow steps E1 E5 below to precipitate protein E1 Add 4 volumes of ice cold acetone to the flow through from the RNeasy MinElute spin column E2 Incubate for 30 min on ice or at 20 C E3 Centrifuge for 10 min at full speed in a benchtop centrifuge Discard the supernatant and air dry the pellet E4 Optional Wash the pellet with 100 pl ice cold ethanol and air dry Do not overdry the pellet as this may make resuspension more difficult E5 Resuspend the pellet in the buffer for your downstream application Sodium dodecyl sulfate SDS causes guanidine salts to precipitate In case the pellet contains traces of guanidine thiocyanate load the sample onto an SDS PAGE gel immediately after heating for 7 minutes at 95 C 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 Supernatant contains guanidine thiocyanate
40. ged incubation which may compromise RNA integrity Buffer RLT Plus Buffer RW1 and Buffer AW1 contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach See page 7 for safety information E Perform all steps of the procedure at room temperature 15 25 C During the procedure work quickly E Perform all centrifugation steps at 20 25 C in a standard microcentrifuge Ensure that the centrifuge does not cool below 20 C Things to do before starting M Mercaptoethanol B ME must be added to Buffer RLT Plus before use Add 10 ul B ME per 1 ml Buffer RLT Plus Dispense in a fume hood and wear appropriate protective clothing Buffer RLT Plus containing B ME can be stored at room temperature 15 25 C for up to 1 month Alternatively add 20 ul of 2 M dithiothreitol DTT per 1 ml Buffer RLT Plus The stock solution of 2 M DTT in water should be prepared fresh or frozen in single use aliquots Buffer RLT Plus containing DTT can be stored at room temperature for up to 1 month M When processing less than about 2 ug tissue carrier RNA may be added to the lysate before homogenization see Carrier RNA page 17 Before using for the first time dissolve the carrier RNA 310 ug in 1 ml RNase free water Store this stock solution at 20 C and use it to make fresh dilutions for each set of preps The concentration of this stock solution is 310 ug ml i e 310 ng ul To make a working
41. igh salt buffer selectively and efficiently binds genomic DNA The column is washed and pure ready to use DNA is then eluted Ethanol is added to the flow through from the AllPrep DNA spin column to provide appropriate binding conditions for RNA and the sample is then applied to an RNeasy MinElute spin column where total RNA binds to the membrane and contaminants are efficiently washed away High quality RNA is then eluted in 14 ul water In this handbook different protocols are provided for different starting materials The protocols differ primarily in the lysis and homogenization of the sample Once the sample is applied to the AllPrep DNA spin column the protocols are similar see flowchart next page Samples with particularly high DNA content may require additional DNase digestion PN AllPrep DNA RNA Micro Handbook 07 2007 9 10 ean ij f a AllPrep DNA RNA Procedure Cells or tissue Lyse and homogenize l T 3 Bind genomic DNA Total RNA Genomic DNA 4 N Add ethanol Wash H Bind total RNA Elute s Total RNA u Eluted DNA ait 4 cmt 4 Wash Elute Eluted RNA AllPrep DNA RNA Micro Handbook 07 2007 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 suppli
42. ile working with RNA General handling Proper microbiological aseptic technique should always be used when working with RNA Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin or from dusty laboratory equipment Change gloves frequently and keep tubes closed whenever possible Keep purified RNA on ice when aliquots are pipetted for downstream applications Disposable plasticware The use of sterile disposable polypropylene tubes is recommended throughout the procedure These tubes are generally RNase free and do not require pretreatment to inactivate RNases Nondisposable plasticware Nondisposable plasticware should be treated before use to ensure that it is RNase free Plasticware should be thoroughly rinsed with 0 1 M NaOH 1 mM EDTA followed by RNase free water see Solutions page 45 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 44 AllPrep DNA RNA Micro Handbook 07 2007 Glassware Glassware should be treated before use to ensure that it is RNase free Glass
43. inefficient lysis and reduced nucleic acid yields For direct lysis of cells grown in a monolayer add 350 pl Buffer RLT Plus to the cell culture dish Collect the lysate with a rubber policeman Pipet the lysate into a microcentrifuge tube not supplied Vortex or pipet to mix and ensure that no cell clumps are visible before proceeding to step 3 If processing lt 1 x 10 cells 75 ul Buffer RLT Plus can be added instead This may be necessary for multiwell plates and cell culture dishes Pipet up and down to lyse the cells 3 Homogenize the lysate according to step 3a 3b or 3c See Disrupting and homogenizing starting material page 15 for more details on homogenization If processing 1 x 10 cells they can be homogenized by vortexing for 1 min After homogenization proceed to step 4 Note If only 75 ul Buffer RLT Plus was used in step 2 transfer the lysate to a new 1 5 ml microcentrifuge tube and adjust the volume to 350 ul with Buffer RLT Plus Vortex for 1 min to homogenize and proceed to step 4 Note If processing 500 cells 20 ng carrier RNA 5 ul of a 4 ng ul solution may be added to the lysate before homogenization Prepare the carrier RNA as described in Things to do before starting Note Incomplete homogenization leads to significantly reduced RNA yields and can cause clogging of the AllPrep and RNeasy MinElute spin columns Homogenization with the TissueRuptor or QlAshredder homogenizer generally res
44. ing and or material page 15 for details on disruption and homogenization homogenization methods Increase g force and centrifugation time if necessary In subsequent preparations reduce the amount of starting material see the individual protocols and or increase the homogenization time b Too much starting Reduce the amount of starting material It is material essential to use the correct amount of starting material see page 12 c Centrifugation The centrifugation temperature should be temperature too low 20 25 C Some centrifuges may cool to below 20 C even when set at 20 C This can cause formation of precipitates that can clog the spin column If this happens set the centrifugation temperature to 25 C Warm the lysate to 37 C before transferring it to the AllPrep DNA spin column Low nucleic acid yield a Insufficient disruption See Disrupting and homogenizing starting and homogenization material page 15 for details on disruption and homogenization methods In subsequent preparations reduce the amount of starting material see the individual protocols and or increase the volume of lysis buffer and the homogenization time b Too much starting Overloading the spin columns significantly material reduces nucleic acid yields Reduce the amount of starting material see the individual protocols 40 AllPrep DNA RNA Micro Handbook 07 2007 Comments and suggestions c RNA still bound to Repeat RNA
45. ion of DNA RNA and protein in tissue samples at room temperature The procedures for tissue harvesting and RNA protection should be carried out as quickly as possible Frozen tissue samples should not be allowed to thaw during handling or weighing After disruption and homogenization in Buffer RLT Plus lysis buffer samples can be stored at 70 C for months Disrupting and homogenizing starting material Efficient disruption and homogenization of the starting material is an absolute requirement for all nucleic acid purification procedures Disruption and homogenization are 2 distinct steps E Disruption Complete disruption of plasma membranes of cells and organelles is absolutely required to release all the nucleic acids contained in the sample Different samples require different methods to achieve complete disruption Incomplete disruption results in significantly reduced nucleic acid yields E Homogenization Homogenization is necessary to reduce the viscosity of the lysates produced by disruption Homogenization shears high molecular weight cellular components to create a homogeneous lysate Incomplete homogenization results in inefficient binding of DNA and RNA and therefore significantly reduced yield and purity of nucleic acids Excessive homogenization on the other hand results in shorter genomic DNA fragments Some disruption methods simultaneously homogenize the sample while others require an additional homogenization step
46. it contains poly A RNA for use as carrier RNA When added to lysates from very small samples the carrier RNA may in some cases improve the recovery of total RNA Carrier RNA is not required when processing more than 500 cells or more than about 2 ug tissue As demonstrated in many different RT PCR systems the small amounts of poly A RNA used as carrier RNA in total RNA purification do not interfere with subsequent RT PCR even when oligo dT is used as a primer for reverse transcription Reverse transcription reactions typically contain an excess of AllPrep DNA RNA Micro Handbook 07 2007 17 oligo dT primers and the small amounts of poly A used as carrier RNA are insignificant in comparison However total RNA purified using poly A RNA as carrier RNA is not compatible with protocols to amplify mRNA transcripts using oligo dT primers These include the Eberwine method and the QuantiTect Whole Transcriptome Kit the kit uses a mix of random and oligo dT primers For the Eberwine method other types of RNA can be purchased separately for use as carrier RNA Note however that tRNA and other RNAs lt 200 nucleotides will not bind to the RNeasy MinElute membrane and cannot be used as carrier RNA For most applications bacterial ribosomal RNA e g from Roche cat no 206938 gives good results and can be used as an alternative to the poly A RNA supplied with this kit However if amplifying mRNA transcripts with the QuantiTect Whole Transcript
47. iwell plates are used varies slightly depending on the supplier t Cell numbers are given for HeLa cells approximate length 15 um assuming confluent growth Cell numbers will vary for different kinds of animal and human cells which vary in length from 10 to 30 um This number of cells exceeds the maximum binding capacity of the RNeasy MinElute spin columns To process this many cells split the lysate into appropriate aliquots x5 x 10 cells each and load them onto separate gDNA Eliminator spin columns M Cells stored in RNAprotect Cell Reagent can also be used in the procedure Transfer the entire sample including any material deposited at the bottom of the storage vessel to a centrifuge tube Pellet the cells by centrifuging for 5 min at 5000 x g and remove the supernatant by pipetting if necessary thaw the sample before centrifuging Proceed immediately to step 2 Buffer RLT Plus Buffer RW1 and Buffer AW1 contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach See page 7 for safety information E Perform all steps of the procedure at room temperature 15 25 C During the procedure work quickly E Perform all centrifugation steps at 20 25 C in a standard microcentrifuge Ensure that the centrifuge does not cool below 20 C 20 AllPrep DNA RNA Micro Handbook 07 2007 Things to do before starting E If purifying RNA from cell lines rich in RNases we rec
48. ix by gently inverting the tube and centrifuge briefly to collect residual liquid from the sides of the tube Buffer RDD is supplied with the RNase Free DNase Set Note DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube Do not vortex F3 Add the DNase incubation mix 80 pl directly to the RNeasy MinElute spin column membrane and place on the benchtop 20 30 C for 15 min Note Be sure to add the DNase incubation mix directly to the RNeasy 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 F4 Add 350 ul Buffer RW1 to the RNeasy MinElute spin column and centrifuge for 15 s at 28000 x g 210 000 rpm Discard the flow through Continue with step 8 of the protocol on page 27 i e the wash with Buffer RPE Reuse the collection tube in step 8 Flow through contains Buffer RW1 and is therefore not compatible with bleach See page 7 for safety information AllPrep DNA RNA Micro Handbook 07 2007 55 Ordering Information Product AllPrep DNA RNA Micro Kit 50 AllPrep DNA RNA Mini Kit 50 Accessories RNAprotect Cell Reagent 250 ml RNAlater RNA Stabilization Reagent 50 ml RNAlater RNA Stabilization Reagent 250 ml RNAlater TissueProtect Tubes 50 x 1 5 ml RNAlater TissueProtect Tubes 20 x 5 ml Allprotect Tissue Reagent 100 ml RNase F
49. lPrep DNA spin column from step 5 Close the lid gently and centrifuge for 15 s at 28000 x g 10 000 rpm to wash the spin column membrane Discard the flow through Reuse the spin column in step 14 Note Buffer AW is supplied as a concentrate Ensure that ethanol is added to Buffer AW1 before use see Things to do before starting Add 500 pl Buffer AW2 to the AllPrep DNA spin column Close the lid gently and centrifuge for 2 min at full speed to wash the spin column membrane Note Buffer AW2 is supplied as a concentrate Ensure that ethanol is added to Buffer AW2 before use see Things to do before starting The long centrifugation dries the spin column membrane ensuring that no ethanol is carried over during DNA elution Residual ethanol may interfere with downstream reactions Note After centrifugation carefully remove the AllPrep DNA spin column from the collection tube If the column contacts the flow through empty the collection tube and centrifuge the spin column again for 1 min at full speed Place the AllPrep DNA spin column in a new 1 5 ml collection tube supplied Add 50 pl Buffer EB preheated to 70 C directly to the spin column membrane and close the lid Incubate at room temperature 15 25 C for 2 min and then centrifuge for 1 min at 28000 x g 10 000 rpm to elute the DNA Repeat step 15 to elute further DNA To prevent dilution of the first DNA eluate use a new 1 5 ml collection tube not sup
50. llPrep DNA spin column M The RNA binding capacity of the RNeasy MinElute spin column When processing samples containing high amounts of DNA or RNA less than the maximum amount of starting material shown in Table 1 should be used so that the binding capacity of the spin columns is not exceeded When processing samples containing average or low amounts of DNA and RNA the maximum amount of starting material shown in Table 1 can be used However even though the binding capacity of the spin columns is not reached the maximum amount of starting material must not be exceeded Otherwise lysis will be incomplete and cellular debris may interfere with the binding of nucleic acids to the spin column membranes resulting in lower yield and purity of DNA and RNA More information on using the correct amount of starting material is given in each protocol Table 2 shows typical DNA and RNA yields from various cells and tissues Note Although the AllPrep DNA spin column can bind a maximum of 100 ug DNA the use of starting materials containing more than 20 ug DNA may lead to the purification of RNA containing small amounts of DNA If the binding capacity of the RNeasy MinElute spin column is exceeded RNA yields will not be consistent and less than expected If lysis of the starting material is incomplete DNA and RNA yields will be lower than expected even if the binding capacity of the spin columns is not exceeded ENR 12 AllPrep DNA RNA Micro H
51. mmediately in liquid nitrogen and grind to a fine powder under liquid nitrogen Transfer the suspension tissue powder and liquid nitrogen into a liquid nitrogen cooled appropriately sized tube and allow the liquid nitrogen to evaporate without allowing the sample to thaw Add lysis buffer and continue as quickly as possible with the homogenization according to one of the 2 methods below Note Grinding the sample using a mortar and pestle will disrupt the sample but will not homogenize it Homogenization must be performed afterwards Homogenization using QlAshredder homogenizers Using QlAshredder homogenizers is a fast and efficient way to homogenize cell and tissue lysates without cross contamination of samples Up to 700 ul of lysate is loaded onto a QlAshredder spin column placed in a 2 ml collection tube and spun for 2 minutes at maximum speed in a microcentrifuge The lysate is homogenized as it passes through the spin column QlAshredder homogenizers typically result in less DNA fragmentation compared with rotor stator homogenizers Homogenization using a syringe and needle Cell and tissue lysates can be homogenized using a syringe and needle Lysate is passed through a 20 gauge 0 9 mm needle attached to a sterile plastic syringe at least 5 10 times or until a homogeneous lysate is achieved Increasing the volume of lysis buffer may be required to facilitate handling and minimize loss Carrier RNA The AllPrep DNA RNA Micro K
52. nd liquid nitrogen into an RNase free liquid nitrogen cooled 2 ml microcentrifuge tube not supplied Allow the liquid nitrogen to evaporate but do not allow the tissue to thaw E Add 350 ul Buffer RLT Plus W Pipet the lysate directly into a QlAshredder spin column placed in a 2 ml collection tube and centrifuge for 2 min at full speed Alternatively pass the lysate at least 5 times through a blunt 20 gauge needle fitted to an RNase free syringe Proceed to step 3 3 Centrifuge the lysate for 3 min at full speed Carefully remove the supernatant by pipetting and transfer it to an AllPrep DNA spin column placed in a 2 ml collection tube supplied Close the lid gently and centrifuge for 30 s at 28000 x g 210 000 rpm In some preparations very small amounts of insoluble material will be present after the 3 min centrifugation making the pellet invisible Note Make sure that no liquid remains on the column membrane after centrifugation If necessary repeat the centrifugation until all liquid has passed through the membrane 4 Place the AllPrep DNA spin column in a new 2 ml collection tube supplied and store at room temperature 15 25 C or at 4 C for later DNA purification in steps 12 15 Use the flow through for RNA purification in steps 5 11 AllPrep DNA RNA Micro Handbook 07 2007 31 Note Do not store the AllPrep DNA spin column at room temperature or at 4 C for long periods Do not freeze the column
53. ns from flash frozen specimens to minimize this problem A wide range of equipment and consumables for sectioning staining and microdissection of specimens is available from Leica www leica microsystems com and P A L M Microlaser Technologies www palm mikrolaser com Important points before starting E If using the AllPrep DNA RNA Micro Kit for the first time read Important Notes page 12 E If preparing RNA for the first time read Appendix A page 44 M To minimize RNA degradation avoid prolonged storage of unstabilized samples at room temperature RNA in tissues is not protected before flash freezing in liquid nitrogen E Tissue lysates from step 3 can be stored at 70 C for several months Incubate frozen lysates at 37 C in a water bath until completely thawed and salts are dissolved before continuing with step 4 Avoid prolonged incubation which may compromise RNA integrity E Buffer RLT Plus Buffer RW1 and Buffer AW contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach See page 7 for safety information M Perform all steps of the procedure at room temperature 15 25 C During the procedure work quickly E Perform all centrifugation steps at 20 25 C in a standard microcentrifuge Ensure that the centrifuge does not cool below 20 C Things to do before starting M Mercaptoethanol B ME must be added to Buffer RLT Plus before use Add 10 pl B ME per
54. od amp Tissue 50 DNeasy Mini Spin Columns 69504 Kit 50 Proteinase K Buffers Collection Tubes RNeasy Fibrous Tissue Mini Kit for purification of total RNA from fiber rich tissues RNeasy Fibrous Tissue 50 RNeasy Mini Spin Columns 74704 Mini Kit 50 Collection Tubes Proteinase K RNase Free DNase RNase Free Reagents and Buffers RNeasy FFPE Kit for purification of high yields of usable RNA from FFPE tissue sections RNeasy FFPE Kit 50 50 RNeasy MinElute Spin Columns 50 74404 gDNA Eliminator Mini Spin Columns Collection Tubes RNase Free Reagents and Buffers REPLI g Kits for highly uniform whole genome amplification from small or precious samples REPLI g Mini Kit 25 DNA Polymerase Buffers and 150023 Reagents for 25 x 50 ul whole genome amplification reactions typical yield 10 Ug per reaction QIAGEN Fast Cycling PCR Kit for fast and specific PCR on any thermal cycler QIAGEN Fast Cycling For 200 x 20 ul reactions 2 x 1 ml 203743 PCR Kit 200 QIAGEN Fast Cycling PCR Master Mix 10x CoralLoad Fast Cycling Dye Q Solution RNase Free Water QuantiTect Whole Transcriptome Kit for unlimited real time PCR analysis from precious RNA samples QuantiTect Whole For 25 x 50 ul whole transcriptome 207043 Transcriptome Kit 25 amplification reactions T Script Enzyme Ligation Enzymes REPLI g DNA Polymerase Reagents and Buffers Larger kit size and or format available please inquire
55. olve 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 AllPrep 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 proper care and check the supplier s instructions AllPrep DNA RNA Micro Handbook 07 2007 45 Appendix B Storage Quantification and Determination of Quality of RNA Storage of RNA Purified RNA may be stored at 20 C or 70 C in RNase free water Under these conditions no degradation of RNA is detectable after 1 year Quantification of RNA The concentration of RNA can be determined by measuring the absorbance at 260 nm A350 in a spectrophotometer see Spectrophotometric quantification of RNA below For small amounts of RNA however it may n
56. ome Kit no carrier RNA of any type should be used in RNA purification Limitations of small samples When purifying nucleic acids from particularly small samples e g laser microdissected samples the amounts of DNA and RNA may be too small for quantification by spectrophotometry or even fluorometric assays In this case quantitative real time PCR or RT PCR should be used for quantification When purifying DNA and RNA from less than 100 cells stochastic problems with respect to copy number can occur For example if 20 cells are processed and DNA is eluted in the recommended minimum volume of 30 ul there will be less than 1 copy of each genomic DNA allele per microliter Similarly some RNA transcripts may be present at very low copy numbers per cell or only in a fraction of all cells in the sample of interest Whole genome amplification or whole transcriptome amplification can be carried out to generate sufficient amounts of DNA or RNA if several downstream assays need to be performed from a single small sample However care should be taken to include a sufficient amount of starting material in the amplification reaction to avoid stochastic problems REPLI g Kits and the QuantiTect Whole Transcriptome Kit provide highly uniform amplification of the genome and transcriptome respectively For details visit www giagen com WGA and www giagen com goto WTA This is not a complete list of suppliers and does not include many importan
57. ometric quantification of RNA page 46 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 the vast majority of cellular DNA will bind to the AllPrep DNA spin column trace amounts may still remain in the purified RNA depending on the amount and nature of the sample For analysis of very low abundance targets any interference by residual DNA contamination can be detected by performing real time RT PCR control experiments in which no reverse transcriptase is added prior to the PCR step To prevent any interference by DNA in real time RT PCR applications such as with Applied Biosystems and LightCycler instruments we recommend designing primers that anneal at intron splice junctions so that genomic DNA will not be amplified QuantiTect Primer Assays from QIAGEN are designed for 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 AllPrep DNA RNA Micro Handbook 07 2007 47 SYBR Green based real time RT PCR analysis of RNA sequences without detection of genomic DNA where possible For real time RT PCR assays where amplification of genomic DNA cannot be avoided the QuantiTect Reverse Tr
58. ommend adding B mercaptoethanol B ME to Buffer RLT Plus before use Add 10 ul B ME per 1 ml Buffer RLT Plus Dispense in a fume hood and wear appropriate protective clothing Buffer RLT Plus containing B ME can be stored at room temperature 15 25 C for up to 1 month Alternatively add 20 ul of 2 M dithiothreitol DTT per 1 ml Buffer RLT Plus The stock solution of 2 M DTT in water should be prepared fresh or frozen in single use aliquots Buffer RLT Plus containing DTT can be stored at room temperature for up to month E When processing 500 cells carrier RNA may be added to the lysate before homogenization see Carrier RNA page 17 Before using for the first time dissolve the carrier RNA 310 ug in 1 ml RNase free water Store this stock solution at 20 C and use it to make fresh dilutions for each set of preps The concentration of this stock solution is 310 ug ml i e 310 ng ul To make a working solution 4 ng Ll for 10 preps add 5 ul stock solution to 34 ul Buffer RLT Plus and mix by pipetting Add 6 ul of this diluted solution to 54 ul Buffer RLT Plus to give a working solution of 4 ng ul Add 5 ul of this solution to the lysate in step 3 Do not add the carrier RNA to the lysate if purifying RNA for use in oligo dT based amplification E Buffer RPE Buffer AW1 and Buffer AW2 are each supplied as a concentrate Before using for the first time add the appropriate volume of ethanol 96 100 as indicated on the
59. omogenizers refer to suppliers guidelines Note Longer homogenization times with the TissueRuptor result in greater DNA fragmentation Therefore the homogenization time should be kept as short as possible if the DNA will be used in downstream applications that require long DNA fragments Disruption and homogenization using the TissueLyser In bead milling tissues can be disrupted by rapid agitation in the presence of beads and lysis buffer Disruption and simultaneous homogenization occur by the shearing and crushing action of the beads as they collide with the cells The TissueLyser disrupts and homogenizes up to 48 tissue samples simultaneously when used in combination with the TissueLyser Adapter Set 2 x 24 which holds 48 x 2 ml microcentrifuge tubes containing stainless steel beads of 5 mm mean diameter The TissueLyser can also disrupt and homogenize up to 192 tissue samples simultaneously when used in combination with the TissueLyser Adapter 16 AllPrep DNA RNA Micro Handbook 07 2007 Set 2 x 96 which holds 192 x 1 2 ml microtubes containing stainless steel beads of 5 mm mean diameter For guidelines on using the TissueLyser refer to the TissueLyser Handbook For other bead mills refer to suppliers guidelines Note Tungsten carbide beads react with Buffer RLT Plus and must not be used to disrupt and homogenize tissues Disruption using a mortar and pestle For disruption using a mortar and pestle freeze the tissue sample i
60. one 03 5547 0811 Fax 03 5547 0818 Technical 03 5547 0811 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 South Korea Orders 1544 7145 Fax 1544 7146 Technical 1544 7145 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 TILT 0000o QIAGEN inp Sample amp Assay Technologies
61. ot be possible to accurately determine amounts photometrically Small amounts of RNA can be quantified using an Agilent 2100 bioanalyzer fluorometric quantification or quantitative real time RT PCR When purifying RNA from particularly small samples e g laser microdissected samples quantitative real time RT PCR should be used for quantification Spectrophotometric quantification of RNA To ensure significance Aygo readings should be greater than 0 15 An absorbance of 1 unit at 260 nm corresponds to 44 ug of RNA per ml Aggo 1 44 ug 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 47 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 45 Use the buffer in which the RNA is diluted to zero the spectrophotometer An example of the calculation involved in RNA quantification is shown below Volume of RNA sample 100 ul Dilution 10 ul of RNA sample 490 ul of 10 mM Tris Cl pH 7 0 1 50 dilution Measure absorbance of diluted sample in a 1 ml cuvette
62. plied to collect the second DNA eluate To combine the first and second DNA eluates reuse the collection tube from step 15 Note To achieve a higher DNA concentration elute with 2 x 30 ul Buffer EB The final DNA yield however will be reduced For PCR and real time PCR with the purified DNA QIAGEN offers a range of optimized ready to use kits that provide highly specific and sensitive results For details visit www qiagen com PCR QIAGEN kits and services for whole genome amplification WGA of limited amounts of DNA are also available For details visit www giagen com WGA Flow through contains Buffer AW1 and is therefore not compatible with bleach See page 7 for safety information 26 AllPrep DNA RNA Micro Handbook 07 2007 Protocol Simultaneous Purification of Genomic DNA and Total RNA from Animal and Human Tissues This protocol is for the purification of genomic DNA and total RNA from easy to lyse animal and human tissues For total RNA purification from frozen microdissected tissue samples see page 35 Determining the correct amount of starting material It is essential to use the correct amount of starting material in order to obtain optimal nucleic acid yield and purity A maximum amount of 5 mg fresh or frozen tissue or 2 3 mg RNAlater or Allprotect stabilized tissue which is partially dehydrated can generally be processed For most tissues the DNA binding capacity of the AllPrep DNA spin column the RNA
63. pplications we recommend performing DNase digestion by following steps F1 F4 Appendix F page 54 instead of step 7 8 Add 500 ul Buffer RPE 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 9 Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use see Things to do before starting Flow through contains Buffer RLT Plus or Buffer RW1 and is therefore not compatible with bleach See page 7 for safety information 32 AllPrep DNA RNA Micro Handbook 07 2007 9 Add 500 ul 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 wash the spin column membrane Discard the collection tube with the flow through Prepare the 80 ethanol with 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 10 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 collection tube with the flow through To avoid damage to their lids place the spin columns into the
64. r 30 s at 28000 x g 210 000 rpm Note Make sure that no liquid remains on the column membrane after centrifugation If necessary repeat the centrifugation until all liquid has passed through the membrane 5 Place the AllPrep DNA spin column in a new 2 ml collection tube supplied and store at room temperature 15 25 C or at 4 C for later DNA purification in steps 13 16 Use the flow through for RNA purification in steps 6 12 Note Do not store the AllPrep DNA spin column at room temperature or at 4 C for long periods Do not freeze the column Total RNA purification 6 Add 1 volume usually 350 pl of 70 ethanol to the flow through from step 5 and mix well by pipetting Do not centrifuge Proceed immediately to step 7 Note The volume of 7096 ethanol to add may be less than 350 ul if some lysate was lost during homogenization and DNA isolation Note Precipitates may be visible after addition of ethanol but this does not affect the procedure 7 Transfer the sample including any precipitate that may have formed to an RNeasy MinElute spin column placed in a 2 ml collection tube supplied Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm Discard the flowthrough Reuse the collection tube in step 8 8 Add 700 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
65. r DTT is added to Buffer RLT Plus before use see Things to do before starting Note If processing lt 2 ug tissue 20 ng carrier RNA 5 ul of a 4 ng ul solution may be added to the lysate before homogenization Prepare the carrier RNA as described in Things to do before starting After storage in RNAlater or Allprotect Reagent tissues may become slightly harder than fresh or thawed tissues Disruption and homogenization using standard methods is usually not a problem Note Incomplete homogenization leads to significantly reduced RNA yields and can cause clogging of the AllPrep and RNeasy MinElute spin columns Homogenization with the TissueRuptor or TissueLyser generally results in higher nucleic acid yields than with other methods However prolonged homogenization with these homogenizers results in greater DNA fragmentation 2a Disruption and homogenization using the TissueRuptor E Place the tissue in a suitably sized vessel Add 350 pl Buffer RLT Plus Note Use a suitably sized vessel with sufficient extra headspace to accommodate foaming which may occur during homogenization Generally round bottomed tubes allow more efficient disruption and homogenization than conical bottomed tubes E Place the tip of the disposable probe into the vessel and operate the TissueRuptor at full speed until the lysate is homogeneous usually 30 s Proceed to step 3 Note To avoid damage to the TissueRuptor and disposable probe d
66. r RW1 Contains ethanol flammable Risk phrase R10 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 R10 Flammable R20 21 22 Harmful by inhalation in contact with skin and if swallowed R22 Harmful if swallowed R32 Contact with acids liberates very toxic gas R36 38 Irritating to eyes and skin S13 Keep away from food drink and animal feedingstuffs S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S36 Wear suitable protective clothing S46 If swallowed seek medical advice immediately and show the container or label AllPrep DNA RNA Micro Handbook 07 2007 7 Introduction The AllPrep DNA RNA Micro Kit is designed to purify genomic DNA and total RNA simultaneously from the same precious sample Lysate is first passed through an AllPrep DNA spin column to selectively isolate DNA and then through an RNeasy MinElute spin column to selectively isolate RNA Pure DNA and RNA are purified from the entire sample in contrast to other procedures where either the biological sample or the purified total nucleic acids is divided into two before being processed separately The kit is compatible with small amounts of a wide range of cultured cells sorted cells tissues and microdissected samples of animal and human origin The AllPrep DNA RNA Micro Ki
67. ranscriptome Kit the QuantiTect Reverse Transcription Kit QuantiTect Primer Assays and QuantiFast Kits are intended for research use No claim or representation is intended to provide information for the diagnosis prevention or treatment of a disease Other kit sizes available please inquire Ere 55 eec LLLI LLLLLILLIILLIZLLILUIL IZLLLLLLLLLLOILLILLIIBIlblILu l LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLILLILAAALLLAALLLLLALLLAAAAALLILLELELZZZZLL AX AllPrep DNA RNA Micro Handbook 07 2007 59 www qiagen com Australia Orders 03 9840 9800 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 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 021 51345678 Fax 021 51342500 Technical 021 51345678 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 02 33430411 Fax 02 33430426 Technical 800 787980 Japan Teleph
68. ree DNase Set 50 Collection Tubes 2 ml Contents 50 AllPrep DNA Mini Spin Columns 50 RNeasy MinElute Spin Columns Collection Tubes RNase Free Reagents and Buffers 50 AllPrep DNA Mini Spin Columns 50 RNeasy Mini Spin Columns Collection Tubes RNase Free Reagents and Buffers 250 ml RNAprotect Cell Reagent For stabilization of RNA in 25 x 200 mg tissue samples 50 ml RNAlater RNA Stabilization Reagent For stabilization of RNA in 125 x 200 mg tissue samples 250 ml RNAlater RNA Stabilization Reagent For stabilization of RNA in 50 x 150 mg tissue samples 50 screw top tubes containing 1 5 ml RNA ater RNA Stabilization Reagent each For stabilization of RNA in 20 x 500 mg tissue samples 20 screw top tubes containing 5 ml RNAlater RNA Stabilization Reagent each For stabilization of DNA RNA protein in 50 x 200 mg tissue samples 100 ml Allprotect Tissue Reagent Allprotect Reagent Pump For 50 RNA minipreps DNase Buffer RDD and Water all RNase Free 1000 x 2 ml Collection Tubes For DNA and RNA purification from up to 1 x 107 cells or 30 mg tissue 56 Cat no 80284 80204 76526 76104 76106 76154 76163 76405 79254 19201 AllPrep DNA RNA Micro Handbook 07 2007 Product Contents Cat no QlAshredder 50 50 disposable cell lysate homogenizers 79654 QlAshredder 250 250 disposable cell lysate 79656 homogenizers TissueRuptor Handheld rotor stator homogenizer 5 Varie
69. s TissueRuptor Disposable Probes TissueRuptor 25 nonsterile plastic disposable probes 990890 Disposable Probes 25 for use with the TissueRuptor TissueLyser Universal laboratory mixer mill Varies disruptor TissueLyser Adapter Set 2 sets of Adapter Plates and 2 racks for 69982 2x24 use with 2 ml microcentrifuge tubes on the TissueLyser Stainless Steel Beads Stainless Steel Beads suitable for use 69989 5 mm 200 with the TissueLyser system TissueLyser Single Bead For dispensing individual beads 5 mm 69965 Dispenser 5 mm diameter Related products AllPrep DNA RNA Protein Mini Kit for simultaneous purification of DNA RNA and protein from cells and tissues AllPrep 50 AllPrep DNA Spin Columns 50 80004 DNA RNA Protein Mini RNeasy Spin Columns Collection Kit 50 Tubes RNase Free Reagents and Buffers AllPrep RNA Protein Kit for simultaneous purification of total RNA and protein from cultured cells AllPrep RNA Protein Kit 50 AllPrep Mini Spin Columns 50 80404 50 RNeasy Mini Spin Columns 50 Protein Cleanup Mini Spin Columns Collection Tubes RNase Free Reagents and Buffers Visit www qiagen com automation to find out more about the TissueRuptor and Tissuelyser and to order AllPrep DNA RNA Micro Handbook 07 2007 57 Product Contents Cat no DNeasy Blood amp Tissue Kit for purification of total DNA from animal blood and tissues and from cells yeast bacteria or viruses DNeasy Blo
70. solution 4 ng ul for 10 28 AllPrep DNA RNA Micro Handbook 07 2007 preps add 5 ul stock solution to 34 ul Buffer RLT Plus and mix by pipetting Add 6 ul of this diluted solution to 54 ul Buffer RLT Plus to give a working solution of 4 ng ul Add 5 ul of this solution to the lysate in step 2 Do not add the carrier RNA to the lysate if purifying RNA for use in oligo dT based amplification Buffer RPE Buffer AW1 and Buffer AW2 are each supplied as a concentrate Before using for the first time add the appropriate volume of ethanol 96 100 as indicated on the bottle to obtain a working solution W Before using the kit for the first time prepare 80 ethanol by mixing 24 ml ethanol 96 100 and 6 ml RNase free water supplied The procedure also requires 7096 ethanol which can be prepared by diluting ethanol 96 10096 with distilled water not supplied E Buffer RLT Plus may form a precipitate upon storage If necessary redissolve by warming and then place at room temperature Wi Preheat Buffer EB to 70 C to ensure optimal DNA elution Procedure Sample disruption and homogenization 1 Excise the tissue sample from the animal or remove it from storage Determine the amount of tissue Do not use more than 5 mg Proceed immediately to step 2 Weighing tissue is the most accurate way to determine the amount If necessary cut the tissue on a clean surface and weigh the piece to be used For RNAlater or Allprote
71. t DNA eluate use a new 1 5 ml collection tube not supplied to collect the second DNA eluate To combine the first and second DNA eluates reuse the collection tube from step 14 Note To achieve a higher DNA concentration elute with 2 x 30 ul Buffer EB The final DNA yield however may be reduced For PCR and real time PCR with the purified DNA QIAGEN offers a range of optimized ready to use kits that provide highly specific and sensitive results For details visit www qiagen com PCR QIAGEN kits and services for whole genome amplification WGA of limited amounts of DNA are also available For details visit www giagen com WGA Flow through contains Buffer AW1 and is therefore not compatible with bleach See page 7 for safety information 34 AllPrep DNA RNA Micro Handbook 07 2007 Protocol Simultaneous Purification of Genomic DNA and Total RNA from Microdissected Cryosections This protocol is for the purification of genomic DNA and total RNA from frozen microdissected samples of animal and human tissues For microdissected formalin fixed samples we recommend purifying RNA with the RNeasy FFPE Kit Laser microdissected tissue specimens present a particular challenge for molecular analysis as nucleic acids must be purified from very small amounts of starting material In addition fixation and staining steps may compromise the integrity of RNA and it may be necessary either to modify fixation protocols or to use cryosectio
72. t allows the parallel processing of multiple samples in less than 40 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 AllPrep DNA RNA procedure Genomic DNA purified with the AllPrep DNA RNA procedure has an average length of 15 30 kb depending on homogenization conditions DNA of this length is particularly suitable for PCR where complete denaturation of the template is important to achieve the highest amplification efficiency The purified DNA is ready to use in any downstream application including M PCR and real time PCR E Southern dot and slot blot analyses E Comparative genome hybridization CGH E Genotyping SNP analysis With the AllPrep DNA RNA procedure all RNA molecules longer than 200 nucleotides are purified The procedure provides an enrichment for mRNA since most RNAs 200 nucleotides such as 5 88 rRNA 5S rRNA and tRNAs which together comprise 15 2096 of total RNA are selectively excluded The purified RNA is ready to use in any downstream application including E RT PCR B Quantitative real time RT PCR W Differential display M cDNA synthesis For purification of high molecular weight DNA up to 150 kb we recommend using either QIAGEN Genomic tips or Blood amp Cell Culture DNA Kits For details visit www giagen com DNA t For purification of miRNA and other small RNAs from
73. t vendors of biological supplies When working with chemicals always wear a suitable lab coot disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 18 AllPrep DNA RNA Micro Handbook 07 2007 Protocol Simultaneous Purification of Genomic DNA and Total RNA from Animal and Human Cells Determining the correct amount of starting material It is essential to use the correct amount of starting material in order to obtain optimal nucleic acid yield and purity The maximum amount depends on M The RNA content of the cell type M The DNA binding capacity of the AllPrep DNA spin column M The RNA binding capacity of the RNeasy MinElute spin column 45 ug RNA M The volume of Buffer RLT Plus required for efficient lysis In addition cellular debris can reduce the binding capacity of the AllPrep DNA and RNeasy MinElute spin columns If processing a cell type not listed in Table 2 page 14 and if there is no information about its RNA content we recommend starting with no more than 5 x 10 cells Do not overload the AllPrep DNA spin column as this will lead to copurification of DNA with RNA Do not overload the RNeasy MinElute spin column as this will significantly reduce RNA yield and purity Counting cells is the most accurate way to quantitate the amount of starting material As a guide the number of HeLa cells obtained in various cultur
74. tain a working solution Storage The AllPrep DNA RNA Micro Kit should be stored dry at room temperature 15 25 C and is stable for at least 9 months under these conditions RNeasy MinElute spin columns should be stored at 2 8 C Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of AllPrep DNA RNA Micro Kit is tested against predetermined specifications to ensure consistent product quality AllPrep DNA RNA Micro Handbook 07 2007 Product Use Limitations The AllPrep DNA RNA Micro Kit is intended for research use No claim or representation is intended to provide information 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 Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not
75. tantial DNA contamination try processing smaller cell numbers rr LLELLELLELLLLLLILLLILILLLLLLLLLLIZLLLI ILLLLIILULLLLILLILULAAALLALLAALLLALLLLLLLLLLLLALLAAAAAAALAAAAAAALAAAAUMLLZZLLLZZZZLL ae AllPrep DNA RNA Micro Handbook 07 2007 41 Comments and suggestions b Incomplete removal of Be sure to remove any excess cell culture cell culture medium or medium or stabilization reagent to prevent stabilization reagent significant dilution of the lysis buffer The AllPrep DNA spin column will not bind DNA effectively if the lysis buffer is diluted c Tissue has high DNA For certain tissues with extremely high DNA content content e g thymus trace amounts of DNA may pass through the AllPrep DNA spin column Try using smaller samples Alternatively perform DNase digestion on the RNeasy MinElute spin column membrane see Appendix F page 54 Low A so A5so value in RNA eluate Water used to dilute Use 10 mM Tris Cl pH 7 5 not RNase free RNA for A o A s0o water to dilute the sample before measuring measurement purity see Appendix B page 46 RNA degraded a Inappropriate handling Ensure that tissue samples are properly stabilized of starting material and stored in RNAlater RNA Stabilization Reagent or Allprotect Tissue Reagent For frozen cell pellets or frozen tissue samples ensure that they were flash frozen immediately in liquid nitrogen and properly stored at 70 C Perform the AllPrep DNA RNA procedure quickly espe
76. trifuge at full speed for 5 min Discard the collection tube with the flow through 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 Place the RNeasy MinElute spin column in a new 1 5 ml collection tube supplied Add 14 ul 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 As little as 10 ul RNase free water can be used for elution if a higher RNA concentration is required but the yield will be reduced by approximately 20 Do not elute with less than 10 ul RNase free water as the spin column membrane will not be sufficiently hydrated The dead volume of the RNeasy MinElute spin column is 2 ul elution with 14 ul RNase free water results in a 12 pl eluate 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 qiagen com PCR For whole transcriptome amplification WTA of limited
77. ults in higher nucleic acid yields than with a syringe and needle 3a Pipet the lysate directly into a QlAshredder spin column not supplied placed in a 2 ml collection tube and centrifuge for 2 min at full speed Proceed to step 4 3b Place the tip of the TissueRuptor disposable probe into the lysate and operate the TissueRuptor at full speed until the lysate is homogenous usually 30 s Proceed to step 4 Note To avoid damage to the TissueRuptor and disposable probe during operation make sure the tip of the probe remains submerged in the buffer 3c Pass the lysate at least 5 times through a blunt 20 gauge needle 0 9 mm diameter fitted to an RNase free syringe Proceed to step 4 4 Transfer the homogenized lysate to an AllPrep DNA spin column placed in a 2 ml collection tube supplied Close the lid gently and centrifuge for 30 s at 28000 x g 10 000 rpm AllPrep DNA RNA Micro Handbook 07 2007 23 Note Make sure that no liquid remains on the column membrane after centrifugation If necessary repeat the centrifugation until all liquid has passed through the membrane 5 Place the AllPrep DNA spin column in a new 2 ml collection tube supplied and store at room temperature 15 25 C or at 4 C for later DNA purification in steps 13 16 Use the flow through for RNA purification in steps 6 12 If purification of total RNA containing small RNAs such as miRNA is desired follow steps D1 D6 in Appendix D on page 51 instead of
78. uring operation make sure the tip of the probe remains submerged in the buffer Foaming may occur during homogenization If this happens let the homogenate stand at room temperature for 2 3 min until the foam subsides before continuing with the procedure 2b Disruption and homogenization using the TissueLyser E Place the tissues in 2 ml microcentrifuge tubes containing one stainless steel bead 5 mm mean diameter If handling fresh or frozen tissue samples keep the tubes on dry ice 30 AllPrep DNA RNA Micro Handbook 07 2007 E Place the tubes at room temperature Immediately add 350 pl Buffer RLT Plus per tube E Place the tubes in the TissueLyser Adapter Set 2 x 24 E Operate the TissueLyser for 2 min at 20 Hz The time depends on the tissue being processed and can be extended until the tissue is completely homogenized E Rearrange the collection tubes so that the outermost tubes are innermost and the innermost tubes are outermost Operate the TissueLyser for another 2 min at 20 Hz Rearranging the tubes allows even homogenization E Carefully pipet the lysates into new microcentrifuge tubes not supplied Proceed to step 3 Do not reuse the stainless steel beads 2c Disruption using a mortar and pestle followed by homogenization using a GlAshredder homogenizer or a needle and syringe E Immediately place the weighed tissue in liquid nitrogen and grind thoroughly with a mortar and pestle E Decant tissue powder a
79. vice Departments or local distributors see back cover or visit www qiagen com 6 AllPrep DNA RNA Micro Handbook 07 2007 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 QIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Buffer AW1 contains guanidine hydrochloride Buffer RLT Plus contains guanidine thiocyanate and Buffer RW 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 196 v v sodium hypochlorite The following risk and safety phrases apply to the components of the AllPrep DNA RNA Micro Kit Buffer AW1 Contains guanidine hydrochloride harmful irritant Risk and safety phrases R22 36 38 S13 26 36 46 Buffer RLT Plus Contains guanidine thiocyanate harmful Risk and safety phrases R20 21 22 32 13 26 36 46 Buffe
80. ware 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 ethanolt and allowed to dry Solutions Solutions water and other solutions should be treated with 0 1 DEPC DEPC is a strong but not absolute inhibitor of RNases It is commonly used at a concentration of 0 1 to inactivate RNases on glass or plasticware or to create RNase free solutions and water DEPC inactivates RNases by covalent modification Add 0 1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution Let the solution incubate for 12 hours at 37 C Autoclave for 15 minutes to remove any trace of DEPC DEPC will react with primary amines and cannot be used directly to treat Tris buffers DEPC is highly unstable in the presence of Tris buffers and decomposes rapidly into ethanol and CO When preparing Tris buffers treat water with DEPC first and then diss
81. wash with Buffer RW1 Washing with Buffer RPE and elution are then performed according to the protocol on page 27 Important points before starting E Do not vortex the reconstituted DNase I DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the vial Things to do before starting E Prepare DNase stock solution before using the RNase Free DNase Set for the first time Dissolve the solid DNase 1500 Kunitz units in 550 pl of the RNase free water provided To avoid loss of DNase lI 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 M For long term storage of DNase I 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 54 AllPrep DNA RNA Micro Handbook 07 2007 Procedure Carry out the protocol starting on page 27 up to and including step 6 Instead of peforming step 7 the wash with Buffer RW1 follow steps F1 F4 below F1 Add 350 ul Buffer RW1 to the RNeasy MinElute spin column 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 F4 F2 Add 10 pl DNase stock solution see above to 70 pl Buffer RDD M

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