Home

InviTrap Spin Universal RNA Mini Kit User manual

1. Table 1 shows an example of one kind of tissue material following the theoretical predictions In other tissues it may be different The best method for disruption and homogenization depends on the kind of tissue used The tissues differ in cell size cell shape DNA RNA and protein contents as well as cell components Also several tissue materials are very rigid like aorta cartilage and bones etc These materials obviously differ in behavior during homogenization from tissues like for example liver etc Brain tissue in several regions contains a lot axonal material which has no nucleus respectively no DNA A homogenization always is influenced by the DNA contents of the sample Therefore here an other example of the influence of different homogenization methods with liver tissue is shown below fig 1 table 2 Liver tissue contains very high contents of DNA and RNA and is simple to disrupt so that here a treatment with mortar and pestle disrupt this material completely During the further lysis step under shaking the InviTrap RNA kit specific carrier material in the Lysis Solution shears efficient the high molecular weight cellular components and create a homogeneous lysate so that the RNA yield is also high when mortar and pestle will be used Total RNA was isolated from around 20 mg rat liver each using the InviTrap Spin Universal RNA Mini Kit in combination with different method for the sample homogenization Lane 1 marker Lane 2
2. Note To maximize the final yield of total RNA a complete disruption of tissue sample is important DNA in the sample may be sheared by passing it through a 20 gauge needle This may increase the lysis efficiency and the yield 2 Removal of genomic DNA Centrifuge the reaction tubes containing the tissue lysates for 2 min at maximum speed in a microcentrifuge and transfer the supernatant app 800 ul carefully into a 2 0 ml Receiver Tube not provided Add 500 ul 96 100 ethanol and mix thoroughly by pipetting up and down It is important to mix the lysate completely with ethanol 3 Binding of the total RNA to the RTA Spin Filter Transfer app 750 ul of the lysate to a RTA Spin Filter Incubate for 1 min and centrifuge for 2 min at 11 000 x g 11 000 rpm Discard the flow through and place the RTA Spin Filter back into the Receiver Tube Reload the RTA Spin Filter with the residual volume of lysate and centrifuge again for 1 min Discard the flow through and place the RTA Spin Filter back into the Receiver Tube 21 InviTrap Spin Universal RNA Mini Kit 1015 4 First washing of the RTA Spin Filter Add 600 ul Wash Buffer R1 onto the RTA Spin Filter and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the flow through and the RTA Receiver Tube Transfer the RTA Spin Filter in anew RTA Receiver Tube 5 Second washing of the RTA Spin Filter Add 700 ul Wash Buffer R2 onto the RTA Spin Filter and centrifuge for 1 min at 11 00
3. A maximum amount of 1 5 ml whole blood from healthy adults typically 4 000 7 000 leukocytes per ul can be processed on a single RTA spin column For blood with elevated numbers of leukocytes less than 1 5 ml must be used The maximum number of leukocytes that can be processed is 1 x 10 per spin column If more leukocytes are processed they will not be fully lysed and contaminants will not be completely removed even if the volume of the Lysis Solution TR is increased Maximum RNA yields using the InviTrap Spin RNA Kits are generally determined by two criteria lysis volume and binding capacity of the spin column Using the maximum amount of leukocytes that can be processed in the procedure 1 x 10 however the binding capacity of the RTA Spin Filter is not usually attained due to the low RNA content of leukocytes Note Ifthe RNA binding capacity of the RTA Spin Filter is exceeded yields of total RNA will not be consistent and less than the maximum possible total RNA may be recovered If the starting material is incompletely lysed the yield of total RNA will be lower than expected even if the binding capacity of the RTA Spin Filter is not exceeded Yields can also vary due to the developmental stage growth conditions etc of the sample source Lysis of whole blood Blood cells are lysed in two separate procedures erythrocyte lysis and leukocyte lysis Erythrocytes red blood cells of human blood do not contain nuclei and are therefore n
4. Spin Blood RNA Mini Kit starting with step 2 from protocol 1 The content of leukocytes should not be higher than 1 x 10 Note Lysis Solution TR contains carrier to bind the genomic DNA The carrier with the bound DNA looks gel like Don t confound this with clumps If the lysate is difficult to pipet too many DNA is bound See in the section Troubleshooting to solve the problem 3 Frozen leukocyte pellet Lysed leukocytes stored under Lysis Solution TR at 80 C can be used for RNA isolation Follow protocol from step 3 in protocol 1 Also shock frozen leukocyte pellets liquid nitrogen stored at 80 C may be used Follow protocol from step 2 in protocol 1 Important Material frozen at 20 C musin t be used 28 InviTrap Spin Universal RNA Mini Kit 1015 Troubleshooting Problem Probable cause Comments and suggestions clogged RTA Spin Filter too much starting material incomplete erythrocyte lysis inefficient lysis and homogenization of the starting material Increase g force and centrifugation time in subsequent preparations reduce the amount of starting material and or increase volume of Lysis Solution TR after lysis spin lysate to pellet debris and continue with the protocol using the supernatant all centrifugation steps should be conducted at room temperature extend incubation on ice to 20 min the leukocyte pellet should be white and may contain residual traces of erythrocytes
5. Sstratecee molecular 3 User manual InviTrap Spin Universal RNA Mini Kit for purification of total RNA from human and animal cells bacteria and yeast cells tissue samples paraffin embedded tissue whole blood samples with the common anticoagulants EDTA citrate RNA cleanup amp simultaneous protein isolation 1060100x0 MAA sTRATEC Molecular GmbH D 13125 Berlin Instruction for the InviTrap Spin Universal RNA Mini Kit The InviTrap Spin Universal RNA Mini Kit is a universal system for reliable and fast isolation of high quality total RNA from fresh or frozen samples of human or animal cell cultures max 1 x 10 cells from up to 20 mg fresh or frozen human and animal tissue sample and from up to 1 5 ml whole blood samples with anticoagulants like citrate and EDTA except heparin stabilized blood in a spin filter format The patented STRATEC Molecular technology combines an efficient lysis of the starting material with the rapid inactivation of RNases the stabilization of the total RNA and the almost complete separation of the DNA The kit can be used further for simultaneous isolation of total RNA and proteins as well as of total RNA and for RNA CleanUp in Trizol phases and paraffin embedded tissue DNase digestion to remove contaminating genomic DNA is not necessary The InviTrap Spin Universal RNA Mini Kit supplemental protocols are intended for life science research use only Prior to using it for other
6. 4 Adjust RNA binding conditions Add 250 ul lt 5 x 10 cells or 500 pl 5 x 10 1 x 10 cells 1 volume of 70 ethanol to the RNA containing lysate and mix thoroughly by pipetting up and down 5 Binding of the total RNA to the RNA RTA Spin Filter Pipet the RNA containing sample resulting from step 4 directly onto the RNA RTA Spin Filter Incubate for 1 min and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the flow through and reuse the RTA Receiver Tube If the volume of the RNA containing sample exceeds 700 ul divide the sample and load aliquots into the RNA RTA Spin Filter 6 First washing of the RNA RTA Spin Filter Add 600 ul Wash Buffer R1 onto the RNA RTA Spin Filter and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the flow through and the RTA Receiver Tube Put the RNA RTA Spin Filter in a new RTA Receiver Tube in step 7 7 Second washing of the RNA RTA Spin Filter Add 700 ul Wash Buffer R2 onto the RNA RTA Spin Filter and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the flow through and reuse the RTA Receiver Tube Repeat this washing step once 8 Drying of the RNA RTA Spin Filter membrane Discard the flow through and put the RNA RTA Spin Filter back to the RTA Receiver Tube To eliminate any traces of ethanol centrifuge for 4 min at maximum speed Discard the RTA Receiver Tube 9 Elution of total RNA Transfer the RNA RTA Spin Filter into a RNase free Elution Tube and pipet
7. DEPC diethyl pyrocarbonate The glassware has to stand 12 hours at 37 C and then autoclave or heat to 100 C for 15 min to remove residual DEPC o Electrophoresis tanks should be cleaned with detergent solution e g 0 5 SDS thoroughly rinsed with RNase free water and then rinsed with ethanol and allowed to dry o Non disposable plastic ware should be treated before use to ensure that it is RNase free Plastic ware should be thoroughly rinsed with 0 1 M NaOH 1 mM EDTA followed by RNase free water You can also take chloroform resistant plastic ware rinsed with chloroform to inactivate RNases o All buffers must be prepared from DEPC treated RNase free ddH20 When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Change gloves frequently and keep tubes closed All centrifugation steps are carried out at room temperature To avoid cross contamination cavity seams shouldn t be moisted with fluid Reduce the preparation time as much as possible Use only sterile disposable polypropylene tubes throughout the procedure these tubes are generally RNase free Keep isolated RNA on ice o Do not use kit components from other kits with the kit you are currently using unless the lot numbers are identical o Tominimize the risk of infections from potentially infectious material we recommend working under laminar air flow until the samples are lysed OTO O OTO O O This kit should on
8. However if erythrocyte lysis is incomplete the white pellet may not be visible and large amounts of erythrocytes will form a red pellet If this happens incubate for an additional 5 10 min on ice after addition of Buffer EL a second time very viscous lysate like a gel after addition of Lysis Solution TR to much starting material if too many leukocytes cells or tissue sample including DNA have been used the carrier with the bound DNA will be too viscous to pipet after homogenization in this case divide the sample into two aliquots and adjust the volumes of each aliquot to 900 ul with Lysis Solution TR Continue with the procedure from DNA removal little or no total RNA eluted incomplete removal of cell culture medium carryover of erythrocytes insufficient disruption or homogenization Incomplete elution make sure that the cell culture medium is complete removed after the cell harvest see Incomplete erythrocyte lysis above reduce the amount of starting material Overloading the kit reduces the yield prolong the incubation time with Elution Buffer R to 5 10 min or repeat elution step once again elute a second time with 100 ul of Elution Buffer R incubate RTA Spin Filter at room temperature 15 25 C for 5 min with RNase free water prior to centrifugation 29 InviTrap Spin Universal RNA Mini Kit 1015 Problem Probable cause Comments and suggestions DNA contamination
9. too much starting material no optimal suspension of DNA binding carrier reduce amount of starting material DNase digestion of the eluate containing the total RNA Shake Lysis Solution TR carefully before use RNA degraded inappropriate handling of the starting material age of blood or tissue sample lysis buffer does not contain DTT handling RNase contamination the RNA purification protocol should be performed quickly see also General notes on handling RNA page 31 cell pellets stored at 80 C for later processing should be immediately frozen after cell harvest by liquid nitrogen treatment blood or tissue samples stored for too long prior to RNA isolation See Sample storage page 9 ensure that DTT has been added to the Lysis Solution TR ensure that the protocol especially during the first few steps has been performed quickly See Appendix General Notes on handling RNA page 31 check for RNase contamination of buffers Be certain not to introduce any RNase throughout the procedure or during further handling for analysis See Appendix General Notes on handling RNA page 31 total RNA does not perform well in downstream applications e g RT PCR Ethanol carryover during elution salt carryover during elution perform the recommended additional centrifugation step in the protocol to remove all traces of ethanol before eluting increase g force or centrifugation time when
10. 40 100 ul of Elution Buffer R directly onto the membrane of the RNA RTA Spin Filter Incubate for 2 min and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the RNA RTA Spin Filter and place the eluted total RNA immediately on ice 16 InviTrap Spin Universal RNA Mini Kit 1015 Scheme Total RNA extraction from up to 20 mg tissue Please work quickly and perform all extraction steps at room temperature RT Please read the protocols carefully prior to the start of the preparation procedure transfer the sample into an appropriate vial homogenize completely the tissue sample using Zirconia beads Note Homogenize the carrier in the Lysis Solution TR by shaking and mix it with DTT add 600 ul DTT containing Lysis Solution TR in case of spleen kidney or lung tissue add 900 ul Lysis Solution TR transfer the lysate in a 2 ml Receiver Tube incubate the sample for an appropriate time under continuously shaking centrifuge for 2 min at maximum speed in a microcentrifuge transfer 500 ul of the supernatant carefully into a new 2 ml Receiver Tube and discard the DNA binding mineral particles to adjust the RNA binding conditions add 330 ul of 96 100 ethanol to the supernatant and mix thoroughly by pipetting up and down transfer the lysate completely to the RTA Spin Filter Set incubate for 1 min and centrifuge for 2 min at 11 000 x g 11 000 rpm discard the flow through and place the RTA Spin Filter back into the
11. Discard the flow through and reuse the RTA Receiver Tube If the volume of the RNA containing sample exceeds 700 ul divide the sample and load aliquots into the RTA Spin Filter 6 First washing of the RTA Spin Filter Add 600 ul Wash Buffer R1 onto the RTA Spin Filter and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the flow through and use a new RTA Receiver Tube in step 7 7 Second washing of the RTA Spin Filter Add 700 ul Wash Buffer R2 onto the RTA Spin Filter and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the flow through and reuse the RTA Receiver Tube Repeat this washing step once Discard the flow through and reuse the RTA Receiver Tube 8 Drying of the RTA Spin Filter membrane To eliminate any traces of ethanol centrifuge for 4 min at maximum speed Discard the RTA Receiver Tube 9 Elution of total RNA Transfer the RTA Spin Filter into a RNase free Elution Tube and pipet 40 100 ul of Elution Buffer R directly onto the membrane of the RTA Spin Filter Incubate for 2 min and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the RTA Spin Filter and place the eluted total RNA immediately on ice 40 InviTrap Spin Universal RNA Mini Kit 1015 Supplemental protocol for RNA clean up from liquids 1 Starting the cleanup procedure for samples containing no DNA This step is useful if your RNA is not contaminated by DNA A Purification of 100 ul sample from contaminations Pipet 350 ul
12. Spin Universal RNA Mini Kit 1015 Supplemental protocol for simultaneous isolation of RNA and DNA The starting material for RNA DNA purifications is sometimes limited and in some cases the isolation of DNA from the same sample is necessary Following the given protocol below the simultaneous preparation of RNA and DNA is possible using the InviTrap Spin Universal RNA Mini Kit Important Note The Wash Buffer R1 and R 2 as well 2 0 ml Receiver Tubes and 1 5 ml Elution Tubes for RNA purification provided with the kit are calculated for RNA isolation The calculated amount doesn t include the needed buffers and plastic simultaneous DNA isolation Please order additional Wash Buffers and tubes Simultaneous DNA isolation from cells See Protocol 1 Step 3 Binding of genomic DNA to the DNA Binding Spin Filter Pipet the lysate resulting from step 2 including any precipitate which may have formed directly onto the DNA Binding Spin Filter placed in a 2 ml Receiver Tube with lid Incubate the sample for 1 min and centrifuge at 11 000 x g 11 000 rpm for 2 min The eluate contains the total RNA Transfer the DNA Binding Spin Filter with the separated DNA bound to the carrier material into a new 2ml Receiver Tube and start with the washing steps A page 30 Simultaneous DNA isolation from tissue samples 1 Protocol 2 step 2 Removal of genomic DNA and of beads Centrifuge the 2 ml Receiver Tube containing the tissue lysates at 11 000 x
13. Wash Buffer R1 Add 48 ml 96 100 ethanol to each bottle Wash Buffer R2 Add 30 ml Buffer EL Concentrate to 970 ml H30 Label the bottle with Buffer EL Store Buffer EL at 4 C 250 total RNA extractions Adjust Lysis Solution TR with 1 100 Volume of 1 M DTT Add 80 ml 96 100 ethanol to the bottle Wash Buffer R1 Add 160 ml 96 100 ethanol to each bottle Wash Buffer R2 Add 30 ml Buffer EL Concentrate to 970 ml H20 Label the bottle with Buffer EL Store Buffer EL at 4 C It is possible to replace DTT by B Mercaptoethanol Adjust Lysis Solution TR with 1 100 volume of Mercaptoethanol 11 InviTrap Spin Universal RNA Mini Kit 1015 2 DTT Adjust Lysis Solution TR with 1 100 volume of 1 M DTT Due to the instability of dissolved DTT under oxidative conditions do not mix the whole Lysis Solution TR with DTT in case of the kits with 50 and 250 preparations We recommend the preparation of a volume DTT containing Lysis Solution TR shortly before carrying out the purifications adapted to the number of samples that will be processed Store the remaining Lysis Solution TR and DTT separately in accordance to the storage instructions see Storage Page 4 It is possible to replace DTT by B Mercaptoethanol In that case adjust Lysis Solution TR with 1 100 volume of B Mercaptoethanol as described above 3 Lysis Buffer TR Shake Lysis Solution TR gently before use to homogenize the DNA binding mineral carrier par
14. achieve complete disruption The homogenization means the reduction of the viscosity of the lysate after disruption Contaminating genomic DNA and other cellular components of high molecular weight are 9 InviTrap Spin Universal RNA Mini Kit 1015 sheared to form a homogenous lysate Incomplete homogenization results in inefficient binding of RNA to the RTA Spin Filter membrane and therefore significantly reduced RNA yields It is possible to use a commercially available bead mills in combination with or without beads for the disruption and homogenization of the starting material Alternatively the starting material can be reduced to a fine powder in liquid nitrogen using a mortar and pestle A mixture of Zirconia beads with a diameter of 0 7 mm Zirconia Beads and 1 2 mm Zirconia Beads II and a mixture of them in a ratio in number of 2 1 Beads Beads II are optimal to use for tissue in combination with the InviTrap Spin Universal RNA Mini Kit All other disruption parameters should be determined empirically for each application Disruption and homogenization using a mortar and pestle To disrupt tissue using a mortar and pestle freeze the sample immediately in liquid nitrogen and grind to a fine powder under liquid nitrogen Transfer the suspension tissue powder and liquid nitrogen into an RNase free tube useful for this temperature range Allow the liquid nitrogen to evaporate but do not allow the sample to thaw Add Lysis Soluti
15. drying the RTA Spin Filter ensure that Wash Buffer R1 and R2 are at room temperature check up Wash Buffer R1 and R2 for salt precipitates If there are any precipitates solve these precipitates by careful warming low A260 A280 value RNA sample is diluted in H20 do not use RNase free water to dilute the sample for measuring the RNA purity The use of a neutral buffer 10 mM Tris HCI pH 7 0 is recommended 30 InviTrap Spin Universal RNA Mini Kit 1015 Appendix General notes on handling RNA RNA is far less stable than DNA It is very sensitive to degradation by endogenous RNases in the biological material and exogenous RNases which are permanently present everywhere in the lab To achieve satisfactory qualitative and quantitative results in RNA preparations contaminations with exogenous RNases has to be reduced as much as possible Avoid handling bacterial cultures cell cultures or other biological sources of RNases in the same lab where the RNA purification is to be carried out All glassware should be treated before use to ensure that it is RNase free Glassware should be cleaned with detergent thoroughly rinsed and oven baked at 240 C for four or more hours before use Autoclaving alone will not completely inactivate many RNases Oven baking will both inactivate RNases and ensure that no other nucleic acids such as Plasmid DNA are present on the surface of the glassware You can also clean glassware with 0 1
16. for 1 min Discard the flow through and reuse the RTA Receiver Tube Repeat this washing step once 8 Drying of the RNA RTA Spin Filter membrane Discard the flow through and put the RNA RTA Spin Filter back to the RTA Receiver Tube To eliminate any traces of ethanol centrifuge for 4 min at maximum speed Discard the RTA Receiver Tube 9 Elution of total RNA Transfer the RNA RTA Spin Filter into a RNase free Elution Tube and pipet 40 100 ul of Elution Buffer R directly onto the membrane of the RNA RTA Spin Filter Incubate for 2 min and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the RNA RTA Spin Filter and place the eluted total RNA immediately on ice 42 InviTrap Spin Universal RNA Mini Kit 1015 Sstratecee molecular STRATEC Molecular GmbH Robert Rossle Str 10 13125 Berlin Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09 E mail info berlin stratec com www stratec com 1A5001 10 2015
17. g 11 000 rpm for 1 min in a microcentrifuge and transfer 500 ul of the supernatant carefully into a new 2 0 ml Receiver Tube for further RNA isolation Remove the residual supernatant to waste Add 600 ul Wash Buffer R1 to the pellet and resuspend the pellet by pipetting up and down Put a DNA Binding Spin Filter in a new 2 ml Receiver Tube Transfer the resuspended DNA containing sample without the ziconia beads to the DNA Binding Spin Filter and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the flow through and put the DNA Binding Spin Filter back to the 2 ml Receiver Tube Start with the washing steps B page 30 For big amounts of unlysed material which could clog the membrane the following procedure is recommended ll Before starting Protocol 2 step 1 Please transfer 600 ul DTT containing Lysis Solution TR vigorously mixed before added in a 2 ml Receiver Tube not provided and spin down the included carrier for 30 sec at 6 000 x g 8000 rpm Store the carrier in the closed tube Use the carrier free Lysis Solution TR and proceed step 1 and 2 of the protocol 2 Add 500 ul of the cleared supernant to the tube with the carrier and resuspend the carrier with the lysate 37 InviTrap Spin Universal RNA Mini Kit 1015 Centrifuge the 2 ml Receiver Tube containing the tissue lysates with carrier for 2 min at maximum speed in a microcentrifuge and transfer the complete supernatant wihtout carrier carefully into a new 2 0 ml
18. ml 96 100 ethanol Add 160 ml 96 100 to each bottle Wash Buffer ethanol to each bottle R2 Wash Buffer R2 Add 30 ml Buffer EL Add 30 mi Buffer EL Add 30 mi Buffer EL concentrate to 970 ml H20 Label or sign the bottle with Buffer EL Store Buffer EL at 4 C For protocol 1 RNA from cells Preparation of 10 ml 50ml 200 ml 70 ethanol Adjust Lysis Solution TR with 1 100 volume of 1M DTT Due to the instability of dissolved DTT under oxidative conditions do not mix the whole Lysis Solution TR with DTT in case of the kits with 50 and 250 preparations We recommend the preparation of a volume DTT containing Lysis Solution TR shortly before carrying out the purifications adapted to the number of samples that will be processed Store the remaining Lysis Solution TR and DTT separately in accordance to the storage instructions see Storage page 4 It is possible to replace DTT by B Mercaptoethanol In that case adjust Lysis Solution TR with 1 100 volume of B Mercaptoethanol as described above InviTrap Spin Universal RNA Mini Kit 1015 Symbols pal Manufacturer Eor Lot number Catalogue number Expiry date Consult operating instructions Temperature limitation ox HKE Do not reuse Storage All buffers except the 1 M DTT solution not provided and kit components of the InviTrap Spin Universal RNA Mini Kit should be stored well sealed and dry at room temperature RT and are stable for at l
19. onto the DNA Binding Spin Filter incubate for 1 min at RT and centrifuge for 1 min at 11 000 x g 11 000 rpm discard the DNA Binding Spin Filter add the recommended volume of ethanol 1 volume of 70 ethanol see Protocol 1 transfer the sample on the RNA RTA Spin Filter incubate for 1 min and centrifuge for 2 min at 1 000 x g 11 000 rpm discard the flow through and reuse the RTA Receiver Tube pipet 600 ul Wash Buffer R1 onto the RNA RTA Spin Filter and centrifuge for 1 min at 1 000 x g 11 000 rpm discard the flow through and put the RNA RTA Spin Filter in a new RTA Receiver Tube pipet 700 ul Wash Buffer R2 onto the RNA RTA Spin Filter and centrifuge for 1 min at 1 000 x g 11 000 rpm discard the flow through and reuse the RTA Receiver Tube Repeat this washing step to eliminate any traces of ethanol centrifuge for 4 min at maximum speed discard the RTA Receiver Tube transfer the RNA RTA Spin Filter into a RNase free Elution Tube pipet 30 100 ul of Elution Buffer R onto the membrane of the RTA Spin Filter incubate for 2 min at RT and centrifuge for 1 min at 1 000 x g 11 000 rpm discard the RNA RTA Spin Filter place the eluted total RNA immediately on ice 13 InviTrap Spin Universal RNA Mini Kit 1015 Sample quantity The maximal amount of pure total RNA that will be isolated depends on the applied starting material its age storage conditions and intracellular RNA level To assure a high
20. results are obtained using fresh blood samples that were collected in the presence of an anticoagulant like EDTA or citrate except heparin stabilized blood to get a real picture of the variety of RNA in the cell For optimal results blood samples should be processed within a few hours after collection mRNAs from blood cells have different stabilities e g shows the mRNA of some regulatory genes shorter half live the mRNAs from most housekeeping genes To ensure that the isolated RNA contains a representative distribution of mRNAs the blood sample should not be stored for long periods before isolating RNA Note The InviTrap Spin Universal RNA Mini Kit mustn t be used for frozen blood samples STRATEC Molecular will be released of its responsibilities if other sample materials than described in the Intended Use are processed or if the sample preparation protocols are changed or modified Sample preparation Disrupting and homogenizing of the tissue material are 2 distinct steps only in case of processing tissue samples The efficient and complete disruption and homogenization of the tissue samples is absolute essential for isolation of a high yield of total RNA The disruption procedure the breakage of intercellular matrix like cells walls organelles and plasma membranes is necessary to release the nucleic acids contained in the cell thus inefficient disruption decreases the RNA yield Different samples require different methods to
21. the same sample is necessary Following the given protocol below the simultaneous preparation of RNA and proteins is possible using the InviTrap Spin Universal RNA Mini Kit Important Note The buffer solutions for RNA purification provide very denaturing conditions due to the contained salts Proteins isolated according to the protocol below are denatured and can be analyzed by SDS PAGE or Western Blot Experiments requiring the native state of the proteins e g interaction assays are excluded 1 Collection of protein containing material The protein fraction is found in the flow through of the RTA Spin Filter in the purification protocol see Protocol 1 2 step 3 Measure the volume of the flow through 2 Precipitation of proteins Add the three fold volume of ice cold acetone and mix well by vortexing Centrifuge for 10 min at 11 000 x g 11 000 rpm at 4 C Discard the supernatant Be careful not to remove the pellet 3 Washing step Add 500 ul of ice cold 96 100 ethanol and centrifuge for 4 min at maximum speed at 4 C Discard the supernatant be careful not to remove the pellet 4 Dissolving of proteins Dissolve the protein pellet by suspending in a buffer solution suitable for the subsequent application For SDS PAGE directly dissolve proteins in 1 fold Laemmli Buffer and heat them at 99 C for 5 min ATTENTION Never try a TCA Precipitation from RNA Lysis Solutions poisonous gas will be generated 36 InviTrap
22. yield of pure total RNA the number of cells given in this manual should not be exceeded Direct cell counting is recommended If more than the allowed cell numbers will be processed it is suggested to split the sample In that case the volumes of Wash and Elution Buffer need not to be aligned To adjust the number of cells in a special application to the RNA binding capacity of the InviTrap Spin Universal RNA Mini Kit and avoid overloading the RNA binding membrane an RNA purification experiment with variation of the amount of starting material is recommended The RNA binding membranes have a maximum binding capacity of approx 100 ug The InviTrap Spin Universal RNA Mini Kit purification procedure is optimized for the use of max 1 x 10 human or animal cells Average yield of total RNA in dependence on the type of cell line using the InviTrap Spin Universal RNA Mini Kit Cell line Source Yield yg from 1 x 10 cells HeLa human cervical carcinoma 2 0 Jurkat human T cell leukemia 1 7 MRC 5 human 5 0 NIH3T3 human 6 5 Growth area and number of HeLa cells in various cell culture plates Cell culture plate Growth area in cm Number of cell 96 well 0 3 0 6 4 5x10 48 well 1 0 1 3 x 10 12 well 4 0 5 0 x 10 6 well 9 5 1 2 x 10 Growth area varies slightly depending on the supplier Confluent growth is assumed Values are reported per well Depending on the used cell lines lysates may beco
23. 0 x g 11 000 rpm Discard the flow through and reuse the RTA Receiver Tube Repeat this washing step once Discard the flow through and reuse the RTA Receiver Tube 6 Drying of the RTA Spin Filter To eliminate any traces of ethanol centrifuge for 4 min at maximum speed Discard the RTA Receiver Tube 7 Elution of total RNA Transfer the RTA Spin Filter into a RNase free Elution Tube and add 30 60 ul of Elution Buffer R directly onto the membrane of the RTA Spin Filter Incubate for 2 min and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the RTA Spin Filter and place the eluted total RNA immediately on ice Note Depending on the extracted yield or the needed concentration of total RNA different volumes of Elution Buffer R can be used A lower volume of Elution Buffer R will increase the concentration of RNA and a higher Volume of Elution Buffer R will lead to an increased yield but a lower concentration of total RNA Please note that the minimum of Elution Buffer R should be 30 ul 22 InviTrap Spin Universal RNA Mini Kit 1015 Protocol 4 Total RNA extraction from paraffin embedded tissue treated with formalin paraffin slices Starting material Paraffin embedded tissue Deparaffination 1 Transfer the starting material into a 1 5 ml reaction tube not provided Add 0 5 ml Octane or Xylene and vortex carefully to dissolve the paraffin 2 Centrifuge for 2 min at maximum speed to pellet down the tissue sample
24. 1015 4 Adjust RNA binding conditions Add 750 ul 96 100 ethanol to the supernatant Mix the suspension by pipetting several times Note A precipitate may form after the addition of ethanol This will not affect the InviTrap procedure 5 Binding of the total RNA to the RTA Spin Filter Transfer 800 ul of the lysate into a RTA Spin Filter without moistening the rim Incubate for 1 min at RT Centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the flow through and place the RTA Spin Filter back into the RTA Receiver Tube Transfer the residual sample into the same RTA Spin Filter incubate for 1 min and centrifuge again at 11 000 x g 11 000 rpm for 1 min Discard the flow through and place the RTA Spin Filter back into the RTA Receiver Tube 6 First washing of the RTA Spin Filter Add 600 ul Wash Buffer R1 onto the RTA Spin Filter and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the flow through and place the RTA Spin Filter into a new RTA Receiver Tube 7 Second washing of the RTA Spin Filter Add 700 ul Wash Buffer R2 onto the RTA Spin Filter and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the flow through and reuse the RTA Receiver Tube Repeat this washing step 8 Drying of the RTA Spin Filter membrane To eliminate any traces of ethanol from the RNA centrifuge for 4 min at maximum speed Discard the RTA Receiver Tube 9 Elution of total RNA Transfer the RTA Spin Filter into a RNase free El
25. 3 homogenization with mortar and pestle under liquid nitrogen A Lane 4 5 homogenization using zirconia beads on a Gyrator similar to a mixer mill B Lane 6 7 homogenization using a Gyrator C Lane 8 9 homogenization using zirconia beads on a vortex D 1 2 H r 5 6 7 8 9 1011 Lane 10 11 homogenization using a vortex E fig 1 The direct comparison shows that best result will be realized by using the mortar and pestle to homogenize the sample under liquid nitrogen for this tested sample RNA yield from rat liver in in comparison to the Table S MEINE oF Homogenization homogenization using mortar pestle 100 and liquid N2 Gyrator Bead Mill Zirconia Beads 90 5 Gyrator Bead Mill 45 7 Vortex Zirconia Beads 64 5 Vortex 39 1 18 InviTrap Spin Universal RNA Mini Kit 1015 Protocol 2 Total RNA extraction from up to 20 mg tissue Important For extraction of total RNA from spleen kidney or lung tissue please use protocol 2 1 Disruption and lysis of the starting material see page 9ff It is possible to use a commercially available rotor stator homogenizer or bead mills for the disruption of the starting material or to disrupt the starting material using mortar and pestle in liquid nitrogen and grind the tissue sample to a fine powder Note Incomplete disruption and homogenization will lead to significantly reduced yields and can cause clogging of the RTA spin column Homogenizat
26. 60 A2s0 provides an estimate of the purity of RNA with respect to the contaminants that absorb in the UV such as protein However the Azeo Azgo ratio is influenced considerably by pH Since water is not buffered the pH and the resulting Azgo Azgo ratio can vary greatly Lower pH results in lower Azgo Azgo ratio and reduced sensitivity to protein contaminations For accurate values it is recommend to measure absorbance in 10 mM Tris Cl pH 7 5 Pure RNA has an A260 A2s0 ratio of 1 9 2 1 in 10 mM Tris HCl pH 7 5 Always be sure to calibrate the spectrophotometer with the same solution For determination of the RNA concentration however it is recommend to dilute the sample in a buffer with neutral pH since the relationship between absorbance and concentration Azgo reading of 1 40 ug ml of RNA is based on an extinction coefficient calculated for RNA at neutral pH Wilfinger W W Mackey M and Chomczynski P 1997 Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity Bio Techniques 22 474 Values up to 2 3 are routinely obtained for pure RNA in 10 mM Tris HCl pH 7 5 with some photospectrometers 32 InviTrap Spin Universal RNA Mini Kit 1015 Denaturing agarose gel electrophoresis of RNA The overall quality of an RNA preparation may be assessed by electrophoresis on a denaturing agarose gel this will also give some information about RNA yield A denaturing gel system is suggested becaus
27. Discard the supernatant very careful This step should be repeated if any paraffin is still remained in the sample Follow the dissolution until the tissue sample looks transparent while paraffin is still white A final washing step with 96 100 ethanol is recommended then dry the sample 3 Centrifuge shortly and remove of the ethanol by aspiration with pipette Then incubate the open reaction tube at 52 C to evaporate the residual ethanol 4 Doa lysis step with Proteinase K Add 10 ul Proteinase K 40 mg ml and 90 ul of RNase free TE Buffer under the presence of 10 mM DTT to the sample mix completely by pipetting ap and down and incubate at 48 C for 10 min Mechanical grinding or a cutting of the material is recommended before or during lysis The amount of proteinase depends on the proteinase used in the protocol this protocol is giving an example also consider the manufacturers recommendations 5 10 minutes incubation under continuously agitation at 80 C of the mixture partially reverses formalin crosslinking of the released nucleic acids This improves RNA yield and quality as well as RNA performance in downstream enzymatic assays 6 The sample then has to be used complete the liquid and the solid phase as sample in step 1 protocol 2 Cause this kind of sample depending of the fixation in several cases gives no useable RNA we just can give several advice Following advices can be given Non pH adjusted formalin
28. Filter Pipet the whole lysate 450 ul or 2 x 450 ul including any precipitate which may have formed directly onto the DNA Binding Spin Filter placed in a 2 ml Receiver Tube Incubate for 1 min and centrifuge at 11 000 x g 11 000 rpm for 2 min Discard the DNA Binding Spin Filter Note On the surface of the membrane of DNA Binding Spin Filter is a gelly material visible This is the DNA bound to the Carrier 4 Adjust RNA binding conditions Add 250 ul 100 ul sample volume respectively 500 pl 200ul sample volume 96 100 ethanol to the RNA containing lysate and mix thoroughly by pipetting and down 5 Binding of the RNA to the RNA RTA Spin Filter Transfer the whole RNA containing sample resulting from step 4 directly into the RNA RTA Spin Filter Incubate for 1 min and centrifuge at 11 000 x g 11 000 rpm for 2 min Discard the flow through and reuse the RTA Receiver Tube If the volume of the RNA containing sample exceeds 700 ul divide the sample and load aliquots into the RNA RTA Spin Filter 6 First washing of the RNA RTA Spin Filter Add 600 ul Wash Buffer R1 onto the RNA RTA Spin Filter and centrifuge at 11 000 x g 11 000 rpm for 1 min Discard the flow through and put the RNA RTA Spin Filter in a new RTA Receiver 41 InviTrap Spin Universal RNA Mini Kit 1015 7 Second washing of the RNA RTA Spin Filter Add 700 ul Wash Buffer R2 onto the RNA RTA Spin Filter followed by centrifugation at 11 000 x g 11 000 rpm
29. RTA Receiver Tube pipet 600 ul Wash Buffer R1 onto the RTA Spin Filter centrifuge for 1 min at 11 000 x g 11 000 rpm discard the flow through and the RTA Receiver Tube place the RTA Spin Filter into a new RTA Receiver Tube pipet 700 ul Wash Buffer R2 onto the RTA Spin Filter centrifuge for 1 min at 11 000 x g 11 000 rpm discard the flow through and reuse the RTA Receiver Tube repeat this washing step discard the flow through and reuse the RTA Receiver Tube to eliminate any traces of ethanol centrifuge for 4 min at maximum speed discard the RTA Receiver Tube transfer the RTA Spin Filter into the RNase free Elution Tube pipet 30 60 ul of Elution Buffer R onto the membrane of the RTA Spin Filter incubate for 2 min and centrifuge for 1 min at 11 000 x g 11 000 rpm discard the RTA Spin Filter place the eluted total RNA immediately on ice 17 InviTrap Spin Universal RNA Mini Kit 1015 Influence of the different methods of sample homogenization on the yield using the InviTrap Spin Universal RNA Mini Kit with rat brain Total RNA was isolated from around 20 mg rat brain using the InviTrap Spin Universal RNA Mini Kit in combination with different method for the sample homogenization see below the comparison of the yield Table 1 Method of homogeizaion eee aaa Beate 00 Vortex Zirconia Beads 97 0 Gyrator without Zirconia Beads 94 7 Vortex 95 1 Mortar Pestle and liquid N2 71 0
30. Receiver Tube 6 Drying of the RTA Spin Filter To eliminate any traces of ethanol centrifuge for 4 min at maximum speed Discard the RTA Receiver Tube 7 Elution of total RNA Transfer the RTA Spin Filter into a RNase free Elution Tube and add 30 60 ul of Elution Buffer R directly onto the membrane of the RTA Spin Filter Incubate for 2 min and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the RTA Spin Filter and place the eluted total RNA immediately on ice Note Depending on the yield or the needed concentration of total RNA with different volumes of Elution Buffer R can be used A lower volume of Elution Buffer R will increase the concentration of RNA and a higher volume of Elution Buffer R will lead to an increased yield but a lower concentration of total RNA Please note that the minimum of Elution Buffer R should be 30 ul 20 InviTrap Spin Universal RNA Mini Kit 1015 Protocol 3 Total RNA extraction from up to 20 mg spleen kidney or lung tissue 1 Disruption and lysis of the starting material see page 9ff It is possible to use a commercially available rotor stator homogenizer or bead mills for the disruption of the starting material or to disrupt the starting material using mortar and pestle in liquid nitrogen and grind the tissue sample to a fine powder Note Incomplete disruption and homogenization will lead to significantly reduced yields and can cause clogging of the RTA spin column Homogenization
31. Receiver Tube For DNA isolation resuspend the carrier in 600 ul Wash Buffer R1 and follow from the Washing step A in the protocol below page 30 For RNA isolation use the supernatant and follow the protocol 2 step 3 Binding of the total RNA to the RTA Spin Filter page 19 Simultaneous DNA isolation from blood samples Protocol 3 step 3 Binding of genomic DNA to the Carrier Vortex the mixture from step 2 for 10 sec and incubate for 5 min at RT vortex 3 5 times during incubation Put a DNA Binding Spin Filter in a new 2 ml Receiver Tube Pipet the lysate resulting from step 2 including any precipitate which may have formed directly onto the DNA Binding Spin Filter placed in a 2 ml Receiver Tube with lid Incubate the sample for 1 min and centrifuge at 11 000 x g 11 000 rpm for 2 min The eluate contains the total RNA Transfer the DNA Binding Spin Filter with the separated DNA bound to the carrier material into a new 2 ml Receiver Tube and start with the washing steps A page 30 Washing Steps Washing step A First washing of the DNA Binding Spin Filter Add 600 ul Wash Buffer R1 onto the DNA Binding Spin Filter and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the flow through and the 2 ml Receiver Tube Put the DNA Binding Spin Filter in a new 2 ml Receiver Tube Washing step B Second washing of the DNA Binding Spin Filter Add 700 ul Wash Buffer R2 onto the DNA Binding Spin Filter and centrifuge for 1 min at 11 000
32. Receiver Tube add 750 ul 96 100 ethanol to the supernatant and mix by pipetting up and down transfer 800 ul onto the RTA Spin Filter incubate for 1 min and centrifuge for 1 min at 11 000 x g 11 000 rpm discard the flow through and place the RTA Spin Filter back into the RTA Receiver Tube transfer the residual sample into the same RTA Spin Filter and centrifuge again for 1 min at 11 000 x g 11 000 rpm discard the flow through and place the RTA Spin Filter back into the RTA Receiver Tube pipet 600 ul Wash Buffer R1 onto the RTA Spin Filter and centrifuge 1 min at 11 000 x g 11 000 rpm discard the flow through and place the RTA Spin Filter into a new RTA Receiver Tube pipet 700 ul Wash Buffer R2 onto the RTA Spin Filter and centrifuge for 1 min at 11 000 x g 11 000 rpm discard the flow through and reuse the RTA Receiver Tube Repeat this washing step to eliminate any traces of ethanol centrifuge for 4 min at maximum speed discard the RTA Receiver Tube transfer the RTA Spin Filter into a RNase free Elution Tube pipet 30 60 ul of Elution Buffer R onto the membrane of the RTA Spin Filter incubate for 2 min and centrifuge for 1 min at 11 000 x g 11 000 rpm discard the RTA Spin Filter place the eluted total RNA immediately on ice 24 InviTrap Spin Universal RNA Mini Kit 1015 Starting amounts of samples It is essential to use the correct amount of starting material in order to obtain optimal result
33. Solution TR The particle oound DNA is removed from the solution by subsequent centrifugation However RNA applications that show an increased sensitivity to contaminations of DNA e g caused by overloading the kit require an optional DNase digestion If the DNase digestion is carried out while running RNA purification protocol the DNase will be removed completely in the wash steps of the RNA binding RTA Spin Filter see supplemental protocols Binding total RNA After removal of the genomic DNA from the lysate by removal of the DNA binding mineral carrier particles the total RNA is found in the supernatant To adjust the RNA binding conditions 96 100 ethanol is added Following carefully mixing the solution is completely transferred onto the RNA binding RTA Spin Filter and the RNA is bound on the membrane by centrifugation Removing residual contaminants Contaminants are efficiently washed away using Wash Buffer R1 and R2 while the RNA remains bound to the membrane Elution Total RNA is eluted from the membrane using Elution Buffer R or RNase free water The eluted RNA is ready for use in different subsequent applications 10 InviTrap Spin Universal RNA Mini Kit 1015 Important notes Important points before starting a protocol Immediately upon receipt of the Product inspect the Product and its components as well as the package for any apparent damages correct quantities and quality If there are any unconformities you have to
34. all buffers are optimized to assure a high yield as well as a high purity of purified total RNA All manual work is reduced to a minimum THE PRODUCT IS INTENDED FOR USE BY PROFESSIONALS ONLY SUCH AS TECHNICIANS PHYSICIANS AND BIOLOGISTS TRAINED IN MOLECULAR BIOLOGICAL TECHNIQUES It is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of RNA followed by signal detection or amplification Any diagnostic results generated by using the sample preparation procedure in conjunction with any downstream diagnostic assay should be interpreted with regard to other Clinical or laboratory findings To minimize irregularities in diagnostic results adequate controls for downstream applications should be used The kit is in compliance with EU Directive 98 79 EC on in vitro medical devices But it is not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Product use limitation The kit is neither validated for the isolation of total RNA from serum plasma nor for viral RNA isolation The performance of the kit in isolating and purifying total RNA from fecal samples has not been evaluated The kit was not tested on its ability to desalinate RNA or for RNA purification from enzymatic reactions like DNase digestion mixtures Proteinase digestion RNA ligation or labeling reactions The included chemicals are only useable
35. at only the small white pellet is left Note Incomplete removal of the supernatant will interfere with lysis and subsequent binding of RNA to the RTA spin column resulting in lower yield and purity All following steps were perform quickly and at RT 2 Leukocyte disruption Important Before starting step 2 vortex Lysis Solution TR to resuspend the mineral carrier for DNA removal Dont forget to add DTT to the Lysis Solution TR Be aware that DTT in buffer is stable for at least 4 weeks see page 12 Add 900 ul Lysis Solution TR containing DTT to the cell pellet Pipet several times to complete remove any clumps of cells Note Lysis Solution TR contains carrier to bind the genomic DNA The carrier with the bound DNA looks gel like Don t confound this with clumps See in the section Troubleshooting 3 Binding of genomic DNA to the Carrier Transfer the cell lysis suspension into a 2 ml Receiver Tube Vortex for 10 sec and incubate for 5 min vortex 3 5 times during incubation Centrifuge the sample for 1 min at 11 000 x g 11 000 rpm a jelly pellet will be visible Transfer the supernatant into a new 2 ml Receiver Tube avoid carry over of the mineral particles with bound DNA Note During the continuously shaking the kit specific carrier material shears more efficient the high molecular weight cellular components and create a homogeneous lysate during vortexing several times 26 InviTrap Spin Universal RNA Mini Kit
36. aterial For more information please consult the appropriate material safety data sheets MSDS These are available online in convenient and compact PDF format at www stratec com for each STRATEC Molecular Product and its components If buffer bottles are damaged or leaking WEAR GLOVES AND PROTECTIVE GOGGLES when discarding the bottles in order to avoid any injuries STRATEC Molecular has not tested the liquid waste generated by the InviTrap Spin Universal RNA Mini Kit procedures for residual infectious materials Contamination of the liquid waste with residual infectious materials is highly unlikely but cannot be excluded completely Therefore liquid waste must be considered infectious and be handled and discarded according to local safety regulations European Community risk and safety phrases for the components of the InviTrap Spin Universal RNA Mini Kit to which they apply are listed below as follows Lysis Solution TR and Wash Buffer R1 contain a chaotropic salt which is an irritant Lysis Solution TR Wash Buffer R1 contains Guanidinthiocyanate contains Guanidinthiocyanate warning Warning H302 312 332 412 EUH032 P273 H302 312 332 412 EUH032 P273 H302 Harmful if swallowed H312 Harmful in contact with skin H332 Harmful if inhaled H412 Harmful to aquatic life with long lasting effects EUH032 Contact with acids liberates very toxic gas P273 Avoid release to the environment Emergency medical information can be obtained 24 h
37. by Ficoll density gradient centrifugation can be processed with the InviTrap Spin Universal RNA Mini Kit The erythrocyte lysis and Ficoll density centrifugation procedures both rely upon intact blood cells so fresh blood must be used Leukocyte lysis After complete removal of the of the erythrocytes by centrifugation the leukocytes are resuspended with the DTT containing Lysis Solution TR and thoroughly mixed Shake the Lysis Solution TR to resuspend the mineral carrier particles prior to use Due to the highly denaturing lysis conditions leukocytes are quickly broken and RNases are inactivated simultaneously allowing isolation of intact RNA the RNA is secured DTT is added to inactivate the RNases by cleaving intramolecular disulfide bridges Isolation of total cellular RNA from leukocytes requires efficient disruption of cells and homogenization of lysate for optimal yield and purity Traditional homogenization methods usually require a rotor stator homogenizer or a syringe and needle However the InviTrap Spin Universal RNA Mini Kit includes the carrier material in the Lysis Solution TR The sample will be incubated with Lysis Solution TR 10 min shaking continuously This treatment allows fast and simple homogenization of cell lysates without risk of cross contamination 25 InviTrap Spin Universal RNA Mini Kit 1015 Protocol 5 Total RNA isolation from 0 5 ml up to 1 5 ml whole blood 1 Erythrocyte lysis Invert carefully the fre
38. e During the InviTrap procedure for purification of RNA from tissue samples the tissue material will be efficiently lysed using Zirconia beads to homogenize the tissue material under highly denaturating conditions In the procedure of the InviTrap Spin Universal RNA Mini Kit if a whole blood sample is processed Buffer EL is provided that selectively lyses the red blood cells and leukocytes are recovered by centrifugation This pellet later on is processed like other cell pellets Since the procedure relies on intact leukocytes frozen blood cannot be used The InviTrap RNA isolation technology simplifies total RNA isolation by combining efficient lysis of the starting material and the inactivation of exogenous and endogenous RNases The genomic DNA is removed efficiently with the speed and convenience of a spin technology The genomic DNA is fixed at the surface of the nucleic acid binding mineral carrier particles provided within the Lysis Solution TR simultaneously to the cell lysis of the sample Selective binding of the DNA is assured by optimized buffer conditions that reduce the undesired binding of the RNA to a minimum The mineral carrier particles loaded with the genomic DNA are removed by centrifugation or by centrifugation on spin filter surface The RNA remains in the supernatant or filtrate and is after the adjustment of special binding conditions bound onto the membrane of the RNA RTA Spin Filter Contaminants are removed by re
39. e will lack the sharp rRNA bands or will not exhibit a 2 1 ratio Completely degraded RNA will appear as a very low molecular weight smear Figure 1 lane 2 Inclusion of RNA size markers on the gel will allow the size of any bands or smears to be determined and will also serve as a good control to ensure the gel was run properly Figure 1 lane 1 Note Poly A selected samples will not contain strong rRNA bands and will appear as a smear from approximately 6 kb to 0 5 kb resulting from the population of mRNAs and depending on exposure times and conditions with the area between 1 5 and 2 kb being the most intense this smear is sometimes apparent in total RNA samples as well Un NPW AUO in uw Figure 1 Intact vs Degraded RNA Two pg of degraded total RNA and intact total RNA were run beside Ambion s RNA Millennium Markers on a 1 5 denaturing agarose gel The 18S and 28S ribosomal RNA bands are clearly visible in the intact RNA sample The degraded RNA appears as a lower molecular weight smear Generally at least 200 ng of RNA must be loaded onto a denaturing agarose gel in order to be visualized with ethidium bromide Some RNA preparations such as those from needle biopsies or from laser capture microdissected samples result in very low yields In these cases it may be impossible to spare 200 ng of RNA to assess integrity Alternative nucleic acid stains such as SYBR Gold and SYBR Green II RNA gel stain fr
40. e comprises the following steps erythrocyte lysis only in case of processing a whole blood sample or disruption and homogenization of tissue samples using beads of different size or other methods or o cell lysis in case of processing cells o lysis of leukocytes only in case of processing a whole blood sample o selective binding of the genomic DNA to a specific carrier and separation of the carrier bound contaminating DNA o adjusting of RNA binding conditions o transfer of the sample into the RNA binding RTA Spin Filter Set o binding of the total RNA to the membrane optional purification of proteins o washing of the membrane and elimination of contaminants and ethanol o elution of high pure total RNA Repeated wash steps make sure that contaminations and enzyme inhibitors are efficiently removed and high purified RNA is eluted in Elution Buffer R or water This manual contains 6 protocols and 5 supplemented protocols 8 InviTrap Spin Universal RNA Mini Kit 1015 Sampling and storage of starting material Cell samples Best results are obtained using freshly extracted starting material e g cells As long as the samples are not shock frosted with liquid nitrogen or are incubated with RNase inhibitors or denaturing reagents the RNA is not secured and the gene expression profiles based on the RNA isolation and quantification will provide a false result Therefore it is essential that cells are immediately flash frozen subseque
41. e most RNA forms extensive secondary structure via intramolecular base pairing and this prevents it from migrating strictly according to its size Be sure to include a positive control RNA on the gel so that unusual results can be attributed to a problem with the gel or a problem with the RNA under analysis RNA molecular weight markers RNA sample known to be intact or both can be used for this purpose The given denaturing agarose gel method for RNA electrophoresis is modified from Current Protocols in Molecular Biology Section 4 9 Ausubel et al eds 1 Prepare the gel a Heat 1 g agarose in 72 ml water until dissolved then cool to 60 C b Add 10 ml 10X MOPS running buffer and 18 ml 37 formaldehyde 12 3 M WARNING Formaldehyde is toxic through skin contact and inhalation of vapors Manipulations involving formaldehyde should be done in a chemical fume hood 10X MOPS running buffer 0 4 M MOPS pH 7 0 0 1 M sodium acetate 0 01 MEDTA c Pour the gel using a comb that will form wells large enough to accommodate at least 25 ul d Assemble the gel in the tank and add enough 1X MOPS running buffer to cover the gel by a few millimeters Then remove the comb 2 Prepare the RNA sample Heat denature samples at 65 70 C for 5 15 min Denaturation for 5 min is typically sufficient for simply assessing RNA on a gel but a 15 min denaturation is recommended when running RNA for a Northern blot The longer incubation may be nece
42. eaction mix is for example 1 ul of DNase 50 u Enzyme in 9 ul 1 x DNase reaction buffer but here follow the advice of the manufacturer Do not exceed the reaction mix volume to more than 10 ul Incubate the RTA Spin Filter at room temperature for 10 min Add 600 ul Wash Buffer R2 onto the RTA Spin Filter incubate for 1 minute and centrifuge for 1 min at 11 000 x g 11 000 rpm Reuse the Receiver Tube Repeat the second washing step once again 39 InviTrap Spin Universal RNA Mini Kit 1015 Supplemental protocol for RNA clean up from Trizol aqueous phase 1 Starting the cleanup procedure Add an equal volume of DTT containing Lysis Solution TR to up to 350 ul of the Trizol aqueous phase in a 2 ml Receiver Tube Mix thoroughly by pipetting up and down Important Shake Lysis Solution TR gently before use Wait a short time because of foam formation 2 Binding of genomic DNA Incubate the sample for 1 min and centrifuge at 11 000 x g 11 000 rpm for 2 min 3 Harvesting of supernatant Pipet the supernatant into a new 2 ml Receiver Tube Discard the Receiver Tube with the pellet 4 Adjust RNA binding conditions Add 1 volume of 96 100 ethanol to the RNA containing lysate and mix thoroughly by pipetting up and down 5 Binding of the total RNA to the RTA Spin Filter Pipet the RNA containing sample resulting from step 4 directly onto the RTA Spin Filter Incubate for 1 min and centrifuge at 11 000 x g 11 500 rpm for 1 min
43. east 12 months under these conditions Store 1 M DTT solution not provided at 20 C to prevent oxidative damage Under this condition the solution of 1 M DTT is stable for 12 months If the kit components are consumed in more than one run it is recommended to aliquot the 1 M DTT solution and minimize the number of freezing and thawing cycles Wash Buffer R1 and R2 charged with ethanol should be appropriate sealed Before every use make sure that all components have room temperature If there are any precipitates within the provided solutions solve these precipitates by warming carefully Room temperature RT is defined as range from 15 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the InviTrap Spin Universal RNA Mini Kit for applications as described in this manual Purchaser must determine the suitability of the Product for its particular use Should any Product fail to perform the applications as described in the manual STRATEC Molecular will check the lot and if STRATEC Molecular investigates a problem in the lot STRATEC Molecular will replace the Product free of charge STRATEC Molecular reserves the right to change alter or modify any Product to enhance its performance and design at any time In accordance with STRATEC Molecular s ISO 9001 2000 and ISO EN 13485 certified Quality Management System the performance of all components of the InviTrap Spin Universal RNA Mini Ki
44. flick the tube and add DTT containing Lysis Solution TR volume as given in table 1 Mix thoroughly by pipetting up and down No cell clumps should be visible before proceeding with the next step table 1 Lysis Solution TR Number of pelleted cells 350 ul lt 5x 10 cells 700 ul 5 x 10 1 x 10 cells Monolayer cells Add DTT containing Lysis Solution TR to the monolayer cells volume as given in table 2 Collect the cell lysate with a rubber policeman Mix thoroughly by pipetting up and down No cell clumps should be visible before proceeding with the next step table 2 Lysis Solution TR Size of the culture vessels 350 pl 96 24 12 well plates 700 ul 6 well plates 35 mm dishes 12 5 cm flasks Important Shake Lysis Solution TR gently before use Wait a short time because of foam formation Note DNA in the sample may be sheared by passing it through a 20 gauge needle This may increase the lysis efficiency and the yield 15 InviTrap Spin Universal RNA Mini Kit 1015 3 Binding of genomic DNA to the DNA Binding Spin Filter Pipet the lysate resulting from step 2 including any precipitate which may have formed directly onto the DNA Binding Spin Filter placed in a 2 ml Receiver Tube with lid Incubate the sample for 1 min and centrifuge at 11 000 x g 11 000 rpm for 2 min Discard the DNA Binding Spin Filter or use the DNA Binding Spin Filter for simultaneous DNA isolation see page 37
45. ion with a bead mill etc generally results in higher RNA yields than with other methods Important Shake Lysis Solution TR gently before use Wait a short time because of foam formation Disruption using a rotor stator homogenizer vortexer bead mill gyrator 1 Transfer the weighed amount of fresh or frozen starting material in a suitable reaction vesicle for the homogenizer not provided and add about 10 Zirconia beads to the sample Zirconia Beads small Zirconia Beads II big ratio 2 1 Add 600 ul DTT containing Lysis Solution TR mixed before added Homogenize the sample Transfer the sample into the 2 ml Receiver Tube and place the sample under Lysis Solution TR for longer storage at 20 C or use the sample immediately for isolation of total RNA following the protocol step 2 ARON Disruption of the starting material using a mortar and pestle and liquid nitrogen 1 Transfer the weighed amount of fresh or frozen starting material under liquid nitrogen and grind the material to a fine tissue powder 2 Transfer the powder into the 2 ml Receiver Tube Do not allow the sample to thaw 3 Add 600 ul DTT containing Lysis Solution TR mixed before added and incubate the sample for an appropriate time under continuously shaking until a homogenic lysate is visible 4 Finally place the sample under Lysis Solution TR for longer storage at 20 C or use the sample immediately for isolation of total RNA following protocol s
46. l protocol for simultaneous isolation of RNA and DNA 37 Supplemental protocol for digestion on the RTA Spin Filter 39 Supplemental protocol for RNA clean up from Trizol aqueous phase 40 Supplemental protocol for RNA clean up from liquids 41 2 InviTrap Spin Universal RNA Mini Kit 1015 Kit contents of the InviTrap Spin Universal RNA Mini Kit Store 1 M DTT not provided at 20 C All other kit components are stable at room temperature RT 10 total RNA extractions 50 total RNA extractions 250 total RNA extractions Wash Buffer R2 ready to use final volume 2 x 60 ml Catalogue No 1060100900 1060100200 1060100300 Buffer EL 30 ml conc 30 ml conc 4 x 30 ml conc Lysis Solution TR 10 ml 50 ml 250 ml Zirconia Beads 1 1 5 Zirconia Beads Il 1 1 5 MaS TRUNET faa ee final TEF ml final R ml 15 ml 2x12ml 2x40 ml final volume 2 x 200 ml concentrate to 970 ml H20 Label or sign the bottle with Buffer EL Store Buffer EL at 4 C concentrate to 970 ml H20 Label or sign the bottle with Buffer EL Store Buffer EL at 4 C Elution Buffer R 2ml 15ml 30 ml DNA Binding Spin Filter 10 50 5x50 RNA RTA Spin Filter Set 10 50 5x50 2 0 ml Receiver Tubes 20 2x50 10 x 50 RTA Receiver Tubes 10 50 5x50 Elution Tubes 10 50 5x50 Manual 1 1 1 Add 20 ml 96 100 ethanol Add 80 ml 96 100 Initial Steps to the bottle Wash Buffer ethanol to the bottle Wash R1 Buffer R1 Add 48
47. ly be used by trained personnel Storage of RNA Purified viral RNA can be stored at 80 C and is stable for several years at this condition Quantification of RNA The concentration of RNA should be determinate by measuring the absorbance at 260 nm A2s0 in a spectrophotometer Readings should be greater than 0 10 to ensure significance An absorbance of 1 unit at 260 nm corresponds to 40 ug of RNA per ml This relation is valid only for measurements at neutral pH The ratio between absorbance values at 260 nm and 280 nm gives an estimate of RNA purity see below When measuring RNA samples make sure that cuvettes are RNase free esp if the RNA is to be recovered after spectroohotometry This can be accomplished by washing cuvettes with 0 1 NaOH 1 mM EDTA followed by washing with RNase free water Use the buffer in which the RNA is diluted for calibration of the spectrophotometer 31 InviTrap Spin Universal RNA Mini Kit 1015 An example of the calculation involved in RNA quantification o Volume of RNA sample 100 ul o Dilution 20 ul of RNA sample 180 ul of 10 mM Tris HCl pH 7 0 1 10 dilution o Measure absorbance of diluted sample in a 0 2 ml cuvette RNase free Azs 0 2 Concentration of the RNA sample 40 ug ml Azgo dilution factor 40 ug ml 0 2 10 80 ug ml Total amount concentration volume of sample in ml 80 ug ml 0 1 ml 8 ug of RNA Purity of RNA The ratio of the readings at 260 nm and 280 nm A2
48. me viscous if a large number of cells is lysed High viscosity always causes clogged membranes and leads to a reduced yield and quality of the isolated RNA Therefore it is suggested to perform a preliminary viscosity analysis of the desired cell line depending on the cell number 14 InviTrap Spin Universal RNA Mini Kit 1015 Important indications The following notes are valid for all protocols Note The centrifugation steps were made with the Centrifuge 5415 D from Eppendorf The indicated rom amounts are referring to this centrifuge Protocol 1 Total RNA extraction from cell culture 1 Harvesting cells see also Sample quantity page 14 Cells grown in suspension Spin up to 1 x 10 cells for 5 min at 240 x g 1 500 rpm Discard the supernatant and remove all media completely Cells grown in a monolayer In large culture vessels dishes gt 35 mm flasks gt 12 5 cm detach cells by trypsination Transfer the cells to a centrifuge tube and sediment by centrifugation at 240 x g 1 500 rpm for 5 min Remove the supernatant completely In small culture vessels 96 24 12 6 well plates 35 mm dishes 12 5 cm flasks discard the media completely and continue with the lysis immediately Important Incomplete removal of the cell culture media will inhibit the lysis and dilute the lysate which will affect the binding of RNA to the RNA RTA Spin Filter 2 Cell Disruption Cell pellet To loosen the cell pellet
49. notify STRATEC Molecular in writing with immediate effect upon inspection thereof If buffer bottles are damaged contact the STRATEC Molecular Technical Services or your local distributor In case of liquid spillage refer to Safety Information see page 6 Do not use damaged kit components since their use may lead to poor kit performance o Always change pipet tips between liquid transfers To avoid cross contamination we recommend the use of aerosol barrier pipet tips o All centrifugation steps are carried out at room temperature When working with chemicals always wear a Suitable lab coat disposable gloves and protective goggles Discard gloves if they become contaminated Do not combine components of different kits unless the lot numbers are identical Avoid microbial contamination of the kit reagents To minimize the risk of infections from potentially infectious material we recommend working under laminar air flow until the samples are lysed o This kit should only be used by trained personnel O O O0 0 Preparing reagents and buffers 1 Preparing buffers 10 total RNA extractions Adjust Lysis Solution TR with 1 100 volume of 1 M DTT Wash Buffer R1 and R2 are ready to use Add 30 ml Buffer EL Concentrate to 970 ml H30 Label the bottle with Buffer EL Store Buffer EL at 4 C 50 total RNA extractions Adjust Lysis Solution TR with 1 100 Volume of 1 M DTT Add 20 ml 96 100 ethanol to the bottle
50. nt to the cell harvest using liquid nitrogen and are stored at 80 C RNA contained in such deep frozen samples is stable for months RNA purification should be processed as soon as possible The starting material can be stored also in Lysis Solution TR at 80 C after cell lysis Tissue samples Best results are obtained using fresh tissue samples As long as samples are not shock frosted or are incubated with RNase inhibitors or denaturing reagents the RNA is not secured Therefore it is essential that cells are immediately flash frozen after cell harvest and are stored at 80 C RNA from deep frozen samples is stable for months Frozen tissue should not be allowed to thaw while handling RNA purification should be processed as soon as possible Cells can be stored also in Lysis Solution TR at 80 C after cell lysis Formalin fixation and paraffin embedding procedures according to standard protocols always cause a degradation of nucleic acids The use of freshly cut sections with a thickness of up to 10 um is recommended Do not apply more than 8 sections with a surface area of 250 mm in one purification run Tissues with a high DNA content e g thymus request the use of fewer sections per preparation to avoid overloading the kit To adjust the number of sections used in one preparation to the amount of nucleic acid bound by the InviTrap Spin Universal RNA Mini Kit we recommend starting with one or 2 sections Whole blood samples Best
51. of DTT containing Lysis Solution TR directly onto the DNA Binding Spin Filter without lid placed in a 2 ml Receiver Tube with lid Centrifuge at 13 400 x g 12 000 rpm for 2 min Discard the DNA Binding Spin Filter Take the flow through for the following cleanup procedure Just add 100 ul of the sample reaction mixture to the flow through Lysis Solution TR Then continue with step 4 B Purification of 200 ul sample from contaminations Pipet 700 ul of DTT containing Lysis Solution TR directly onto the DNA Binding Spin Filter without lid placed in a 2 ml Receiver Tube with lid Centrifuge at 13 400 x g 12 000 rpm for 2 min Discard the DNA Binding Spin Filter Take the flow through for the following cleanup procedure Just add 200 ul of the sample reaction mixture to the flow through Lysis Solution TR Then continue with step 4 2 Starting the cleanup procedure for samples containing contaminating DNA This procedure useful if your RNA is contaminated by DNA A Purification of 100 ul sample from contaminations Add 350 ul of DTT containing Lysis Solution TR to 100 ul RNA sample Mix thoroughly by pipetting up and down B Purification of 200 ul sample from contaminations Add 700 ul of DTT containing Lysis Solution TR to 200 ul RNA sample Mix thoroughly by pipetting up and down Important Shake Lysis Solution TR gently before use Wait a short time because of foam formation 3 Binding of genomic DNA to the DNA Binding Spin
52. om Molecular Probes offer a significant increase in sensitivity over ethidium bromide Using a 300 nm transilluminator 6 x 15 watt bulbs and a special filter as little as 1 ng and 2 ng of RNA can be detected with SYBR Gold and SYBR Green II RNA gel stain respectively 34 InviTrap Spin Universal RNA Mini Kit 1015 Ordering information Product Package size Catalogue No InviTrap Spin Universal RNA Mini Kit 50 preps 1060100200 InviTrap Spin Universal RNA Mini Kit 250 preps 1060100300 Single components for InviTrap Spin Universal RNA Mini Kit Buffer EL concentrate 30 ml 1060105600 Lysis Solution TR 30 ml 1060101700 Wash Buffer R1 add 20 ml ethanol 20 ml 1060103500 Wash Buffer R2 add 48 ml ethanol 12 ml 1060103600 Elution Buffer R 15 ml 1060104100 Related products InviTrap Spin Cell RNA Mini Kit 50 preparations 1061100200 InviTrap Spin Cell RNA Mini Kit 250 preparations 1061100300 InviTrap Spin Tissue RNA Mini Kit 50 preparations 1062100200 InviTrap Spin Tissue RNA Mini Kit 250 preparations 1062100300 InviTrap Spin Blood RNA Mini Kit 50 preparations 1063100200 InviTrap Spin Blood RNA Mini Kit 250 preparations 1063100300 35 InviTrap Spin Universal RNA Mini Kit 1015 Supplemental not validated protocols not for diagnostic use Supplemental protocol for simultaneous isolation of RNA and Proteins The starting material for RNA purifications is often limited and in some cases the isolation of proteins from
53. on and it is recommended to continue as quickly as possible with the homogenization using a shredder column or a 20 gauge needle Erythrocyte lysis only in case of processing whole blood samples The removal of the erythrocytes allows the RNA purification from the leukocytes Human blood collected from healthy people contains approximately 1000 times more erythrocytes than leukocytes while only the latter one contain RNA Due to the increased susceptibility of the erythrocytes to osmotic shock lysis by addition of a hypo tonic buffer system the whole blood sample is directly charged with Buffer EL and thoroughly mixed According to the special buffer conditions the erythrocytes are selectively lysed while the leukocytes still remain Procedure Lysis After complete removal of any supernatant erythrocytes or culture medium or after complete disruption the samples are directly charged with the DTT containing Lysis Solution TR and thoroughly mixed Shake the Lysis Solution TR to resuspend the mineral carrier particles prior to use Due to the strong denaturing lysis conditions cells are quickly broken and RNases are inactivated simultaneously The RNA is secured DTT is added to inactivate the RNases by cleaving intramolecular disulfide bridges Binding and removal of DNA In general DNase digestion is not necessary to remove the genomic DNA Under lysis conditions the DNA is efficiently bound by the mineral carrier particles contained in Lysis
54. once Differing of starting material or flow trace may lead to inoperability therefore neither a warranty nor guarantee in this case will be given neither implied nor express The user is responsible to validate the performance of the STRATEC Molecular Product for any particular use STRATEC Molecular does not provide for validation of performance characteristics of the Product with respect to specific applications STRATEC Molecular Products may be used e g in clinical diagnostic laboratory systems conditioned upon the complete diagnostic system of the laboratory the laboratory has been validated pursuant to CLIA 88 regulations in the U S or equivalents in other countries All Products sold by STRATEC Molecular are subject to extensive quality control procedures according to ISO 9001 2000 and ISO EN 13485 and are warranted to perform as described herein Any problems incidents or defects shall be reported to STRATEC Molecular immediately upon detection thereof The chemicals and the plastic parts are for laboratory use only they must be stored in the laboratory and must not be used for purposes other than intended The Product with its contents is unfit for consumption 5 InviTrap Spin Universal RNA Mini Kit 1015 Safety information When and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid skin contact Adhere to the legal requirements for working with biological m
55. ot important for RNA isolation since they do not synthesize RNA The target of RNA isolation from whole blood is leukocytes white blood cells which are nucleated and therefore synthesize RNA Leukocytes consist of 3 main cell types lymphocytes monocytes and granulocytes Erythrocyte lysis The removal of the erythrocytes allows the RNA purification from pure leukocytes Blood of a healthy human contains approximately 1000 times more erythrocytes than leukocytes while only the latter one contain RNA of interest Due to the increased susceptibility of the erythrocytes to osmotic shock lysis by addition of a hypo tonic buffer system the whole blood sample is directly charged with Buffer EL provided in the kit and thoroughly mixed According to the special buffer conditions the erythrocytes are selectively lysed while the leukocytes still remain Intact leukocytes are then recovered by centrifugation The conditions for selective lysis of erythrocytes in the InviTrap procedure have been optimized to allow fast removal of erythrocytes without affecting the stability of the leukocytes The erythrocyte lysis procedure can be scaled up for volumes of whole blood gt 50 ul A common alternative to erythrocyte lysis is Ficoll density gradient centrifugation In contrast to erythrocyte lysis procedures Ficoll density gradient centrifugation only recovers mononuclear cells lymphocytes and monocytes and removes granulocytes Mononuclear cells isolated
56. ours a day from infotrac outside of USA 1 352 323 3500 in USA 1 800 535 5053 6 InviTrap Spin Universal RNA Mini Kit 1015 Product characteristics of the InviTrap Spin Universal RNA Mini Kit Starting material Yield Time Ratio up to 1 x 10 human or animal up to 100 pg total RNA 15 20 minutes A260 A 280 cells cultured cells 1 6 2 0 e g 10 15 ug total RNA from 5 x 10 NIH 3T3 fibroblasts up to 20 mg fresh or frozen up to 80 ug total RNA about 10 min after tissue depends on type and tissue lysis amount of starting material up to 1 5 ml of whole blood up to 1 5 ug total RNA ml about 45 minutes sample containing common of whole blood anticoagulants depending on the amount of lymphocytes The InviTrap Spin Universal RNA Mini Kit provides a fast and efficient way for reliable isolation of high quality DNA free total RNA from fresh or frozen samples of human or animal cell cultures tissue samples and from whole blood The content of leukocytes that can be processed in one preparation must not exceed 1x10 1 5 ml whole blood of a healthy adult contains approximately 4 000 7 000 leukocytes ml sample volume The kit allows a simultaneous isolation of proteins from the same sample During the InviTrap procedure for purification of RNA from cell samples a cell content higher than 1 x 10 should not be used DNA contaminations and reduced RNA purity would be the consequenc
57. peated wash steps and the purified total RNA can be eluted in a small volume of RNase free water The isolated total RNA is ready to use and should be stored at 80 C A specialized buffering system allows RNA species of sizes down to 200 base to bind to the spin filter membrane The particle size distribution of purified RNA is similar to those gained using a CsCl gradient The extracted RNA contains enriched mRNA 7 InviTrap Spin Universal RNA Mini Kit 1015 Due to the high purity the isolated total RNA is ready to use for a broad panel of downstream applications like Northern Blotting RNA dot blots cDNA library in vitro translation RT PCR DDRT PCR or TaqMan analysis and array technologies o0o000000 The purification procedure is rapid and does not require a phenol chloroform extraction Only minimal interaction by the user is necessary The procedure is designed to avoid sample to sample cross contaminations Traditional time killing procedures can be replaced using the InviTrap Spin Universal RNA Mini Kit STRATEC Molecular offers for each application also separate spin kits kits for use in 96 well format for use on robots manifold or centrifuges as well as magnetic bead based RNA isolation systems The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG Principle and procedure The InviTrap Spin Universal RNA Mini Kit procedur
58. purposes the user must validate the system in compliance with the applicable law directives and regulations The kit is neither validated for the isolation of total RNA from serum plasma nor for viruses The performance of the kit in isolating and purifying total RNA from fecal samples has not been evaluated Compliance with EU Directive 98 79 EC on in vitro medical devices Not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Trademarks InviTrap Invisorb Registered marks trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law The Invisorb technology is covered by patents and patent applications US 6 110363 US 6 043 354 US 6 037 465 EP 0880535 WO 9728171 WO 9534569 EP 0765335 DE 19506887 DE 10041825 2 WO 0034463 InviTrap and Invisorb are registered trademarks of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2015 STRATEC Molecular all rights reserved 1 InviTrap Spin Universal RNA Mini Kit 1015 Contents Kit contents of the InviTrap Spin Universal RNA Mini Kit 3 Symbols 4 Storage 4 Quality control 4 Intended use 5 Product use limitation 5 Safety information 6 Product characteristics of the InviTrap Spin Universal RNA Mini Kit 7 Principle and proced
59. sh sample minimum 15 times until all is one homogenous fraction Don t vortex Add 10 ml of cold 4 C prepared Buffer EL to 0 5 ml 1 5 ml of whole blood max 1x 10 leukocytes in a 15 ml tube e g 15 ml Falcon Tubes not provided Mix shortly but completely by inverting Don t vortex Incubate on ice for 15 min Mix briefly by inverting 2 times during incubation Note The cloudy suspension becomes translucent during incubation indicating lysis of erythrocytes If necessary incubation time can be extended to 20 min Please note if you work with a fresh blood sample up to 3 hours after taking of the sample extend the lysis time of erythrocytes to 45 min Please note that up to 1 5 ml of whole blood can be processed If the expected amount of leukocytes is more than 1 x 10 reduce starting volume of the blood ample Centrifuge at 960 x g 3 000 rpm for 5 min at 4 C remove the supernatant completely but very carefully leukocytes will form a visible cell pellet Note Leukocytes will form a pellet after centrifugation Ensure that supernatant is completely removed e g by aspiration Trace amounts of erythrocytes which give the pellet the red tint will be eliminated in the following wash steps Add 5 ml of the cooled Buffer EL to the cell pellet mix by snipping with the finger and centrifuge again for 5 min at 960 x g 3 000 rpm at 4 C Remove the supernatant as complete as possible including the red interface th
60. ssary to completely denature the RNA To 1 3 ug RNA add 0 5 3 x volumes Formaldehyde Load Dye To simply check the RNA on a denaturing gel as little as 0 5 X Formaldehyde Load Dye can be used but to completely denaturate the RNA e g for Northern blots use 3 X volumes of Formaldehyde Load Dye Ethidium bromide can be added to the Formaldehyde Load Dye at a final concentration of 10 ug ml Some size markers may require significantly more than 10 ug ml ethidium bromide for visualization To accurately size your RNA however it is important to use the same amount of ethidium bromide in all the samples including the size marker because ethidium bromide concentration affects RNA migration in agarose gels 3 Electrophoresis Load the gel and electrophorese at 5 6 V cm until the bromophenol blue the faster migrating dye has migrated at least 2 3 cm into the gel or as far as 2 3 the length of the gel 33 InviTrap Spin Universal RNA Mini Kit 1015 4 Results Visualize the gel on a UV transilluminator If ethidium bromide was not added to the Formaldehyde Load Dye the gel will have to be post stained and destained Intact total RNA run on a denaturing gel will have sharp 28S and 18S rRNA bands eukaryotic samples The 28S rRNA band should be approximately twice as intense as the 18S rRNA band Figure 1 lane 3 This 2 1 ratio 28S 18S is a good indication that the RNA is intact Partially degraded RNA will have a smeared appearanc
61. t have been tested separately against predetermined specifications routinely on lot to lot to ensure consistent product quality If you have any questions or problems regarding any aspects of InviTrap Spin Universal RNA Mini Kit or other STRATEC Molecular products please do not hesitate to contact us A copy of STRATEC Molecular s terms and conditions can be obtained upon request or are presented at the STRATEC Molecular webpage For technical support or further information please contact from Germany 49 0 30 9489 2901 2910 from abroad 49 0 30 9489 2907 or contact your local distributor 4 InviTrap Spin Universal RNA Mini Kit 1015 Intended use The InviTrap Spin Universal RNA Mini Kit is the universal tool for reliable and fast isolation and purification of high quality total RNA from fresh or frozen samples of human or animal cell cultures max 1 x 10 cells for up to 20 mg of fresh or frozen human and animal tissue samples and for up to 1 5 ml of whole blood sample with anticoagulants like citrate and EDTA except heparin stabilized blood in a spin filter format For reproducible and high yields an appropriate sample storage and quick operation under the rules for RNA operation is essential For some studies a simultaneous investigation of the RNA and of the sample specific proteins is necessary The kit includes an isolation protocol of RNA and intracellular proteins from the same sample The isolation protocol and
62. tep 2 Note To maximize the final yield of total RNA a complete disruption of tissue sample is important Traces of DNA in the sample may be sheared by passing it through a 20 gauge needle This may increase the lysis efficiency and the yield 2 Removal of genomic DNA and of beads Centrifuge the 2 ml Receiver Tube containing the tissue lysates for 2 min at maximum speed in a microcentrifuge and transfer 500 ul of the supernatant carefully into a new 2 0 ml Receiver Tube Add 330 ul 96 100 ethanol and mix thoroughly by pipetting up and down It is important to mix the lysate completely with ethanol 19 InviTrap Spin Universal RNA Mini Kit 1015 3 Binding of the total RNA to the RTA Spin Filter Transfer the lysate completely to the RTA Spin Filter Set Incubate for 1 min and centrifuge for 2 min at 11 000 x g 11 000 rpm Discard the flow through and place the RTA Spin Filter back into the RTA Receiver Tube 4 First washing of the RTA Spin Filter Add 600 ul Wash Buffer R1 onto the RTA Spin Filter and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the flow through and the RTA Receiver Tube Transfer the RTA Spin Filter in a new RTA Receiver Tube 5 Second washing of the RTA Spin Filter Add 700 ul Wash Buffer R2 onto the RTA Spin Filter and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the flow through and reuse the RTA Receiver Tube Repeat this washing step once Discard the flow through and reuse the RTA
63. ticles Wait a short time because of foam formation Cell lysates stored in Lysis Solution TR are stable at 80 C for several months Important tips Blood and body fluids of all human subjects are considered potentially infectious All necessary precautions recommended by the Food amp Drug Administration in the USA the Bundesseuchen gesetz in Germany or the appropriate regulatory authorities in the country of use should be taken when working with whole blood The maximum amount of human whole blood that can be processed 1 5 ml has been determined for blood from healthy adults approximately 4 000 7 000 leukocytes per microliter Reduce amount appropriately if using blood with elevated numbers of leukocytes A maximum of 1 x 10 leukocytes can be processed on a RTA Spin Filter After erythrocyte lysis all steps of this protocol should be performed at room temperature RT as quickly as possible Homogenized cell lysates from step 2 can be stored lysed under Lysis Solution TR 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 shortly prior isolation Avoid prolonged incubation which may compromise RNA integrity Continue with step 3 The use of frozen leukocyte pellets without addition of Lysis Solution TR is not recommended Frozen whole blood mustn t be used Equipment and reagents to be supplied by user When working with chemicals al
64. ure 8 Sampling and storage of starting material 9 Sample preparation 9 Procedure 10 Important notes 11 Important points before starting a protocol 11 Preparing reagents and buffers 11 Equipment and reagents to be supplied by user 12 Scheme Total RNA extraction from cell culture 13 Sample quantity 14 Important indications 15 Protocol 1 Total RNA extraction from cell culture 15 Scheme Total RNA extraction from up to 20 mg tissue 17 Influence of the different methods of sample homogenization on the yield using the InviTrap Spin Universal RNA Mini Kit with rat brain 18 Protocol 2 Total RNA extraction from up to 20 mg tissue 19 Protocol 3 Total RNA extraction from up to 20 mg spleen kidney or lung tissue 21 Protocol 4 Total RNA isolation from paraffin embedded tissue treated with formalin paraffin slices 23 Scheme Total RNA extraction from up to 1 5 ml of whole blood 24 Starting amounts of samples 25 Protocol 5 Total RNA isolation from 0 5 ml up to 1 5 ml of whole blood 26 Protocol 6 Total RNA isolation from buffy coat from up to 2 ml whole blood max 0 3 ml or from lt 1 x 10 leukocytes 28 Troubleshooting 29 Appendix 31 General notes on handling RNA 31 Storage of RNA 31 Quantification of RNA 31 Purity of RNA 32 Denaturing agarose gel electrophoresis of RNA 33 Ordering information 35 Supplemental not validated protocols not for diagnostic use 36 Supplemental protocol for simultaneous isolation of RNA and Proteins 36 Supplementa
65. ution Tube and add 30 60 ul of Elution Buffer R directly onto the membrane of the RTA Spin Filter Incubate for 2 min and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the RTA Spin Filter and place the eluted total RNA immediately on ice Note Depending on the yield or the needed concentration of total RNA you can also elute with different volumes of Elution Buffer R A lower volume of Elution Buffer R increases the concentration of RNA and a higher volume of Elution Buffer R leads to an increased yield but a lower concentration of total RNA Please note that the minimum of Elution Buffer R should be 30 ul 27 InviTrap Spin Universal RNA Mini Kit 1015 Protocol 6 RNA extraction from buffy coat from up to 2 ml whole blood max 0 3 ml or from lt 1 x 10 leukocytes 1 Treatment of buffy coat Add fresh prepared buffy coat with max 1 x 10 leukocytes in a 1 5 ml RNase free Receiver Tube not provided and spin the leukocytes down at 3000 x g for 3 min Remove carefully the supernatant and follow than the protocol 1 from step 2 Leukocyte disruption 2 Leukocyte pellet A common alternative to erythrocyte lysis is Ficoll density gradient centrifugation In contrast to erythrocyte lysis procedures Ficoll density gradient centrifugation only recovers mononuclear cells lymphocytes and monocytes and removes granulocytes Mononuclear cells isolated by Ficoll density gradient centrifugation can be processed with the InviTrap
66. ways wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDS See our webpage www stratec com o Microcentrifuge 12 000 rpm o Ethanol 96 100 o Tubes for erythrocytes lysis e g 15 ml Falcon Tubes o Sterile RNase free pipet tips o Disposable gloves o DIT Possible Eppendorf AG SIGMA Laborzentrifugen GmbH microcentrifuge 22331 Hamburg Germany 37507 Osterode am Harz Germany suppliers Phone 49 0 40 53801 0 Phone 49 5522 5007 0 Fax 49 0 40 53801 556 Fax 49 5522 5007 12 E Mail eppendorf eppendorf com E Mail info sigma zentrifugen de Internet www eppendorf com Internet www sigma zentrifugen de 12 InviTrap Spin Universal RNA Mini Kit 1015 Instructions Scheme Total RNA extraction from human and animal cell culture Please work quickly and perform all extraction steps at room temperature RT Please read the protocols carefully prior to the start of the preparation procedure Note Homogenize the Carrier in the Lysis Solution TR by shaking and mix it with DTT transfer the sample into a 1 5 ml reaction tube not provided and add 350 ul DTT containing Lysis Solution TR Note For 5 x 10 1 x 10 human or animal cells add 700 ul DTT Rey containing Lysis Solution TR mix thoroughly by pipetting up and down place a DNA Binding Spin Filter into a 2 0 ml Receiver Tube transfer the whole lysate
67. will lead to no RNA the same problem occurs if the sample was not fresh or was not stored correctly before the fixation procedure Also incorrect fixation of the sample for example residual water in the sample leads to no RNA Incorrect storage of the sample for example high temperatures humid surrounding lead to the same result 23 InviTrap Spin Universal RNA Mini Kit 1015 Scheme Total RNA extraction from up to 1 5 ml whole blood Please work quickly and perform all extraction steps at room temperature RT Please read the protocols carefully prior to the start of the preparation procedure invert the blood sample 15 times don t vortex add 10 ml pre cooled 4 C Buffer EL to 0 5 1 5 ml of whole blood in a 15 ml Tube not provided invert incubate on ice for 15 min invert 2 times during incubation centrifuge 960 x g 3 000 rpm 5 min 4 C and remove the supernatant add 5 ml Buffer EL to the pellet of leukocytes mix by snipping with the finger and centrifuge again 5 min at 960 x g 3 000 rpm at 4 C remove the supernatant that the white pellet is left Note Homogenize the carrier in the Lysis Solution TR by shaking and mix it with DTT resuspend the cell pellet in 900 ul Lysis Solution TR pipet several times transfer the sample into a 2 ml Receiver Tube incubate for 5 min vortex several times during incubation centrifuge for 1 min at 11 000 x g 11 000 rpm transfer the supernatant into a new 2 ml
68. with a bead mill etc generally results in higher RNA yields than with other methods Important Shake Lysis Solution TR gently before use Wait a short time because of foam formation Disruption using a rotor stator homogenizer vortexer bead mill gyrator 1 Transfer the weighed amount of fresh or frozen starting material in a suitable reaction vesicle for the homogenizer not provided and add about 10 Zirconia beads to the sample Zirconia Beads small Zirconia Beads II big ratio 2 1 Add 900 ul DTT containing Lysis Solution TR mixed before added Homogenize the sample Transfer the sample into the 2 ml Receiver Tube and place the sample under Lysis Solution TR for longer storage at 20 C or use the sample immediately for isolation of total RNA following the protocol step 2 RON Disruption of the starting material using a mortar and pestle and liquid nitrogen 1 Transfer the weighed amount of fresh or frozen starting material under liquid nitrogen and grind the material to a fine tissue powder 2 Transfer the powder into the 2 ml Receiver Tube Do not allow the sample to thaw 3 Add 900 ul DTT containing Lysis Solution TR mixed before added and incubate the sample for an appropriate time under continuously shaking until a homogenic lysate is visible 4 Finally place the sample under Lysis Solution TR for longer storage at 20 C or use the sample immediately for isolation of total RNA following protocol step 2
69. x g 11 000 rpm Discard the flow through and reuse the 2 ml Receiver Tube Repeat this washing step once Drying of the DNA Binding Spin Filter membrane Discard the flow through and put the DNA Binding Spin Filter back to the 2 ml Receiver Tube To eliminate any traces of ethanol centrifuge for 4 min at maximum speed Discard the 2 ml Receiver Tube Elution of genomic DNA Transfer the DNA Binding Spin Filter into a 1 5 ml Elution Tube and pipet 40 100 ul of Elution Buffer R or water directly onto the membrane of the DNA Binding Spin Filter Incubate for 2 min and centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the DNA Binding Spin Filter and place the eluted genomic DNA in a refrigerator 38 InviTrap Spin Universal RNA Mini Kit 1015 Supplemental protocol for digestion on the RTA Spin Filter If you want to get a totally DNA free sample for RT qPCR Applications or other protocols where smallest amounts of DNA might disturb you may follow this protocol and do a DNase digestion directly on the Spin Filter Therefore you have to change Step 7 in protocol 1 Second washing of the RNA RTA Spin Filter according to the following procedure Add 600 ul Wash Buffer R2 onto the RTA Spin Filter and centrifuge for 30 sec at 10 000 x g 10 500 rpm Discard the flow through and put the RTA Spin Filter back into the RTA Receiver Tube Add 10 ul of a DNase Reaction Mixture directly in the center of the Spin Filter membrane A typical r

InviTrap Spin Universal RNA Mini Kit User manual

Contents

Download Pdf Manuals

Related Search

Related Contents