Invisorb® Spin Cell RNA Mini Kit
Contents
1. 50 total RNA extractions adjust Lysis Solution R with 1 100 volume of 1 M DTT preparation of 50 ml 70 ethanol add 20 ml 96 100 ethanol to the bottle Wash Buffer R1 add 48 ml 96 100 ethanol to each bottle Wash Buffer R2 preparation of TE Buffer 10 mM Tris HCI 1 mM EDTA pH 8 0 only for bacteria preparation of a 1 M Sorbitol 0 1 M EDTA solution pH 7 4 only for yeast 250 total RNA extractions adjust Lysis Solution R with 1 100 volume of 1 M DTT preparation of 200 ml 70 ethanol add 80 ml 96 100 ethanol to the bottle Wash Buffer R1 add 160 ml 96 100 ethanol to each bottle Wash Buffer R2 preparation of TE Buffer 10 mM Tris HCI 1 mM EDTA pH 8 0 only for bacteria preparation of a 1 M Sorbitol 0 1 M EDTA solution pH 7 4 only for yeast 11 InviTrap Spin Cell RNA Mini Kit 0515 DTT Adjust Lysis Solution R 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 R with DTT in case of the kits with 50 and 250 preparations We recommend the preparation of a volume DTT containing Lysis Solution R shortly before carrying out the purifications adapted to the number of samples that will be processed Store the remaining Lysis Solution R and DTT separately in accordance to the storage instructions see Storage page 6 It is possible to replace DTT by B Mercaptoethanol In that case adjust Lysis Solution R with 1 100 v2. Oo 0 0 0 0 O O This kit should only be used by personnel trained in in vitro diagnostic laboratory practice Storage of RNA Purified viral RNA can be stored at 80 C and is stable for several years at this condition 27 InviTrap Spin Cell RNA Mini Kit 0515 Quantification of RNA The concentraton of RNA should be determinated 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 spectrophotometry This can be accomplished by washing cuvettes with 0 1 N 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 An example of the calculation involved in RNA quantification o volume of RNA sample 60 ul o example for dilution 5 ul of RNA 45 ul of ddH2O 1 10 dilution for measurement in a 50 ul cuvette Example Azgo 0 2 Concentration of the RNA sample 40 ug ml A260 dilution factor 40 ug ml 0 2 10 80 ug ml Total amount concentration volume of sample in ml 80 ug ml 0 06 ml 4 8 ug of RNA Purity of RNA Th
3. is a good indication that the RNA is intact Partially degraded RNA will have a smeared appearance 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 N NW AUO un Oo un Figure 1 Intact vs Degraded RNA Two yg 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
4. 5x 10 48 well 1 0 1 3 x 10 12 well 4 0 5 0x 10 6 well 9 5 1 2x 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 become 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 Bacteria cells from up to 1 x 10 The achievable yield of purified total RNA depends strongly on the type of bacteria the culture medium and the growth phase Up to 1 x 10 cells taken from minimal media can be used The use of higher numbers of cells results in inefficient lysis and a reduced yield of isolated RNA The determination of the cell number in a given bacteria culture is often done photometric by measurement of the optical density OD The measured OD should be below 0 3 to exclude a loss of significance and therefore incorrect values of cell density Due to the varying effects of the bacterial species the growth phase and the type of culture medium to the light scattering properties of the sample the measured OD values and calculated cell numbers should be ensured by parallel counting of viable cells on agar plates before starting the first RNA purification procedure 9 InviTrap Spin Cell RNA Mini Kit 05
5. DTT containing Lysis Solution R 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 trough for the following cleanup procedure Just add 200 ul of the sample reaction mixture to the flow through Lysis Solution R 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 R 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 R to 200 ul RNA sample Mix thoroughly by pipetting up and down Important Shake Lysis Solution R gently before use Wait a short time because of foam formation 3 Binding of genomic DNA to the DNA Binding Spin 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 yellow without lid placed in a 2 ml Receiver Tube with lid 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 b
6. R1 Add 48 ml 96 100 Add 160 ml 96 100 ethanol ethanol to each bottle to each bottle Wash Buffer Wash Buffer R2 R2 Adjust Lysis Solution R with 1 100 volume of 1M DTT Due to the instability of dissolved DTT under oxidative conditions do not mix the whole Lysis Solution R with DTT in case of the kits with 50 and 250 preparations We recommend the preparation of a volume DTT containing Lysis Solution R shortly before carrying out the purifications adapted to the number of samples that will be processed Store the remaining Lysis Solution R and DTT separately in accordance to the storage instructions see Storage page 6 It is possible to replace DTT by B Mercaptoethanol In that case adjust Lysis Solution R with 1 100 volume of B Mercaptoethanol as described above Preparation of TE buffer 10 mM Tris HCI 1 mM EDTA pH 8 0 only for bacteria Preparation of 1 M Sorbitol 0 1 M EDTA solution pH 7 4 only for yeast 4 InviTrap Spin Cell RNA Mini Kit 0515 Symbols Ass Manufacturer Lot Lot number Catalogue number Expiry date Consult operating instructions Temperature limitation ox BRE Do not reuse Storage All buffers except the 1 M DTT solution and kit components of the InviTrap Spin Cell RNA Mini Kit should be stored well sealed and dry at room temperature and are stable for at least 12 months under these conditions Store the 1 M DTT solution at 20 C to prevent oxida
7. a 15 min denaturation is recommended when running RNA for a Northern Blot The longer incubation may be necessary to completely denature the RNA To 1 3 ug RNA add 0 5 3x volumes Formaldehyde Load Dye To simply check the RNA on a denaturing gel as little as 0 5x Formaldehyde Load Dye can be used but to completely denaturate the RNA e g for Northern blots use 3X 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 29 InviTrap Spin Cell RNA Mini Kit 0515 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
8. at 2 500 x g 5 000 rpm in a centrifuge tube Discard supernatant and remove all media completely 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 flick the tube and add DTT containing Lysis Solution R 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 R Number of pelleted cells 350 ul lt 5x 10 cells 700 ul 5 x 10 1x 10 cells Monolayer cells Add DTT containing Lysis Solution R 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 R Size of the culture vessels 350 ul 96 24 12 well plates 700 ul 6 well plates 35 mm dishes 12 5 cm flasks Important Shake Lysis Solution R 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 14 InviTrap Spin Cell RNA Mini Kit 0515 3 Binding of the genomic DNA to the DNA Binding Spin Filter Pipet the lysate resulting from step 2 includin
9. 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 Cell 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 Cell RNA Mini Kit to which they apply are listed below as follows Lysis Solution R Wash Buffer R1 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 hours a day from infotrac outside of USA 1 352 323 3500 in USA 1 800 535 5053 7 InviTrap Spin Cell RNA Mini Kit 0515 Product characteristics of the InviTrap Spin Cell RNA Mini Kit Time for Starting material preparation up to 1 x 10 human or animal cells up to 150 yg total RNA 15 20 minutes A260 A 280 cultured cells 1 7 2 1
10. 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 9 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 10 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 11 100 x g 11 000 rpm Discard the RNA RTA Spin Filter and place the eluted total RNA immediately on ice 19 InviTrap Spin Cell RNA Mini Kit 0515 Protocol 4 RNA Cleanup 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 of DTT containing Lysis Solution R 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 R Then continue with step 4 B Purification of 200 ul sample from contaminations Pipet 700 ul of
11. 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 21 InviTrap Spin Cell RNA Mini Kit 0515 Protocol 5 RNA Cleanup from Trizol aqueous phase 1 Starting the cleanup procedure Add 350 ul DTT containing Lysis Solution R to up to 350 ul of the Trizol aqueous phase Mix thoroughly by pipetting up and down Important Shake Lysis Solution R gently before use Wait a short time because of foam formation 2 Binding of the genomic DNA to the DNA Binding Spin Filter Pipet the whole lysate resulting from step 1 including any precipitate which may have formed directly onto the DNA Binding Spin Filter yellow without lid 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 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 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 2 min Discard the flow through and reuse the RTA Receiver Tube If the volume of the RNA containing sample exceeds
12. quantities and quality If there are any unconformities you have to 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 7 Do not use damaged kit components since their use may lead to poor kit performance o Always change pipet tips between liquid transfer to avoid cross contaminations 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 O 0 0 Preparing reagents and buffers 5 total RNA extractions adjust Lysis Solution R with 1 100 volume of 1 M DTT preparation of 10 ml 70 ethanol wash Buffer R1 and R2 are ready to use preparation of TE Buffer 10 mM Tris HCI 1 mM EDTA pH 8 0 only for bacteria preparation of a 1 M Sorbitol 0 1 M EDTA solution pH 7 4 only for yeast
13. see appendix General Notes on handling RNA page 34 total RNA does not perform well in downstream applications RT PCR ethanol carryover during elution salt carryover during elution increase g force or centrifugation time when 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 26 InviTrap Spin Cell RNA Mini Kit 0515 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 glasswa
14. up to 1 x 10 cells of Gram positive or a NT Rbi from Gram negative bacteria X ibroblasts up to 5 x 10 yeast cells The InviTrap Spin Cell RNA Mini Kit provides a fast and efficient way for reliable isolation of high quality total RNA as well as the isolation of the proteins from samples of human or animal cells cell culture bacteria or yeast The InviTrap Spin Cell RNA Mini Kit can also be used for an efficient RNA cleanup and the simultaneous isolation of total RNA and proteins The InviTrap Spin Cell RNA Kit simplifies total RNA isolation by combining efficient lysis of the starting material and the inactivation of exogenous and endogenous RNases with the efficient removal of the genomic DNA The genomic DNA is fixed at the surface of the mineral carrier particles provided within the Lysis Solution R simultaneously to the cell lysis Selective binding of the DNA is assured by optimized buffer conditions and the mineral carrier particles loaded with the genomic DNA are removed by centrifugation on the spin filter surface The RNA remains in the filtrate and is after the adjustment of special binding conditions bound onto the membrane of the RNA RTA Spin Filter RNA Binding Spin Filter Contaminants are removed by repeated 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 The extracted RNA contains enriched mRNA The par
15. 100 7061300200 7061300300 7061300400 1460100 1061114 7460300 1060100900 1060100200 1060100300 InviTrap Spin Cell RNA Mini Kit 0515 stratecee molecular STRATEC Molecular GMbH Robert R ssle 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 1A5c01 05 2015
16. 15 Yeast cells from up to 5 x 10 In strong connection to the type of strain and the growth conditions 30 100 ug RNA can be purified from 5 x 10 yeast cells To get optimal results in RNA purification yeast cells should be harvested in the log phase growth Although the photometrical determination of the cell density of yeast cultures is common an additional plating experiment is recommended to ensure the measured values of light scattering before starting the first RNA purification procedure An enzymatic or mechanical disruption of yeast cell is possible This manual contains a protocol for enzymatic breakage of the yeast cell wall using Lyticase Principle and procedure The InviTrap Spin Cell RNA Mini Kit procedure comprises the following steps o cell lysis o selective binding of the genomic DNA to a specific carrier and additional separation of the bound DNA on the surface of the DNA Binding Spin Filter e adjustment of the RNA binding conditions and transfer of the sample into the RNA RTA Spin Filter RNA Binding Spin Filter o binding of the total RNA to the membrane while proteins remain in the filtrate o washing of the membrane and elimination of contaminants and ethanol o elution of highly 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 RNase free water This manual contains 6 protocols 2 supplemen
17. 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 put the RNA RTA Spin Filter in a new RTA Receiver Tube 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 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 1 100 x g 11 000 rpm Discard the RNA RTA Spin Filter and place the eluted total RNA immediately on ice 22 InviTrap Spin Cell RNA Mini Kit 0515 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 the same sample is necessary Following the given protocol below the simultaneous preparation of RNA an
18. A DNase digestion is not necessary to remove the genomic DNA Under the provided lysis conditions the DNA is efficiently bound by the mineral carrier particles contained in Lysis Solution R The DNA containing carrier remains at the surface of the membrane of the DNA Binding Spin Filter by subsequent centrifugation while the RNA remains in the filtrate If a DNase digestion is required e g caused by overloading the kit this step is carried out after the binding of the RNA to the spin filter All DNA fragments and the DNase will be removed completely in the following wash steps see page 26 10 InviTrap Spin Cell RNA Mini Kit 0515 Binding total RNA To adjust the RNA binding conditions 70 ethanol is added to the RNA containing filtrate Following carefully mixing the solution is completely transferred onto the RNA 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 of the RNA RTA Spin Filter Elution High quality total RNA is eluted from the membrane using 40 100 ul Elution Buffer R or RNase free water The eluted RNA is ready to use in different subsequent applications 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
19. A Spin Filter For special needs applications or other protocols where smallest amounts of DNA might disturb please follow this protocol and do a DNase digestion directly on the RNA RTA Spin Filter Therefore you have to change Step 7 in protocol 1 Second Washing Step or you have to change Step 8 in Protocol 2 and 3 according to the following procedure Second washing of the Spin Filter Add 550 ul Wash Buffer R2 onto the RNA RTA Spin Filter with the ound RNA and centrifuge for 30 sec at 11 000 x g 11 000 rpm Discard the flow through and put the RNA 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 reaction mix is for example 1 ul of DNase I 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 RNA RTA Spin Filter at room temperature for 10 min Add again 550 ul Wash Buffer R2 onto the RNA RTA Spin Filter incubate for 1 minute and centrifuge for 1 min at 11 000 x g 11 000 rpm Reuse the RTA Receiver Tube and repeat this washing step once again 24 InviTrap Spin Cell RNA Mini Kit 0515 Troubleshooting Problem Probable cause Comments and suggestions clogged Spin Filter too much starting material insufficient disruption or homogenisation of the starting material very viscous lysate like a gel
20. ANS 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 blood tissue nor for viruses 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 Proteinase digestion RNA ligation or labeling reactions The included chemicals are only useable 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 o
21. after addition of Lysis Solution R reduce the amount of starting material preparations increase g force and or centrifugation time in subsequent after lysis spin lysate to pellet debris and continue with the protocol using the supernatant all centrifugation steps should be conducted at room temperature if too many cells including DNA has 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 700 ul with Lysis Solution R continue with the procedure from step 3 Aaseo A2go Value below 1 7 RNA sample is diluted in H2O with wrong pH RNA sample is to strong diluted use less sample repeat washing step one do not use RNase free water to dilute the sample for measuring the RNA purity the use of a neutral buffer 10 mM Tris HCl pH 7 0 is recommended see also Quantification of RNA page 35 sample concentration is out of the range of the photometer little or no total RNA eluted sample stored incorrectly incomplete removal of cell culture medium insufficient disruption or homogenisation incomplete elution the sample should be stored at 80 C make sure that the cell culture medium is complete removed after the cell harvest reduce the amount of starting material overloading the kit reduces the yield prolong the incubation time with Elution Buffer R to 5 10 m
22. ard the RTA Receiver Tube transfer the RNA RTA Spin Filter into a RNase free Elution Tube pipet 40 100 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 11 000 x g 11 000 rpm discard the RNA RTA Spin Filter place the eluted total RNA immediately on ice Note The centrifugation steps were made with the Centrifuge 5415 D from Eppendorf The indicated rpm amounts are referring to this centrifuge 13 InviTrap Spin Cell RNA Mini Kit 0515 Instructions Protocol 1 Total RNA extraction from human and animal cell culture 1 Harvesting cells see also Sample quantity page 10 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 Bacteria Spin up to 1 x 10 bacteria cells for 5 min at 2 500 x g 5 000 rpm in a centrifuge tube Discard the supernatant and remove all media completely Yeast cells Spin up to 5 x 10 yeast cells for 5 min
23. d proteins is possible using the InviTrap Spin Cell 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 RNA RTA Spin Filter in the purification protocol see Protocol 1 step 5 Protocol 2 3 step 6 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 13 400 x g 12 000 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 3 min at 11 000 x g 11 000 rpm 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 Buffers poisonous gas will be generated 23 InviTrap Spin Cell RNA Mini Kit 0515 Supplemental Protocol for DNA digestion on the RNA RT
24. e ratio of the readings at 260 nm and 280 nm A260 A280 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 Axgo Azgo 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 diluting the sample ina buffer with neutral pH since the relationship between absorbance and concentration Ass reading of 1 40 ug ml of RNA is based on an extinction coefficient calculated for RNA at neutral pH Using an Agilent Bioanalyzer RNA of good quality shows RIN of 7 10 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 spectrophotometers 28 InviTrap Spin Cell RNA Mini Kit 0515 Denaturing agarose gel electrophoresis of RNA The overall quality of an RNA preparation may be assessed by electrophoresis on a denaturing a
25. edical devices is not recognized Trademarks InviTrap Invisorb Eppendorf 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 2 InviTrap Spin Cell RNA Mini Kit 0515 Contents Kit contents of the InviTrap Spin Cell RNA Mini Kit Symbols Storage Quality control Intended use Product use limitation Safety information Product characteristics of the InviTrap Spin Cell RNA Mini Kit Important indications Principle and procedure Sampling and storage of starting material Procedure Important notes Important points before starting a protocol Preparing reagents and buffers Equipment and reagents to be supplied by user Scheme InviTrap Spin Cell RNA Mini Kit Instructions Protocol 1 Total RNA extraction from human and animal cell culture Protocol 2 Total RNA extraction from Gram positive or Gram negative bacteria Protocol 3 Total RNA extraction from yeast Protocol 4 RNA Cleanup from liquid enzy
26. f 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 6 InviTrap Spin Cell RNA Mini Kit 0515 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 material 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
27. g any precipitate which may have formed directly onto the DNA Binding Spin Filter yellow without lid 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 4 Adjust RNA binding conditions Add 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 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 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 el
28. garose gel this will also give some information about RNA yield A denaturing gel system is suggested because 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
29. iminate 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 15 InviTrap Spin Cell RNA Mini Kit 0515 Protocol 2 Total RNA extraction from Gram positive or Gram negative bacteria 1 Harvest bacteria see also Sample quantity page 10 Spin up to 1 x 10 bacteria cells for 5 min at 2 500 x g 5 000 rpm in a centrifuge tube Discard the supernatant and remove all media completely 2 Lysis of the bacterial cell wall with Lysozyme Important Immediately before use dissolve Lysozyme concentration according to table 3 in TE buffer 10 mM Tris HCl 1 mM EDTA pH 8 0 To loosen the bacteria cell pellet flick the tube and add 100 ul Lysozyme containing TE buffer Mix gently by vortexing and incubate at RT Different Lysozyme concentrations and incubation times are required for Gram positive and Gram negative bacteria see table 3 The amount of Lysozyme and the incubation time may also be modified depending on the bacterial strain A careful optimization of cell wall digestion is the basis for high total RNA yield table 3 Lysozyme concentration
30. in or repeat elution step once again elute a second time with 100 ul Elution Buffer R incubate RTA Spin Filter at room temperature 15 25 C for 5 min with RNase free water or Elution Buffer R prior centrifugation no alcohol added to the Wash Buffer R1 and or Wash Buffer R2 check that Wash Buffer R1 and or Wash Buffer R2 concentrates were diluted with correct volume of 96 100 ethanol Do not use denatured alcohol which contains other substances such as methanol or methylethylketone repeat the purification procedure with a new sample 25 InviTrap Spin Cell RNA Mini Kit 0515 Problem Probable cause Comments and suggestions DNA contamination too much starting material no optimal homogenization of DNA binding carrier reduce amount of starting material DNase digestion of the eluate containing the total RNA shake Lysis Solution R carefully before use RNA degraded inappropriate handling of the starting material Lysis Solution does not contain DTT RNase contamination the RNA purification protocol should be performed quickly see also General notes on handling RNA page 34 cell pellets stored at 80 C for later processing should be immediately frozen after cell harvest by liquid nitrogen treatment or under Lysis Solution check for RNase contamination of buffers be certain not to introduce any RNase throughout the procedure or during further handling for analysis
31. in TE Buffer Incubation time at RT Gram negative bacteria 400 ug ml 5 10 min Gram positive bacteria 3 000 pg ml 10 30 min 3 Disrupt bacteria cells by adding Lysis Solution R Add 350 ul DTT containing Lysis Solution R to the Lysozyme treated sample Mix thoroughly by pipetting up and down No cell clumps should be visible before proceeding with the next step Important Shake Lysis Solution R 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 4 Binding of genomic DNA to the DNA Binding Spin Filter Pipet the lysate 450 ul including any precipitate which may have formed directly onto the DNA Binding Spin Filter yellow without lid placed in a 2 ml Receiver Tube with lid Incubate for 1 min and centrifuge at 11 100 x g 11 000 rpm for 2 min Discard the DNA Binding Spin Filter 5 Adjust RNA binding conditions Add 250 ul 96 100 ethanol to the RNA containing lysate and mix thoroughly by pipetting and down 6 Binding of the total RNA to the RNA RTA Spin Filter Transfer the RNA containing sample resulting from step 5 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 aliqu
32. inding conditions Add 250 ul 100 ul sample volume respectively 500 ul 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 20 InviTrap Spin Cell RNA Mini Kit 0515 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 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 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
33. integrity Alternative nucleic acid stains such as SYBR Gold and SYBR Green II RNA gel stain from 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 30 InviTrap Spin Cell RNA Mini Kit 0515 Ordering information Product InviTrap Spin Cell RNA Mini Kit InviTrap Spin Cell RNA Mini Kit InviTrap Spin Cell RNA Mini Kit Package size 5 preparations 50 preparations 250 preparations Single components for InviTrap Spin Cell RNA Mini Kit Lysis Solution R Wash Buffer R1 add 20 ml ethanol Wash Buffer R2 add 48 ml ethanol Elution Buffer R Related products InviTrap RNA Cell HTS 96 Kit C InviTrap RNA Cell HTS 96 Kit C InviTrap RNA Cell HTS 96 Kit C InviMag RNA Universal Mini Kit InviMag RNA Universal Mini Kit KFmL InviMag RNA Universal Mini Kit KF96 KFflex InviTrap Spin Universal RNA Mini Kit InviTrap Spin Universal RNA Mini Kit InviTrap Spin Universal RNA Mini Kit 30 ml 20 ml 12 ml 15 ml 2 x 96 preparations 4 x 96 preparations 24 x 96 preparations 10 50 250 preps 15 75 preps 1 x 96 5 x 96 preps 10 preparations 50 preparations 250 preparations 31 Catalogue No 1061100100 1061100200 1061100300 1061101700 1061103500 1061103600 1061104
34. ly prior to the start of the preparation procedure Note Homogenize the carrier in the Lysis Solution R by shaking Mix it with DTT transfer the sample into a 1 5 ml reaction tube not provided and add 350 ul DTT containing Lysis Solution R Note For 5 x 10 1 x 10 human or animal cells add 700 ul DTT containing Lysis Solution R mix thoroughly by pipetting up and down place a DNA Binding Spin Filter into a 2 0 ml Receiver Tube transfer the whole lysate onto the DNA Binding Spin Filter incubate for 1 min at RT and centrifuge for 2 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 1 250 ul 96 100 ethanol see Protocol 2 and 3 to the filtrate and mix thoroughly by pipetting up and down transfer the sample on the RNA 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 reuse the RTA Receiver Tube pipet 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 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 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 disc
35. me reactions Protocol 5 RNA Cleanup from Trizol aqueous phase Supplemental protocol for simultaneous isolation of RNA and proteins Supplemental protocol for DNA digestion on the RNA RTA Spin Filter Troubleshooting Appendix General notes on handling RNA Storage of RNA Quantification of RNA Purity of RNA Denaturing agarose gel electrophoresis of RNA Ordering information ONODOD Aa ao RA O CO ON NN OPWBN OWA DA HPWNH H H HO CO SO 3 InviTrap Spin Cell RNA Mini Kit 0515 Kit contents of the InviTrap Spin Cell RNA Mini Kit Store 1 M DTT not provided at 20 C All other kit components are stable at room temperature RT 5 total RNA extractions 50 total RNA extractions 250 total RNA extractions 1061100100 1061100200 1061100300 Catalogue No Lysis Solution R 4 ml 40 ml 190 ml 15 ml 20 ml 80 ml eee ready to use final volume 40 ml final volume 160 ml 15 ml 2x12 ml 2 x 40 ml Was BURRE ready to use final volume 2x60 ml final volume 2 x 200 ml Elution Buffer R 2ml 15 ml 60 ml DNA Binding Spin Filter 5 50 2X30 RNA RTA Spin Filter Set 3 oe FEN 2 0 ml Receiver Tubes 5 50 5 x 50 RTA Receiver Tubes 5 50 5 x 50 Elution Tubes 5 50 5 x 50 Manual 1 1 1 1 1 i O Initial steps Preparation of 10 ml 70 Preparation of 50 ml 70 Preparation of 200 ml 70 ethanol ethanol ethanol Add 20 ml 96 100 Add 80 ml 96 100 ethanol to ethanol to the bottle the bottle Wash Buffer R1 Wash Buffer
36. olume of B Mercaptoethanol as described above Lysis Solution R Shake Lysis Solution R gently before use to homogenize the DNA binding mineral carrier particles Wait a short time because of foam formation Cell lysates stored in Lysis Solution R are stable at 80 C for several months Equipment and reagents to be supplied by user When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDS See our webpage www stratec com Microcentrifuge 2 13 400 x g 12 000 rpm Ethanol 96 100 Tris only for bacteria EDTA only for bacteria and yeast Lysozyme only for bacteria Sorbitol only for yeast 8 Mercaptoethanol only for yeast Lyticase only for yeast Thermoshaker only for yeast Pipettes and filter tips DTT O 0 0 0 0 0 0 0 0 0 0 Possible suppliers Eppendorf AG 22331 Hamburg Germany Phone 49 0 40 53801 0 Fax 49 0 40 53801 556 E Mail eppendorf eppendorf com Internet www eppendorf com SIGMA Laborzentrifugen GmbH 37507 Osterode am Harz Germany Phone 49 5522 5007 0 Fax 49 5522 5007 12 E Mail info sigma zentrifugen de Internet www sigma zentrifugen de InviTrap Spin Cell RNA Mini Kit 0515 Scheme of the InviTrap Spin Cell RNA Mini Kit Please work quickly and perform all extraction steps at room temperature RT Please read the protocols careful
37. ots into the RNA RTA Spin Filter 16 InviTrap Spin Cell RNA Mini Kit 0515 7 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 put the RNA RTA Spin Filter in a new RTA Receiver Tube in step 8 8 Second washing of the RNA RTA Spin Filter Add 700 ul Wash Buffer R2 onto the RNA RTA Spin Filter followed by centrifugation 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 9 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 10 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 17 InviTrap Spin Cell RNA Mini Kit 0515 Protocol 3 Total RNA extraction from yeast 1 Harvest yeast cells see also Sample quantity page 10 Spin up to 5 x 10 yeast cells for 5 min at 2 500 x g 5 000 rpm in a centrifuge tube Discard the supernatant and remove all media comple
38. pin Cell RNA Mini Kit 0515 Important indications Sample quantity The maximal amount of pure total RNA that will be isolated depends on the applied starting material storage conditions and intracellular RNA level To assure a high 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 Cell RNA Mini Kit and to avoid an overloading the RNA binding membrane an RNA purification experiment with variation of the amount of starting material is recommended The RNA Mini kit binding membranes have a maximum binding capacity of approx 100 ug The InviTrap Spin Cell RNA Mini Kit purification procedure is optimized for the use of Human and animal cells max from 1 x 10 Average yield of total RNA in dependence on the type of cell line using the InviTrap Spin Cell RNA Mini Kit Cell Line Source Yield ug from 1 x 10 cells HeLa human cervical carcinoma 2 0 Jurkat human T cell leukemia 1 7 MRC 5 human 5 0 NIH 3T3 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
39. re You can also clean glassware with 0 1 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 plasticware 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 ddH O 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 To minimize the risk of infections from potentially infectious material we recommend working under laminar air flow until the samples are lysed
40. 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 4 Binding of genomic DNA to the DNA Binding Spin Filter Transfer the lysate from step 3 450 ul including any precipitate which may have formed directly onto the DNA Binding Spin Filter yellow without lid placed in a 2 ml Receiver Tube with lid Incubate for 1 min and centrifuge at 11 400 x g 11 000 rpm for 2 min Discard the DNA Binding Spin Filter 5 Adjust RNA binding conditions Add 250 ul 96 100 ethanol to the RNA containing lysate and mix thoroughly by pipetting up and down 6 Binding of the total RNA to the RNA RTA Spin Filter Pipet the RNA containing sample resulting from step 5 directly onto the RNA RTA Spin Filter Incubate for 1 min and centrifuge at 11 400 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 7 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 put the RNA RTA Spin Filter in a new RTA Receiver Tube 18 InviTrap Spin Cell RNA Mini Kit 0515 8 Second washing of the RNA RTA Spin Filter Add 700 ul Wash Buffer R2 onto the RNA RTA Spin Filter and
41. stratecee molecular User manual hviTr p Spin Cell RNA Mini Kit for purification of total RNA from up to1x 10 human amp animal cells up to 1 x 10 bacteria cells up to 5 x 10 yeast cells RNA clean up and simultaneous protein isolation 1061100x00 taal STRATEC Molecular GmbH D 13125 Berlin Instruction for the InviTrap Spin Cell RNA Mini Kit The InviTrap Spin Cell RNA Mini Kit is the ideal tool for the isolation of high quality total RNA from samples of human or animal cell cultures max 1 x 10 cells bacteria suspensions max 1 x 10 cells and yeast suspensions max 5 x 10 cells in a convenient 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 RNA Cleanup and simultaneous isolation of total RNA and proteins A DNase digestion step is not necessary The purified total RNA is ready to use for in vitro diagnostic use The kit is neither validated for the isolation of total RNA from serum plasma blood nor for viruses The performance of the kits in isolating and purifying total RNA from fecal samples has not been evaluated C 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 m
42. tal protocols see page 16 30 Sampling and storage of starting material Best results are obtained using freshly extracted human or animal cells bacteria or yeast 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 subsequent 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 Cells can be stored also in Lysis Solution R at 80 C after cell lysis 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 Procedure Lysis After complete removal of the culture medium the cells are directly charged with the DTT containing Lysis Solution R and thoroughly mixed Shake the Lysis Solution R to resuspended 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 intermolecular disulfide bridges Binding and removal of genomic DN
43. tely Note For enzymatic lysis of yeast the use of freshly harvested cells is recommended 2 Lysis of the yeast cell wall with Lyticase preparation of spheroplasts Important Immediately before use dissolve 50 U Lyticase 1 x 10 yeast cells and add 0 1 B Mercaptoethanol in 1 M Sorbitol 0 1 M EDTA solution pH 7 4 To loosen the yeast pellet flick the tube and add 100 ul Lyticase containing 1 M Sorbitol 0 1M EDTA solution pH 7 4 Mix gently by vortexing and incubate for 10 30 min at 30 C with gentle shaking thermoshaker The enzymatic lysis of yeast cells is based on the digestion of the cell wall with Lyticase optional Zymolase and the generation of spheroplasts in an isotonic buffer system Due to considerable differences among the various yeast strains the concentration of Lyticase the incubation time and the buffer composition given here can only be an example The creation of soheroplasts protects the total RNA from degradation until the transfer of the sample in RNA stabilizing conditions provided by Lysis Solution R see step 3 A careful optimization of soheroplast preparation is the basis for high total RNA yield 3 Disrupt yeast cells by adding Lysis Solution R Add 350 ul DTT containing Lysis Solution R to the Lyticase containing sample Mix thoroughly by pipetting up and down No cell clumps should be visible before proceeding with the next step Important Shake Lysis Solution R gently before use Wait a
44. ticle size distribution of purified RNA is similar to those gained using a CsCl gradient 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 0000000 The purification procedure is rapid and does not require a phenol chloroform extraction Only a minimum of manual work 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 Cell RNA Mini Kit The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG If you are interested in using the kit on a laboratory workstation please do not hesitate to contact our technical support 49 0 30 9489 2907 STRATEC Molecular also offers a system for the purification of RNA from cells in a 96 well format The InviTrap RNA Cell HTS 96 Kit C is designed for centrifuge use The InviTrap RNA Cell HTS 96 Kit R has been developed for use on a 96 well format using laboratory workstation For use with magnetic beads STRATEC Molecular offers the InviMag Universal RNA Mini Kit For technical support or further information please contact 49 0 30 9489 2901 2907 2910 or your local distributor 8 InviTrap S
45. tive 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 Cell 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 Cell RNA Mini Kit have been tested separately against predetermined specifications routinely on lot to lot to ensure consistent product q
46. uality If you have any questions or problems regarding any aspects of InviTrap Spin Cell 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 5 InviTrap Spin Cell RNA Mini Kit 0515 Intended use The InviTrap Spin Cell RNA Mini Kit is the ideal tool for reliable and fast manual isolation and purification of high quality total RNA from samples of human or animal cells cell culture bacteria or yeast suspensions For reproducible high yields an appropriate sample storage and quick operation under the rules for RNA operation is essential The purified RNA is ready to use for in vitro diagnostic analysis only For some studies a simultaneous investigation of the RNA and of the cell bacteria or yeast strain specific proteins synthesized during the cultivation period is necessary The kit includes an isolation protocol of RNA and intracellular proteins from the same sample The isolation protocol and 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 TECHNICI
Download Pdf Manuals
Related Search